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KINETICS OF ENZYMATIC DEHAIRING USING PROTEASE ENZYME & MANUFACTURE OF MALEIC ANHYDRIDE
A PROJECT REPORT
Submitted by BHOOMA MADHAVAN (41502203005) MATHURA JEYARATNASINGAM (41502203008) In partial fulfillment for the award of the degree Of BACHELOR OF TECHNOLOGY In CHEMICAL ENGINEERING
S.R.M. ENGINEERING COLLEGE, KATTANKULATHUR-603 203, KANCHEEPURAM DISTRICT. ANNA UNIVERSITY: CHENNAI-600 025 MAY 2006 ANNA UNIVERSITY: CHENNAI 600 025
BONAFIDE CERTIFICATE
Certified that this project report KINETICS OF ENZYMATIC DEHAIRING USING PROTEASE ENZYME & MANUFACTURE OF MALEIC ANHYDRIDE is the bonafide work of BHOOMA MADHAVAN(41502203005), MATHURA JEYARATNASINGAM(41502203008) supervision. who carried out the project work under our
Dr. R. KARTHIKEYAN PROFESSOR HEAD OF THE DEPARTMENT CHEMICAL ENGINEERING S.R.M Engineering College Kattankulathur 603203 Kancheepuram District
Dr. B. S. M. KUMAR PROFESSOR SUPERVISOR CHEMICAL ENGINEERING S.R.M Engineering College Kattankulathur 603203 Kancheepuram District
ACKNOWLEDGEMENT The authors would like to record their deep sense of gratitude to Mr N K Chandra Babu, Scientist, Central Leather Research Institute for their valuable guidance and encouragement throughout the course of this project work. The support of Mrs R Venba and Mr Jothi, Tannery division, CLRI, are gratefully acknowledged.
We would also like to thank Ms G Rajeshwari for her constant help, support and encouragement. We thank Dr T Ramasami, director, Central Leather Research Institute, for his kind permission to carry out this project in this institute. We acknowledge the invaluable help and guidance provided by the head of the Department, Dr R Karthikeyan and our internal guide, Dr B S M Kumar whose advice and suggestions have helped us to do our project successfully.
ABSTRACT Of all tannery operations; it is fleshing, dehairing and liming processes, that take place in the beamhouse, which are highly polluting. Conventional chemical dehairing methods contribute to 60-70% of the pollution problems arising in tanneries. Advances in biotechnology, mainly in the production of proteolytic enzymes have led to the development of the enzymatic dehairing process, which succeeds in effectively controlling the BOD and COD of the effluent. This is because all the protein in the effluent is broken down to nitrogen, water, carbon dioxide and other volatile end products. This project deals with the study of kinetics of the enzymatic dehairing process using casein as the substrate. Enzymes are biocatalysts and their use should be promoted in the tannery. In this project, the effects of pH, temperature, time and substrate concentration on protease activity were studied. The kinetics studies reveal that the enzyme-catalyzed reaction is dependent on the substrate concentration.
TABLE OF CONTENTS CHAPTER NO. TITLE PAGE NUMBER ACKNOWLEDGEMENT ABSTRACT LIST OF TABLES LIST OF FIGURES LIST OF SYMBOLS Introduction 1.1 Background 1.1.1 Tannery production process 1.2 Pollution in tanneries 1.3 Dehairing 1.3.1 Clipping process 1.3.2 Scalding process 1.3.3 Sweating process 1.3.4 Chemical process 1.3.5 Enzymatic unhairing process 1.3.5.1 Biocatalysts 1.3.5.1.1 Classification of enzymes 1.3.5.2 Proteins alone act as enzymes 1.3.5.2.1 General properties 1.3.5.3 Proteases 1.4 Advantages of Enzymatic Dehairing 2 Experimental work 14 14 15 17 18 iii iv vii ix xi 1 1 1 5 6 7 7 7 8 9 10 11
2.1 Aim 2.2 Materials and Methods 2.3 Assay for Protease 2.3.1 Tyrosine standard graph 2.3.2 Method for determination of Protease Activity 2.4 Experimental Procedures 2.4.1 Effect of protease activity at different time intervals 2.4.2 Effect of Substrate Concentration on Protease Activity 2.4.3 Effect of pH on Protease Activity 2.4.4 Effect of Temperature on Protease Activity 2.4.5 Determination of Km and Vmax of protease catalyzed reaction using Kinetic Studies 2.5 Results and Discussion 2.5.1 Effect of Time on Protease Activity 2.5.2 Effect of Substrate Concentration on Protease Activity 2.5.3 Effect of pH on Protease Activity 2.5.4 Effect of Temperature on Protease Activity 2.5.5 Enzyme Kinetics 2.5.5.1 Kinetic Study of Protease 3 4 Conclusion Bibliography
18 18 18 21 22 23 23 23 24 24 24 25 26 36 37 39 41 43 52 53
LIST OF TABLES
TABLE NO. 1.1 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17
TITLE Tanning process Effect of time for 15 minute time interval Effect of time for 30 minute time interval Effect of time for 45 minute time interval Effect of time for 60 minute time interval Effect of time for 120 minute time interval Effect of time for 180 minute time interval Effect of time for 240 minute time interval Effect of time for 300 minute time interval Effect of time for 360 minute time interval Effect of pH on protease activity Effect of temperature on protease activity Effect of substrate concentration for 15 minute time interval Effect of substrate concentration for 30 minute time interval Effect of substrate concentration for 45 minute time interval Effect of substrate concentration for 60 minute time interval Effect of substrate concentration for 75 minute time interval Effect of substrate concentration for 90 minute time interval
PAGE NO. 4 27 28 29 30 31 32 33 34 35 38 40 44 45 46 47 48 49
2.18 2.19
Effect of substrate concentration for 105 minute time interval Effect of substrate concentration for 120 minute time interval LIST OF FIGURES
50 51
FIGURE NO. 1.1 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 2.19 2.20 2.21 TITLE Tannery process description Tyrosine standard graph Effect of time on protease activity at different concentration Effect on activity for 15 minutes Effect on velocity for 15 minutes Effect on activity for 30 minutes Effect on velocity for 30 minutes Effect on activity for 45 minutes Effect on velocity for 45 minutes Effect on activity for 60 minutes Effect on velocity for 60 minutes Effect on activity for 120 minutes Effect on velocity for 120 minutes Effect on activity for 180 minutes Effect on velocity for 180 minutes Effect on activity for 240 minutes Effect on velocity for 240 minutes Effect on activity for 300 minutes Effect on velocity for 300 minutes Effect on activity for 360 minutes Effect on velocity for 360 minutes Effect of substrate concentration on protease activity PAGE NO. 3 21 26 27 27 28 28 29 29 30 30 31 31 32 32 33 33 34 34 35 35 37
2.22 2.23 2.24 2.25 2.26 2.27 2.28 2.29 2.30 2.31 2.32 2.33 2.34 2.35 2.36 2.37 2.38 2.39 2.40 2.41
Effect of pH on protease activity Effect of pH on velocity Effect of temperature on protease activity Effect of temperature on velocity Effect on velocity for 15min time interval Lineweaver-Burk plot for 15min time interval Effect on velocity for 30min time interval Lineweaver-Burk plot for 30min time interval Effect on velocity for 45min time interval Lineweaver-Burk plot for 45min time interval Effect on velocity for 60min time interval Lineweaver-Burk plot for 60min time interval Effect on velocity for 75min time interval Lineweaver-Burk plot for 75min time interval Effect on velocity for 90min time interval Lineweaver-Burk plot for 90min time interval Effect on velocity for 105min time interval Lineweaver-Burk plot for 105min time interval Effect on velocity for 120min time interval Lineweaver-Burk plot for 120min time interval
38 39 41 41 44 44 45 45 46 46 47 47 48 48 49 49 50 50 51 51
LIST OF SYMBOLS
S. NO. 1 2 3 4
SYMBOL Km V [S] T
EXPANSION Michaelis-Menten constant Velocity Substrate concentration Temperature
CHAPTER 1 INTRODUCTION 1.1 BACKGROUND The tannery operation consists of converting the raw hide or skin, a highly putrescible material, into leather, a stable material, which can be used in the manufacture of a wide range of products. The whole process involves a sequence of complex chemical reactions and mechanical processes. Leather tanning is the process of converting raw hides or skins into leather. The surface of hides and skins contains the hair and oil glands and is known as the grain side. The flesh side of the hide or skin is much thicker and softer. Hides and skins have the ability to absorb tannic acid and other chemical substances that prevent them from decaying, make them resistant to wetting, and keep them supple and durable. 1.1.1 TANNERY PRODUCTION PROCESSES The production processes in a tannery can be split into four main categories: Hide and skin storage and beamhouse operations Tanyard operations Post-tanning operations and Finishing operations. After the hides and skins are flayed from the carcass at the abattoirs, they are delivered to the hide and skin market, directly to the tannery or to the fellmongery. Where necessary, hides and skins are cured before transport to the tannery in order to prevent the hides and skins from putrefying. Upon delivery to the site, hides and skins can be sorted, trimmed, cured and stored pending operations in the beamhouse. The following processes are typically carried out in the beamhouse of a tannery: soaking, unhairing, liming, fleshing and splitting. Typically, the following processes are carried out in the tanyard: deliming, bating, pickling, and tanning. The tanned hides and skins are tradable intermediate products (wet-blue) as they have been converted to a non-putrescible material called leather. Processes typically carried out in 9
post-tanning operations are: samming, setting, splitting, shaving, retanning, dyeing, fatliquoring and drying. At this stage the leather is called 'crust'. Crust is also a tradable intermediate product. Finishing operations include several mechanical treatments as well as the application of a surface coat. The selection of finishing processes depends on the specifications of the final product. Tanneries generally use a combination of the following processes: conditioning, staking, buffing, applying a finish, milling, plating and embossing.
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Figure 1.1 Tanning Process
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Flaying Curing Soaking Liming Dehairing Fleshing Splitting Deliming Bating Pickling Degreasing Tanning Dyeing Draining Sammaying Shaving Sorting
Removal of the skin from the animal Short term preservation of skins for transport or storage Re-hydration of cured skins to loosen hair, fat and flesh and open up the structure of the skin Removal of unwanted hair Removal of unwanted fat and flesh Into a grain and a flesh layer Reduction of pH To clean and soften the skin Adjustment of pH for transportation, storage or tanning Removal of unwanted grease Process which convert the raw hides into leather using vegetable, mineral, synthetic or combination methods The leather is dyed in desired colour. Removal of unwanted waste Reduction of water content Adjustment to required substance Leather material are graded according to the tanning process and hides
Table 1.1 Tanning process description 1.2 POLLUTION IN TANNERIES A considerable potential impact of tanning and associated activities on air, on surface and ground water, soil and resources arises from the chemicals applied, the raw materials used, and the effluents, waste and off-gas releases generated in the process. Wide attention is being paid to leather in the fashion world, and this has caused an
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increase in the production of leather. As a result, leather-developing techniques have been improved but insufficient attention has been paid to the environmental consequences of processing and manufacturing of leather. Leather processing and manufacturing involves a variety of adhesive chemicals and produces organic waste that frequently end up in the environment despite controlled effects. Being biological materials, the processing of hides and skins discharges a lot of proteinaceous wastes besides chemicals and tanning materials. The disposal of tannery effluent is a serious problem in the leather industry because of the large volume of water that is used in the different processes. Since untreated tannery effluents carry a good deal of toxic chemicals and some pathogenic microorganisms, they slowly penetrate the strata of soil and mix with the ground water. It is a fact that the major portions of proteinaceous waste and some toxic and sludgeforming chemicals come as effluents from pre-tanning processes. Since out of the total discharge of tanning industry, 60-80% of the effluent discharge is from the beamhouse operations, the problem can be solved to a great extent by reducing the volume of discharge. It is therefore necessary to rationalize the pre-tanning processes to solve the problem of tannery effluents and environmental pollution. The solution of the effluent problem has been suggested by using enzymes in soaking, unhairing and bating. This reduces the use of large volume of water, avoids the use of toxic and sludge-forming chemicals and rationalizes the pre-tanning processes. 1.3 DEHAIRING Dehairing is one of the main operations in the beamhouse. The process of unhairing is achieved by one of the two general methods viz. 1. by attacking the hair and reducing it to a pulp and 2. by destroying or modifying the epidermal tissue surrounding bulb, so that the hair is loosened and can be removed mechanically. Hair destruction methods involve the rupture of the disulphide and other bonds that stabilize the hard keratins of the epidermis while hair loosening methods have been observed to involve only a softening of the tissues that hold the hair in place. the hair
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Unhairing process having a commercial potentiality can be broadly classified into 2 main categories: hair destroying process and hair saving process. These are subdivided further as: Hair destroying process: (a) (b) (c) Liming process with lime alone Liming process with sharpeners like sulphides, amines, etc. Oxidative process with sodium chlorite, chlorine dioxide, etc.
Hair saving process: a) Sweating process b) Enzymatic unhairing process. Five methods of dehairing are generally adopted viz. (i) clipping process, (ii) scalding process, (iii) sweating process, (iv) chemical process and (v) enzymatic unhairing process. 1.3.1 Clipping process This is carried out by clipping the wool of the sheep by a suitable machine when the animal is either alive or dead. Although best grade wool is obtained by clipping, it generally results in loss of at least 15-20% wool. 1.3.2 Scalding process Thermal unhairing or scalding process is carried out by the immersion of soaked skins in warm water (55C - 60C) for a few minutes which brings about hair loosening without serious damage to the skin itself. Scalding is used for the removal of bristles from pig skins. The disadvantages of scalding are that it requires exact temperature and time and a slight change in these parameters damages the skin. 1.3.3 Sweating process The fresh or soaked skins are kept either by hanging in the air in piles inside suitable chambers under controlled conditions of temperature and humidity for about 4-5 days until the epidermis and hair roots are attacked by the enzymes secreted by bacteria and the loosening of hair takes place. The sweating process gives the maximum yield of high quality wool, but the process is slow, difficult to control and
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may damage the pelt if the enzyme action is not stopped in time. In actual practice, it is very tough to have a rigid control over the process. 1.3.4 Chemical process The chemical process consists of painting the flesh side of washed and soaked skins with a thin past composed of lime-sulphide mixture. The principal objectives of the chemical process using lime-sodium sulphide are a) To remove the hair by attacking epidermis which forms the outermost sheath in the structure if hides and skins b) To dissolve cementing substance and to loosen it from corium c) To saponify the skin lipids and to remove them d) To split up the fiber bundles and to open up the texture e) To swell the adipose tissue to facilitate removal of the adhering flesh. It is common practice to use sharpeners like sodium sulphide, arsenic sulphide, dimethylamine, sodium borohydride in the lime liquor and painting compositions. The main function of such additives is to help the easy removal of hair. The wool recovered by liming process is not good as it is damaged due to high alkalinity and strong action of sulphide. When lime and sulphide are used for painting, insoluble calcium proteinases are produced in the fibers, which cannot be removed by mere washing. If the alkaline action is not stopped in time, other decomposition reactions may occur and consequently damage the wool/hair. Some of the dyeing defects of the tannery wool are due to the long contact of the wool with alkali. In the lime-sulphide unhairing system, the waste lime sludge and effluent are known for the high degree of alkalinity and toxicity. Effluent problems arising due to LIME high alkalinity and suspended solids SULPHIDE liberation of hydrogen sulphide. Hydrogen sulphide is a serious hazard for both tannery workers and sewermen. Also, hydrogen sulphide is liable to be oxidized to sulphuric acid which causes damage to concrete and iron work. Dissolved sulphide and pulped hair contribute to high C.O.D and B.O.D of the effluent. Hence, to evolve suitable methods for effluent disposal and to protect the environment from pollution hazards, several attempts have been made to modify the
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hair-destroying methods by hair-saving methods like sweating and enzymatic unhairing. Of the two hair-saving processes, sweating process is very difficult to control and runs the risk of the skins being damaged by the action of putrefactive bacteria. Although no effluent disposal problem is encountered, the sweating process is lengthy and different sweating rates are observed in different areas of the skin leading to uneven sweating. 1.3.5 Enzymatic unhairing process With enzyme-assisted dehairing, it is possible to reduce the chemical requirements and obtain a cleaner product and a higher area yield with fewer chemicals in the wastewater. Since the enzyme does not dissolve the hair as the chemicals do, it is possible to filter out the hair, thus reducing the chemical and biological oxygen demand of the wastewater. Enzymes are biocatalysts produced by living organisms for the purpose of assisting chemical changes taking place in living matter. They are proteins by nature and have properties common to proteins. They are specific in action, in other words, each enzyme acts only on one substance or one substrate, as it is generally known. Those enzymes which hydrolyze proteins are called proteases, those which attack fats are lipases, and enzymes which hydrolyze starch are amylases. Proteolytic enzymes are derived from various sources viz. microbial, animal and plant sources. These when applied individually or in combinations produce effective unhairing of hides and skins. 1.3.5.1 BIOCATALYSTS Enzyme is a biological macromolecule that acts as a catalyst. Enzymes increase the rate of chemical reactions taking place within the cells without themselves suffering any overall change. The reactants of enzyme catalysed reactions are termed substrates and each enzyme is quite specific in character, acting on a particular substrate or substrates to produce a particular product or products( Robert 2003). All enzymes are proteins. However, without the presence of a non-protein component called a cofactor, many enzyme proteins lack catalytic activity. When this is the case, the inactive protein component of an enzyme is termed the apoenzyme,
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and the active enzyme, including cofactor, the holoenzyme. The cofactor may be an organic molecule, when it is known as coenzyme, or it may be a metal ion. Some enzymes bind cofactors more tightly than others. When a cofactor is bound so tightly that it is difficult to remove without damaging the enzyme, it is sometimes called a prosthetic group (Price and Stevens 1989, Robert 2003). 1.3.5.1.1 Classification Of Enzymes There are approximately 3000 enzymes, which have been characterized. These are grouped into six main classes according to the type of reaction catalysed (Robert 2003, T.Palmer 1995).
Oxidoreductases
These enzymes catalyse oxidation and reduction reactions involving the transfer of hydrogen atoms or electrons. The following are of particular importance in the design of enzyme electrodes. This group can be further divided into 4 main classes. o Dehydrogenases catalyse hydrogen transfer from the substrate to a nicotinamide adenine dinucleotide cofactor ( NAD+). An example of this is the lactate dehydrogenase which catalyses the following reaction: lactate + NAD+ = pyruvate + NADH + H+ o Oxidases catalyse hydrogen transfer from the substrate to molecular oxygen producing hydrogen peroxide as a by-product. An example of this is FAD dependent glucose oxidase which catalyses the following reaction: b-D-glucose + O2 = gluconolactone + H2O2 o Peroxidases catalyse oxidation of a substrate by hydrogen peroxide. An example of this type of enzyme is horseradish peroxidase which catalyses the oxidation of a number of different reducing substances (dyes, amines, hydroquinones etc.) and the concomitant reduction of hydrogen peroxide. The reaction below illustrates the oxidation of neutral ferrocene to ferricinium in the presence of hydrogen peroxide: 2[Fe(Cp)2] + H2O2 + 2H+ = 2[Fe(Cp)2]+ + 2H2O
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o Oxygenases catalyse substrate oxidation by molecular oxygen. The reduced product of the reaction in this case is water and not hydrogen peroxide. An example of this is the oxidation of lactate to acetate catalysed by lactate-2-monooxygenase. lactate + O2 = acetate + CO2 + H2O
Transferases
These enzymes transfer C, N, P or S containing groups (alkyl, acyl, aldehyde, amino, phosphate or glucosyl) from one substrate to another. Transaminases, transketolases, transaldolases and transmethylases belong to this groups.
Hydrolases
These enzymes catalyses cleavage reactions or the reverse fragment condensations. According to the type of bond cleaved, a distinction is made between peptidases, esterases, lipases, glycosidases, phosphotases and so on. Examples of this class of enzyme include cholesterol esterase, alkaline phosphotase and glucoamylase.
Lyases
These enzymes non-hydrolytically remove groups from their substrates with the concomitant formation of double bonds or alternatively add new groups across double bonds.
o o o o
Isomerases Racemases Epimerases Mutases Cis-trans-isomerases An example of this class of enzyme is glucose isomerase which catalyses the
These enzymes catalyse intramolecular rearrangements and are subdivided into:
isomerisation of glucose to fructose.
Ligases
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Ligases split C-C, C-O, C-N, C-S and C-halogen bonds without hydrolysis or oxidation. The reaction is usually accompanied by the consumption of a highenergy compound such as ATP and other nucleoside triphosphates. An example of this type of enzyme is pyruvate carboxylase which catalyses the following reaction: Pyruvate + HCO3- + ATP = Oxaloacetate + ADP +Pi An important aspect of catalytic action is the requirement by certain enzymes of either co-factors or prosthetic groups. Co-factors receive redox equivalents, protons or chemical groups from the substrate during the course of the enzymatic reaction. They tend to associate with the enzyme in a transient manner and can diffuse away from the active site. Examples of this type of molecule indicate NAD+ and NADP+. Prosthetic groups have similar function to co-factors with the exception that they are tightly bound to the enzyme. When they are released, the enzyme is mostly denatured. Flavin nucleotides and hemes are the most important examples of this class of molecule. 1.3.5.2 PROTEINS ALONE ACT AS ENZYMES The monomers of proteins are amino acids. There are 20 different amino acids. These amino acids have different types of side chains, which are hydrophilic and hydrophobic in nature. These side chains are useful for the formation of catalytic groups present in the enzyme molecule. The monomers of other biopolymers such as carbohydrates ad nucleic acids lack such diverse side chains. Therefore, proteins alone serve as enzymes (Price and Stevens 1989, Robert 2003). 1.3.5.2.1 General Properties They are synthesized only by living cells. They are needed in minute quantities for catalytic action. Their chemical nature is not altered irreversibly during the course of catalytic activity. They accelerate the chemical reactions that are taking place in the biological systems.
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Many enzymes need a non-protein component, which could be either an organic compound or a metal ion.
1.3.5.3 PROTEASES Protease refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown) peptide bonds of proteins. They are also called proteolytic enzymes or proteinases. Proteases differ in their ability to hydrolyze various peptide bonds. Each type of protease has a specific kind of peptide bonds it breaks. Proteolytic enzymes are ubiquitous in occurrence, being found in all living organisms and are essential for cell growth and differentiation. Examples of proteases include: fungal proteasepepsin, trypsin, chymotrypsin, papain, bromelain, and subtilisin. Microbial protease are among the important hydrolytic enzymes and have studied extensively since the advent of enzymology. There is renewed interest in the study of proteolytic enzymes, mainly due to the recognition that these enzymes not only play an important role in the cellular metabolic processes but have also gained considerable attention in the industrial community (Alan et al 2003). Proteolytic enzymes are very important in digestion as they breakdown the protein foods to liberate the amino acids needed by the body. Additionally, proteolytic enzymes have been used for a long time in various forms of therapy. Their use in medicine is gaining more and more attention as several clinical studies are indicating their benefits in oncology, inflammatory conditions, blood rheology control, and immune regulation. Proteolytic enzymes are involved in a great variety of physiological processes and their action can be divided into two different categories: 1. Limited proteolysis, in which a protease cleaves only one or a limited number of peptide bonds of a target protein leading to the activation or maturation of the formerly inactive protein e.g. conversion of prohormones to hormones.
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2.
Unlimited proteolysis, in which proteins are degraded into their amino acid
constituents, the proteins to be degraded are usually first conjugated to multiple molecule of the polypeptide ubiquitin. This modification marks them for rapid hydrolysis by the protease in the presence of ATP. Another pathway consists in the compartmentation of proteases e.g. in lysosomes. Proteins transferred into this compartment undergo a rapid degradation. The widely used term protease is synonymous with peptidase. Peptidases comprise two groups of enzymes: the endopeptidases and the exopeptidases, which cleave peptide bonds at points within the protein and remove amino acids sequentially from either N or C-terminus respectively. Proteinases are classified according to their catalytic mechanisms. Four mechanistic classes have been recognized by the International Union of Biochemistry and Molecular Biology (Alan et al 2003): a) b) c) d) The serine proteinases The cysteine proteinases The aspartic proteinases The metallo proteinases
This classification by catalytic types has been suggested to be extended by a classification by families based on the evolutionary relationships of proteases (Rawlings and Barrett 1993).
1.4 ADVANTAGES OF ENZYMATIC DEHAIRING By using the enzyme unhairing method, the entire length of hair is obtained without any damage and the load on the wastewater system is reduced as compared to the conventional lime-sulphide method. Thus, the wastewater is not contaminated by the dissolved proteins resulting from pulping of the hair. Yet another advantage of the use of enzymes in dehairing is that these enzymes will be carried over to the effluent collection tank where a good deal of proteinaceous substances are already accumulated. The proteinase coming from the beamhouse will
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naturally hydrolyze these proteins into their degradation products of low molecular weight, enabling microorganisms to degrade them further, liberating Carbon dioxide, Nitrogen and other volatile end products. This will indirectly help in reducing the high B.O.D value of the effluent.
CHAPTER 2 EXPERIMENTAL WORK 2.1 AIM The present study was undertaken with the aim of studying the kinetics of the enzymatic dehairing process. This study would explore the optimal activity of the protease and analyze at different conditions (temperature, pH and substrate concentration). The kinetic study on casein has been attempted to obtain the rare parameters. Hence, this study would be beneficial to the global leather industry in developing an enzyme based dehairing technique that ultimately aims at reducing the polluting nature of tannery effluents. 2.2 MATERIALS AND METHODS Protease (from bacterial source) was of commercial grade. All the chemicals used for the reparation of assay and reagents were of analytical grade. 2.3 ASSAY FOR PROTEASE [Casein digestion method (Kunitz 1947)] Activity of protease can be determined by a variety of methods. The methods used here is the spectrophotometric method of Kunitz, in which casein serves as the substrate. PRINICIPLE: This method described by Kunitz involves the digestion of casein by protease enzyme under standard conditions after which undigested protein is precipitated with Trichloro acetic acid (TCA) and is then removed by centrifuagation. The extent of digestion, which is the measure of protease activity, is determined on the supernatant with the aid of Folins phenol reagent. A blue color is produced by a reaction with
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tyrosine and tryptophan of the protein split products in the supernatant. Conversion of the color value to units to protease is readily made from a standard curve. PREPARATION OF REAGENTS: Preparation of Tris buffer (20mM, pH 8) 2.4g of Tris was dissolved in 1 liter of reagent grade water and the pH was adjusted to 8.0 with 0.1N hydrochloric acid. Preparation of Casein Stock Solution (1%) A stock solution of casein was prepared by suspending 1g of casein (M.S.Dunn, 1949) in 100ml of Tris buffer. The pH was adjusted to 8.0 using 0.1N HCl. The suspension was heated to make a complete solution of casein and was stored in the refrigerator. This solution is stable for at least one week. Preparation of Casein Stock Solution (2%) A stock solution of casein was prepared by suspending 2g of casein (M.S.Dunn, 1949) in 100ml of Tris buffer. The pH was adjusted to 8.0 using 0.1N HCl. The suspension was heated to make a complete solution of casein and was stored in the refrigerator. This solution is stable for at least one week. Preparation of TCA Solution (5%) 5ml of TCA was dissolved in 100ml of reagent grade water. NaOH (0.5M) solution 1g of sodium hydroxide was dissolved in 50ml of reagent grade water. Folin and Ciocalteau Reagent (1:1) Folin and Ciocalteau laboratory reagent was used. 1ml of the reagent was mixed with 1ml of reagent grade water. Enzyme Solution (protease) 0.1g of protease was dissolved in 10ml of reagent grade water.
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Preparation of Tyrosine Stock Solution (1%) Tyrosine stock solution was prepared by dissolving 100mg of tyrosine in 10ml reagent grade water. Tyrosine working standard solution (0.01%) 0.1ml of tyrosine stock solution was made up to 10ml using reagent grade water in a standard flask. This is the tyrosine working standard solution. 0.1N HCl (for pH adjustment) 0.1N NaOH (for pH adjustment) 2.3.1 TYROSINE STANDARD GRAPH Tyrosine standard graphs were prepared by taking different concentrations of tyrosine from 0.1ml to 0.5ml of tyrosine working standard solution. Then it was made up to 1ml using Tris buffer. To this solution 2ml of TCA and 0.5ml of Folin and Ciocalteau reagent. The blue color developed was measured at 750nm using an UV Visible spectrophotometer. A graph (Figure 2.1) was plotted between the concentration of tyrosine in moles per ml (X-axis) and the A750 (Y-axis). This graph (Figure 2.1) is used for calculating the tyrosine released by the enzyme and thereby the activity of the enzyme can be calculated. Note: 181g of tyrosine = 1mole of tyrosine.
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Optical Density at 750 nm
1 0.8 0.6 0.4 0.2 0 0 0.05 0.1 0.15 0.2 0.25 0.3 Tyrosine Concentration in micro moles per ml
Figure 2.1 Tyrosine standard graph
2.3.2 METHOD FOR THE DETERMINATION OF PROTEASE ACTIVITY 1ml buffer-substrate solution was added to clean glass test tubes. 0.1ml of various concentrations of enzyme solution (0.0025, 0.005, 0.0075, 0.01g/ml) was added and incubated at room temperature. The reaction was stopped by the addition of 2ml of TCA. The reaction mixture was allowed to precipitate. Then the precipitate was centrifuged at 10,000 rpm for 20 minutes. 1ml of supernatant was drawn from each test tube. 2ml of 0.5N sodium hydroxide solution was added to each test tube containing the supernatant. The contents were mixed well. 0.5ml of Folin and Ciocalteau reagent was added.
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The absorbance was measured at 750nm after the development blue color using UV visible spectrophotometer. A control was also performed by adding the enzyme after the inactivation of substrate by TCA. The tyrosine released by the enzyme was calculated from tyrosine standard graph. Unit: One unit of enzyme releases Folin positive amino acid equivalent to 1mole tyrosine per minute from casein at room temperature, pH 8. Calculation: The moles tyrosine released from the substrate are calculated from the Figure 2.1. Activity of enzyme = 2.4 moles of tyrosine released Enzyme (g) * Enzyme (ml) EXPERIMENTAL PROCEDURES
2.4.1 EFFECT OF PROTEASE ACTIVITY AT DIFFERENT TIME INTERVALS 1ml of 2% casein stock solution was pipetted into the series of test tubes. 0.1ml of different enzyme concentrations (0.005, 0.0075, 0.01g/ml) was added to each test tube. The reaction mixture was incubated for different time intervals (0min, 15 min, 30min, 45min, 60min, 120min, 180min, 240min, 300min, 360min) The assay of protease was carried out as cited in section 2.3.2. 2.4.2 EFFECT OF SUBSTRATE CONCENTRATION ON PROTEASE ACTIVITY Various concentrations of casein solution (0.2%, 0.4%, 0.6%, 1%) were prepared from 1% casein stock solution using Tris buffer. 1ml of substrate of above mentioned concentrations were taken in different test tubes and 0.1ml of enzyme (0.01g/ml) was added. The assay of protease was carried out as mentioned in section 2.3.2.
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2.4.3 EFFECT OF pH ON PROTEASE ACTIVITY Tris buffer was adjusted to different pH (4-10) using 0.1N HCl and 0.1N NaOH. The casein substrate at different pH was prepared by dissolving it in appropriate buffer. 1ml of 2% casein substrate solution at different pH was pipetted out in different test tubes and 0.1ml of enzyme solution (0.005g/ml) was added. The assay of protease was carried out as mentioned in section 2.3.2. 2.4.4 EFFECT OF TEMPERATURE ON PROTEASE ACTIVITY 1ml of 2% casein stock solution was pipetted out in different test tubes and 0.1ml of enzyme (0.01g/ml) was added. Different test tubes were incubated at different temperatures (35, 40, 45, 50 and 55C). Protease assay was carried out as mentioned in section 2.3.2. 2.4.5 DETERMINATION OF Km AND Vmax OF PROTEASE CATALYZED REACTION USING KINETIC STUDIES From 1% casein stock solution, 0.2%, 0.4% and 0.6% casein solutions were prepared using Tris buffer. 1ml of substrate of above mentioned concentrations were taken in different test tubes and 0.1ml of enzyme (0.01g/ml) was added. The reaction mixture was incubated for different time intervals (15, 30, 45, 60, 75, 90 and 120min). The reaction was stopped by the addition of 2ml of TCA. The reaction mixture was allowed to precipitate. Then the precipitate was centrifuged at 10,000 rpm for 20 minutes. 1ml of supernatant was drawn from each test tube.
27
2ml of 0.5N sodium hydroxide solution was added to each test tube containing the supernatant. The contents were mixed well. 0.5ml of Folin and Ciocalteau reagent was added. The absorbance was measured at 75nm after the development of blue color using UV visible spectrophotometer. A control was also performed by adding the enzyme after the inactivation of substrate by TCA. The tyrosine released by the enzyme was calculated from tyrosine standard graph. A graph was plotted between time interval in minutes (X-axis) and tyrosine released in micromoles per ml (Y-axis). The initial velocity V was found out from the above-mentioned graph. Lineweaver and Burk plot was constructed between 1/[S] (X-axis) and 1/V (Y-axis). 2.5 RESULTS AND DISCUSSION 2.5.1 EFFECT OF TIME ON PROTEASE ACTIVITY The activity of the protease at different time intervals (15, 30, 45, 60, 120, 180, 240, 300, 360min) has been carried out to find the optimum time for maximum activity. The activity of the protease on increasing time interval with varying concentration of enzyme is shown in Figure 2.1. It is seen that the activity of protease slowly increases from the initial period (15 min) and reaches the equilibrium at 300min. Figure 2.1 also shows that the activity increases as the concentration of protease increases. This is mainly because more amount of tyrosine is released by higher concentration of protease.
28
10000 9000 8000 7000 6000 activity 5000 4000 3000 2000 1000 0
0 50 100 150 200 250 300 350 400 .0025g/ml enzyme .005g/ml enzyme .0075g/ml enzyme .001g/ml enzyme
time
Figure 2.2 Effect of time on protease activity at different protease concentrations
Enzyme conc 0.0025 0.005 0.0075 0.01
Od 0.178 0.432 0.782 1.15
Amino lib 0.612 1.484 2.687 3.951
acid Activity 2446.391 2968.654 3582.542 3951.333
Velocity 163.093 197.91 238.836 263.422
Table 2.1
Effect of time for 15 minutes time interval
29
activity vs enzyme conc 15min

5000 4000 3000 2000 1000 0 0 0.005 0.01 0.015 enzyme concentration
activity
activity
Figure 2.3 Effect on activity for 15 minutes
velocity 15min
300 250 200 150 100 50 0 0 0.005 0.01 0.015 enzyme conc
velocity
velocity
Figure 2.4 Effect on velocity for 15 minutes Enzyme conc 0.0025 0.005 0.0075 0.01 Od 0.241 0.502 0.915 1.35 Amino acid lib 0.828 1.725 3.144 4.639 Activity 3312.248 3449.686 4191.849 4638.522 Velocity 110.408 114.990 139.728 154.617
Table 2.2 Effect of time for 30 minutes time interval
30
activity vs enzyme conc 30min

5000 4000 3000 2000 1000 0 0 0.005 0.01 0.015 enzyme concentration
activity
activity
velocity 30 min
200 velocity 150 100 50 0 0 0.005 0.01 0.015 enzyme conc velocity
Figure 2.6 Effect on velocity for 30 minutes Enzyme conc 0.0025 0.005 0.0075 0.01 Od 0.232 0.546 0.875 1.345 Amino acid lib 0.797 1.876 3.006 4.621 Activity 3188.554 3752.049 4008.599 4621.342 Velocity 70.857 83.338 89.080 102.696
31
activity 45min
5000 4000 3000 2000 1000 0 0 0.005 0.01 0.015 enzyme conc
activity
activity
velocity 45min
120 100 80 60 40 20 0 0 0.005 0.01 0.015 enzyme conc
velocity
velocity
Figure 2.8 Effect on velocity for 45 minutes Enzyme conc 0.0025 0.005 0.0075 0.01 Od 0.254 0.567 0.925 1.543 Amino liberated 0.873 1.948 3.178 5.302 3490.917 3896.358 4237.662 5301.659 58.182 64.939 70.628 88.361 acid Activity velocity
32
activity 60min
6000 5000 4000 3000 2000 1000 0 0 0.005 0.01 0.015 enzyme conc
activity
activity
velocity 60min
100 80 60 40 20 0 0 0.005 0.01 0.015 enzyme conc
velocity
velocity
Figure 2.10 Effect on velocity for 60 minutes Enzyme conc 0.0025 0.005 0.0075 0.01 Od 0.286 0.671 1.201 1.784 Amino liberated 0.983 2.306 4.127 6.130 3930.718 4611.034 5502.088 6129.721 32.756 38.425 45.851 51.081 acid Activity velocity
33
activity 120min
8000 activity 6000 4000 2000 0 0 0.005 0.01 0.015 enzyme conc activity
velocity 120min
60 50 40 30 20 10 0 0 0.005 0.01 0.015 enzyme conc
velocity
velocity
Figure 2.12 Effect on velocity for 120 minutes Enzyme conc 0.0025 0.005 0.0075 0.01 Od 0.302 0.789 1.332 1.957 Amino liberated 1.038 2.711 4.577 6.724 4150.618 5421.916 6102.233 6724.139 23.059 30.122 33.601 37.356 acid Activity Velocity
34
activity 180min
velocity 180min
35
activity 240min
velocity 240min
36
activity 300min
10000 8000 6000 4000 2000 0 0 0.005 0.01 0.015 enzyme conc
activity
activity
velocity 300min
30 25 20 15 10 5 0 0 0.005 0.01 0.015 enzyme conc
velocity
velocity
37
activity 360min
10000 8000 6000 4000 2000 0 0 0.005 0.01 0.015 enzyme conc
activity
activity
velocity 360min
30 25 20 15 10 5 0 0 0.005 0.01 0.015 enzyme conc
velocity
velocity
Figure 2.20 Effect on velocity for 360 minutes 2.5.2 EFFECT OF SUBSTRATE CONCENTRATION ON PROTEASE ACTIVITY In order to study the effect of substrate concentration on protease activity, the concentration of substrate was varied from 0.2 to 1%. The effect of substrate concentration on protease activity is shown in Figure 2.21.. It could be seen that the activity of protease depends on the concentration of substrate. The activity primarily depends on the moles of tyrosine release from the substrate by the protease. The activity of protease is higher for the 0.6 and 1% than 0.2 and 0.4% of substrate. This is
38
mainly due to the inadequate concentration of substrate in 0.2 and 0.4% for the protease activity. The rate of enzyme catalysed reactions increases with increasing substrate concentrations but above a certain substrate concentration, the rate of enzyme action ceases to increase (Figure 2.21). The shape of the curve is commonly explained on the basis of catalytically active sites on the enzyme that react with the substrate. Maximum velocity, Vmax, of the reaction is reached when all the sites are occupied by the substrate molecules.
600 500 activity 400 300 200 100 0 0 50 time

Figure 2.21 Effect of substrate concentration on protease activity
0.2% subs 0.4% subs 0.6% subs 1% subs
100
150
2.5.3 EFFECT OF pH ON PROTEASE ACTIVITY The protease solution was treated with substrate at different levels pH levels in the range of 4.0 to 10.0 in order to find the optimal activity. The influence of pH on the activity of the enzyme is shown in Figure 2.22. Generally, the activity seems to increase upon increase in the pH and it attains maximum in the alkaline region. It is clearly seen that the selected protease is highly active at pH 8.0. When the enzymatic activity is plotted against pH, a bell shaped curve is obtained. This indicates a marked dependence of an enzyme on the pH of the reaction mixture with 39
enzyme activity decreasing rapidly on either side of the optimum pH (Figure 2.22). The pH optimum of an enzyme is dependent upon a number of experimental parameters including temperature, nature of substrate, concentration of buffer, ionic strength of medium and purity of enzyme preparation. pH 4 6 8 10 Od 0.003 0.055 0.459 0.308 Amino liberated 0.013 0.189 1.058 1.058 20.616 377.954 3154.195 2116.540 0.687 12.598 105.140 70.551 acid Activity Velocity
Table 2.10 Effect of pH on protease activity
effect of pH on activity
4000 3000 activity 2000 activity 1000 0 -1000 0 5 pH Figure 2.22 Effect of pH on protease activity 10 15
40
velocity vs pH
120 100 80 60 40 20 0 -20 0
velocity
velocity
5 pH
10
15
Figure 2.23 Effect of pH on velocity
2.5.4 EFFECT OF TEMPERATURE ON PROTEASE ACTIVITY The effect of temperature on the protease activity has been studied to find the optimum temperature. The enzyme-substrate samples were incubated at various temperatures from 35-55C. The effect of temperature on protease activity is presented in Figure 2.24. The tyrosine release is a measure of activity of the protease. It is observed that the activity increases with increase in temperature up to 50C. In principle, the increase in temperature increases the reaction rate. This is primarily due to the increase in the rate of collision (faster Brownian motion) between enzyme and the substrate upon increase in temperature. It is observed that the activity of protease falls sharply at 55C. Hence, the protease can tolerate up to the temperature of 50C. Enzyme catalysed reactions are similar to other reactions in that the rate is increased by increasing temperatures up to a point. Beyond that temperature, the activity of an enzyme declines sharply as shown in Figure 2.24. As the temperature increases beyond 45-50C, the rate decreases. This decrease is caused by thermal denaturation of the enzyme protein or the inactivation of a thermolabile component in the enzyme system, when thermal energy becomes great enough to cause the rupture of a few bonds, the neighboring bonds are weakens and the whole molecule becomes denatured. The optimum temperature of enzymes under physiological conditions is
41
close to 40C. the maximum velocity of enzyme reaction is obtained around the optimum temperature. Temperature 35 40 45 50 55 Od 0.781 1.076 2.328 2.869 1.169 Amino liberated 2.683 3.697 7.999 9.858 4.017 2683.471 3697.074 7998.873 9857.718 4016.616 89.449 123.236 266.629 328.591 133.887 acid Activity Velocity
Table 2.11 Effect of temperature on protease activity
effect of temperature on activity

12000 10000
activity
8000 6000 4000 2000 0 0 10 20 30 40 50 60
activity
temperature Figure 2.24 Effect of temperature on protease activity
42
velocity vs temperature
400 velocity 300 200 100 0 0 20 40 60 temperature Figure 2.25 Effect of temperature on velocity 2.5.5 ENZYME KINETICS The enzyme-catalysed reaction is usually represented by k2 E+S ES k3 E+P EP) is negligible. The velocity measured velocity
During the initial period of enzyme-catalysed reaction, when measurements are made, the rate of reverse reaction (E +P during this period is called the initial velocity, V. The measurement of V simplifies the interpretation of kinetic data. Initial velocities are obtained from graphs exhibiting either the increase in product concentration or the decrease in substrate concentration over time. The initial velocity is the slope P/t at the origin of the progress curve. The Michaelis-Menten equation for calculating Km and Vmax is Vmax [S] V= Km + [S] The above equation for determining Km is slightly complex and therefore some modifications have been made. A reciprocal of Michaelis-Menten equation gives 1 = Km V Vmax x 1 + 1
[S] Vmax
43
This is known as the Lineweaver Burk equation which was proposed by Hans Lineweaver and Dean Burk in 1934. This equation is in the form of y = mx + c. when 1/V is plotted against 1/[S], a straight line is obtained. The 1/V versus 1/[S] plot gives the value of 1/Vmax and 1/Km respectively. The slope of the line corresponds to Km / Vmax. Since Vmax is determined from Y-axis, the Km is calculated using slope value. The Michaelis-Menten constant, Km, is defined as the substrate concentration at which enzyme shows half of the maximal velocity Vmax. Vmax varies with the total concentration of enzyme present, but Km is independent of enzyme concentration and it is characteristic of the system being investigated. If Km is known, the fraction of the sites, f[ES], occupied at any substrate concentration can be calculated using the following equation. V f[ES] = Vmax = Km+ [S] [S]
2.5.5.1 Kinetic Study for Protease The velocity vs substrate concentration curves for protease were drawn for each substrate concentration of casein used in the kinetic studies (0.2%, 0.4%, 0.6% and 1%), for different time intervals (15, 30, 45, 60, 75, 90, 105, 120minutes). This gives us the effect of substrate concentration on velocity. The Lineweaver-Burk plot (1/[S] vs 1/V) gives a straight-line graph. From this plot, Vmax and Km are determined.
44
Substrate conc % 0.2 0.4 0.6 1.0
Od 0.012 0.016 0.02 0.044
Amino acid lib 0.041 0.055 0.069 0.151
Activity 41.231 54.975 68.719 151.181
Velocity 2.749 3.665 4.581 10.079
1/[S] 5 2.5 1.667 1
1/V 0.364 0.273 0.218 0.099
Table 2.12 Effect of substrate concentration for 15 min time interval
velocity vs subs conc15 min

5 4 3 2 1 0 0 0.2 0.4 0.6 0.8 substrate concentration %
velocity
velocity
Figure 2.26 Effect on velocity for 15 min time interval
Lineweaver-Burk plot
0.4 0.3
1/V
0.2 0.1 0 -5 0
1/[S]
1/v
10
Figure 2.27 Lineweaver-Burk plot for 15 min time interval -1 = -2.5 Km => Km = 0.4 Vmax 1 = 0.15 => Vmax = 6.667
45
Od 0.014 0.021 0.033 0.055
Amino acid lib 0.048 0.072 0.113 0.189
Activity 48.103 72.155 113.386 188.977
Velocity 1.603 2.405 3.780 6.299
1/[S] 5 2.5 1.667 1
1/V 0.624 0.416 0.265 0.159
velocity vs subs conc 30min

8 velocity 6 4 2 0 0 0.2 0.4 0.6 0.8 substrate concentration %
velocity
0.8 0.6 1/V 0.4 0.2 0 -5 0 1/[S] 5 10 1/v
Figure 2.29 Lineweaver-Burk plot for 30 min time interval -1 = -2.5 => Km = 0.4 Km Vmax 1 = 0.2 => Vmax = 5
46
Od 0.017 0.027 0.051 0.079
Amino acid lib 0.058 0.093 0.175 0.271
Activity 58.411 92.770 175.233 271.439
Velocity 1.298 2.062 3.894 6.032
1/[S] 5 2.5 1.667 1
1/V 0.770 0.485 0.257 0.166

3.5 velocity 2.5 1.5 0.5 -0.5 0 0.5 substrate concentration % 1 velocity
1 0.8 0.6 0.4 0.2 0 -5 0 1/[S] 5 10
1/V
1/v
Figure 2.31 Lineweaver-Burk plot for 45 min time interval -1 = -2.5 => Km = 0.4 Km Vmax 1 = 0.22 => Vmax = 4.545
47
Od 0.024 0.036 0.067 0.089
Amino acid lib 0.083 0.124 0.230 0.306
Activity 82.463 123.694 230.208 305.799
Velocity 1.374 2.062 3.837 5.097
1/[S] 5 2.5 1.667 1
1/V 0.728 0.485 0.261 0.196

6 velocity 4 velocity 2 0 0 0.5 1 1.5 substrate concentration %
0.8 0.6 1/V 0.4 0.2 0 -5 0 1/[S] 5 10 1/v
Figure 2.33 Lineweaver-Burk plot for 60 min time interval -1 = -2 Km => Km = 0.5 1 = 0.21 Vmax => Vmax = 4.76
48
Od
Amino acid lib
Activity
Velocity
1/[S]
1/V
0.035 0.048 0.091 0.112
0.120 0.165 0.313 0.385
120.258 164.925 312.671 384.826
1.603 2.199 4.169 5.131
5 2.5 1.667 1
0.624 0.455 0.240 0.195

6 velocity 4
velocity
2 0 0 0.5 1 1.5 substrate concentration%
0.8 0.6 1/V 0.4 0.2 0 -5 0 1/[S] 5 1/v
Figure 2.35 Lineweaver-Burk plot for 75 min time interval -1 = -2.5 Km => Km = 0.4 1 = 0.24 => Vmax = 4.1667 Vmax
49
Od 0.047 0.069 0.103 0.12
Amino acid lib 0.161 0.237 0.354 0.412
Activity 161.489 237.08 353.902 415.313
Velocity 1.794 2.634 3.932 4.581
1/[S] 5 2.5 1.667 1
1/V 0.557 0.379 0.254 0.218

5 4 3 2 1 0 0 0.5 1 1.5 substrate concentration%
velocity
velocity
Figure 2.26 Effect of velocity for 90 min time interval
0.6 1/V 0.4 1/v 0.2 0 -2 0 2 1/[S] 4 6
Figure 2.37 Lineweaver-Burk plot for 90 min time interval -1 = -1.5 Km => Km = 0.667 1 = 0.15 => Vmax = 6.67 Vmax
50
Od 0.058 0.062 0.114 0.139
Amino acid lib 0.199 0.213 0.392 0.478
Activity 199.285 213.028 391.697 477.596
Velocity 1.898 2.029 3.730 4.549
1/[S] 5 2.5 1.667 1
1/V 0.527 0.493 0.268 0.220

velocity
velocity
Figure 2.38 Effect of velocity for 105 min time interval
0.6 1/V 0.4 1/v 0.2 0 -2 0 2 1/[S] 4 6
Figure 2.39 Lineweaver-Burk plot for 105 min time interval -1 = -1.5 => Km = 0.67 Km 1 = 0.15 => Vmax = 6.67 Vmax
51
Od 0.063 0.074 0.114 0.14
Amino acid lib 0.216 0.254 0.392 0.481
Activity 16.464 254.260 391.697 481.032
Velocity 1.804 2.119 3.264 4.009
1/[S] 5 2.5 1.667 1
1/V 0.554 0.472 0.306 0.249

velocity
velocity
0.6 1/V 0.4 1/v 0.2 0 -5 0 1/[S] 5 10
Figure 2.41 Lineweaver-Burk plot for 120 min time interval -1 = -2 => Km = 0.5 Km Vmax 1 = 0.19 => Vmax = 5.263
52
CHAPTER 3 CONCLUSION The kinetics of the enzymatic dehairing process was studied by conducting experiments on casein substrate with protease enzyme. The Michaelis-Menten constant for the reaction was found using the Lineweaver-Burk plot. The Lineweaver-Burk is a more convenient equation to work with for such studies. The plots of protease activity versus time for different substrate concentration shows that activity increases with time and then becomes stable at about 300 minutes of reaction time. Similarly, plot of pH versus activity show that protease activity decreases on either side of the optimum pH of 8.0. The graphs of temperature versus activity show that rate increases with temperature, up to a point and then decreases, optimum temperature is 50C. Enzymatic dehairing gives more lengthy, stronger wool, which requires minimum washing afterwards. Hair gets removed along with epidermal layer and this makes the process of hair-loosening easier. The enzymatic dehairing process is a more environmentally friendly process, and studies of its kinetics will help to improve the process.
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CHAPTER 4 BIBLIOGRAPHY BOOKS R.Puvanakrishnan, Susil C. Dhar, Enzyme Technology in beamhouse practice, 1988 Journal of American Leather Chemists Association, vol 69, 1974, page 50-65 Journal of American Leather Chemists Association, vol 94, 1999, page 51-58, 355362. REFERENCES S. C. Dhar (1977), The use of enzymes in the beamhouse to solve the tannery effluent problems, TGT, 1977 A. Dayanandan, J. Kanagaraj, Lesley Sounderraj, R. Govindaraju and G. Suseela Rajkumar, 2003, Application of an alkaline protease in leather processing: an ecofriendly approach Journal of Cleaner Production, Volume 11, Issue 5, August 2003, Pages 533-536 S. Saravanabhavan, R. Aravindhan, P. Thanikaivelan, J. Raghava Rao, Balachandran Unni Nair and T. Ramasami, 2004, A source reduction approach: Integrated bio-based tanning methods and the role of enzymes in dehairing and fibre opening Clean technologies & Environmental policy, volume 7 no. 1, Dec 2004, Pages 3-14 Palanisamy Thanikaivelan, Jonnalagadda R. Rao, Balachandran U. Nair and Thirumalachari Ramasami, 2004, Progress and recent trends in biotechnological methods for leather processing, Trends in Biotechnology, Volume 22, Issue 4, April 2004, Pages 181-188 WEBSITES a. b. c. www.epa.gov http://ejbiotechnology.info/content/vol8/issue2/full/5/index.html#m_9 http://pubs.acs.org/subscribe/journals/esthag-
1) 2) 3)
1. 2.
3.
4.
w/2005/apr/tech/kb_leather.html
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MANUFACTURE OF MALEIC ANHYDRIDE
ACKNOWLEDGEMENT
It is indeed an immense pleasure and privilege for us to present this report before which we would like to thank all those who assisted and guided us at all stages of the project. Our sincere thanks to our Principal Prof. R. Venkataramani, and Director Dr. T.P.Ganesan for their constant support. We express our heartfelt thanks to Dr R Karthikeyan, Head of the Department of Chemical Engineering, S.R.M Engineering College, for his valuable guidance. We extend our thanks to Dr B S M Kumar, our project guide for his continuous help and encouragement. We express our special thanks to Ms P Suganthi Rani, Ms E Kavitha and Ms Poonguzhali, lecturers, Dept of Chemical Engineering, S.R.M Engineering College, for their patient guidance. We would like to thank all the staff members of our department, friends and our parents for their endless suggestions and guidance for the completion of this project.
ABSTRACT This project essentially deals with the production of maleic anhydride, a compound that has a wide range of applications, from the production of unsaturated polyester resins to agricultural chemicals such as herbicides and insecticides. Resins made from maleic anhydride are known to possess excellent structural strength. Maleic anhydride is also used in the production of malic acid, succinic acid and lube-oil additives. Maleic anhydride is produced by the oxidation of benzene, the reaction proceeds in two steps. First is the reaction step in which maleic anhydride is produced, next is the recovery and purification step, to give better yield. In recent times, benzene is being replaced by n-
55
butane and n-butylene. Improvements in catalyst used have been integral to the advent of a commercially viable manufacturing process.
TABLE OF CONTENTS Chapter no. Page no. xii xiii xv xvi xvii 55 55 56 57 59 62 64 64 69 75 77 82 89 97 102 103
Title Acknowledgement Abstract List of tables List of figures List of symbols
Introduction 1.1 Background 1.2 Physical properties 1.3 Chemical properties 1.4 Applications 1.5 Literature Review
Process 2.1process description and flow sheet 2.2 Material balance 2.3 Energy balance 2.4 Design 2.6 Plant layout 2.7 Cost estimation 2.8 Health and Safety
2.5 Instrumentation and Process control 78
3 4
Conclusion Bibliography
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LIST OF TABLES TABLE NO. 2.1 2.2 2.3 2.4 PAGE NO. 90 90 91 92
TITLE Cost of equipments Direct cost factor Indirect cost factor Auxillary services cost factor
LIST OF FIGURES FIGURE NO. 1.1 2.1 2.2 2.3 2.4 2.5 TITLE Structure of Maleic Anhydride Process flow sheet Material balance of reactor Material balance of absorber Material balance of evaporator Plant layout PAGE NO. 56 68 69 72 74 88
57
LIST OF SYMBOLS S. NO. 1 2 3 4 5 6 7 8 9 10 11 12 13
SYMBOL HR ta tb t M A V ho hi
EXPANSION Heat of reaction Inlet temperature Outlet temperature Change in temperature Mass flow rate Viscosity Area Velocity Density Latent heat of vaporization Outside film coefficient Inside film coefficient Space time
UNITS kJ/kmol K K K kg/hr Kg/m s m2 m/s kg/m3 kJ/kg kcal/hr m2 K kcal/hr m2 K hr
CHAPTER 1 INTRODUCTION 1.1 BACKGROUND Although maleic anhydride and its two acid isomers were prepared and characterized in the 1830s, they were not commercially available until about 100 years later. Prior to 1930, maleic anhydride was formed only in small quantities as a by-product of the phthalic anhydride manufacturing process. In 1919, patents for the catalytic oxidation of benzene to maleic anhydride were issued. The advent of patents for the catalytic oxidation of benzene coupled with improvements in the vanadium oxide catalysts was integral to the creation of a
58
commercially viable process for maleic anhydride manufacture. Today, nearly three billion pounds of maleic anhydride are made each year throughout the world using variations of this process. 1.2 PHYSICAL PROPERTIES Other names: 2,5-furandione, toxilic anhydride, cis-butenedioc anhydride, maleic acid anhydride. Structural Formula:
Figure 1.1 Molecular Weight: 98.06 Melting point: 52.8C Boiling point: 202C Flash point Open cup: 110C Closed cup: 102C Specific gravity (at 20/20C, solid): 1.48 Specific gravity (at 70/70C, molten): 1.3 Heat of combustion, MJ/mol: -1390 Heat capacity, kJ/(kg.K) Solid: 1.21 Liquid: 1.67 Heat of vaporization, kJ/mol: 54.8 Heat of fusion, kJ/mol: 13.65 Heat of hydrolysis, kJ/mol: -34.9 Heat of neutralization, kJ/mol: 126.9
59
Flammable limits, vol% Lower: 1.4-3.4 Upper: 7.1 Autoignition temperature: 447C Vapor density (air=1): 3.38 Crystal structure: orthorhombic Maleic anhydride is nearly planar with the anhydride oxygen tilted 3 pm from the plane of the ring. The ring has one twofold axis of symmetry, belonging to the C2v point group.
1.3 CHEMICAL PROPERTIES Both the ethylenic double bond and the carbonyl groups are susceptible to attack. Because the C=C bond is conjugated with the two C=O bonds, both groups may participate in certain reactions. Polymerization: Maleic anhydride homopolymerizes with some difficulty but copolymerizes with ease. Monomers that are used with maleic anhydride to make commercial copolymers include ethylene, styrene, methyl vinyl ether, and vinyl chloride. These are free-radical-catalysed copolymerizations and they generally prodce materials with alternating units. Unsaturated polyester resin manufacture comprises the largest single use of maleic anhydride. Maleic anhydride is esterified with a glycol forming a linear unsaturated polyester. A vinyl monomer, e.g., styrene, is added, with a free radical catalyst; peroxides commonly used. The result is a cross-linked, three-dimensional structure that imparts rigidity, insolubility, and strength to the material. Physical properties, e.g., degree of flexibility, can be adjusted by using different glycols or by substituting adipic acid or phthalic anhydride for some of the maleic anhydride. Uncatalysed polymerizations based on Diels-Alder reactions are possible by using a diene and a maleic anhydride derivative such as dimaleimide.
60
Reduction The heterogeneous catalytic reduction of maleic anhydride and its derivatives gives succinates, butyrolactone, tetrahydrofuran, or 1,4 butanediol, depending on the catalyst used. Homogeneous reduction also has been accomplished. Diimide provides an especially convenient route to succinates. Sulfonation Sulfomaleic anhydride, an intermediate in the production of sulfo-succinate derivatives, results from the reaction of maleic anhydride and sulfur trioxide. Violent reactions can occur when maleic anhydride contacts alkali or alkaline earth metals, carbonates, hydroxides, or amines. 1.4 APPLICATIONS The dominant end use of maleic anhydride is in the production of unsaturated polyester resins. These laminating resins, which have high structural strength and good dielectric properties, have a variety of applications in automobile bodies, building panels, molded boats, chemical storage tanks, lightweight pipe, machinery housings, furniture, radar domes, luggage and bathtubs. Other end products are fumaric acid, agricultural chemicals, alkyd resins, lubricants, copolymers, plastics, succinic acid, surface active agents, and more. Maleic Anhydride is the principal raw material for fumaric acid. The largest use for fumaric acid is in the manufacture of sizing resins for paper. It also is used in unsaturated polyester resins, alkyd resins, quick-setting inks, and as a food acidulant. Maleic anhydride is used to manufacture lube-oil additives, e.g., ashless dispersants of which an example is the reaction product of an amine and an alkenyl succinate. The latter is made by alkylating maleic anhydride with polyisobutylene. The oil-soluble dispersants are used to prolong oil-change intervals and to improve engine efficiency so as to comply with antipollution laws. Maleic anhydrides use in agricultural products includes herbicides, insecticides, fungicides, and plant growth regulators. Malathion is made from dithiophosphoric acid and diethyl maleate and is an effective insecticide. Maleic hydrazide, the product of the
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reaction of maleic anhydride and hydrazine hydrochloride, is used as growth inhibitor and as a herbicide. Captan, a foliage fungicide, is derived from tetrahydrophthalic anhydride which is produced from the reaction of butadiene and maleic anhydride. Endothall is made by the Diels-Alder condensation of furan and maleic anhydride and is used as a herbicide and soil sterilant. Difolatan is used extensively on potatoes and tomatoes. Alar is a growth regulator used on apples, tomatoes, and grapes. Maleic anhydride is used in the production on malic acid; the anhydride is made by adding the elements of a water molecule across the ethylenic double bond of fumaric acid or maleic acid. It is used as a food and beverage acidulant. Copolymer products of maleic anhydride are made by reaction with styrene, ethylene, and methyl vinyl ether. Chlorendic anhydride and Cloran TM provide polyester resins with fireretardant properties and greater light stability.
APPLICATIONS 1. Unsaturated Polyester Resins 1,4-Butanediol and derivatives Tetrahydrofuran Methyl vinyl ether-maleic anhydride Diisobutylene-maleic anhydride (Acrylic acid-maleic anhydride) 1,3-Butadiene-maleic anhydride 2. Fumaric, Maleic, Malic, and Succinic Acids (A) Fumaric Acid 1) Papermaking 2) Animal Feed 3) Unsaturated Polyester Resins 4) Alkyd Resins (B) Maleic Acid (C) Malic Acid (D) Succinic Acid 3. Lubrication Oil Additives - e.g. Polyisobutylene succinic anhydride 4. Agricultural Chemicals e.g. Malathion
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5. Styrene Copolymers and Other Polymers (a) Styrene Maleic Anhydride (b) Olefin Copolymers (c) Maleic Anhydride Grafted Copolymers (d) Other Polymers I. Bis-Maleimide Resins II. Polybutadiene/Maleic Anhydride Adducts III. Ethylene Vinyl Acetate - Maleic Anhydride Terpolymers (Ultrathene) IV. n-Phenylmaleimide V. Other Polymeric Uses 7. Fine chemicals: - Sulphosuccinic acid esters - Alkenyl succinic anhydrides - Aspartic acid - Succinic anhydride - Succinic acid - Maleic acid ester 6. Other Uses (a) 1,4-Butanediol/THF/gamma-Butyrolactone/NMP (b) Detergents 1.5 LITERATURE REVIEW Maleic anhydride is produced by the oxidation of benzene, n-butane, n-butylene and other hydrocarbons in the presence of different catalysts. Benzene was the raw material in use for a number of years, it is only in recent times that other hydrocarbons are being used. Vanadium phosphorous oxygen (VPO), Fe-Sb-Mo, P-Mo, Sb-Mo, P-Ti, V-Zn/Nb are some of the catalysts used. G.Bignardi et al studied the Influence of the oxidation state of vanadium on the reactivity of V/P/O, catalyst for the oxidation of npentane to maleic anhydride. Higher concentrations of V5+ led to the preferred formation of maleic anhydride. Beatriz T. Pierini et al studied Cr, Mo and W used as VPO promoters in the partial oxidation of n-butane to maleic anhydride. It was found
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that the addition of the promoters invariably increased the catalytic activity but in every case there was an optimum load to achieve the best selectivity. The origin of this maximum could be ascribed to the right balance between the presence of very strong Lewis acid sites and the development of V5+ isolated sites in the matrix of the vanadyl pyrophosphate, by far the major crystalline phase present in the catalyst. The three group VIB promoters affect the balance between acid and redox properties but increase the overall activity in all cases. Arqumedes Cruz-Lpez et al studied the Selective oxidation of butane to maleic anhydride in a catalytic membrane reactor adapted to rich butane feed. In their study, it was found that the addition of cobalt to the VPO catalyst allowed keeping the MA selectivity at a high level (75%). The combination of the Co VPO catalyst and the MFI membrane was used to explore the membrane reactor performance with high butane concentrations in the feed, corresponding to the flammability zone in a conventional reactor. For these conditions, the MA productivity was three times higher than that observed with the conventional reactor.
CHAPTER 2 PROCESS 2.1 PROCESS DESCRIPTION AND FLOW SHEET The predominant commercial route to maleic anhydride is the vapor-phase oxidation of hydrocarbons over a solid catalyst. Benzene, n-butane, and n-butylenes are used. Recovery of maleic anhydride as a by-product from phthalic anhydride manufacture provides small, commercial amounts. Benzene oxidation has been the prevailing commercial procedure. The currently used hydrocarbon oxidation processes may be divided into two steps. The reaction step involves oxidation of a relatively low concentration of hydrocarbon in air to form maleic anhydride, carbon oxides, water, and smaller amounts of partially oxidized by-products. The second step involves the recovery of the maleic anhydride from the dilute reactor off-gas and purification of the resultant crude product.
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The large exotherm and thermal sensitivity of the hydrocarbon oxidation, i.e., the first step, requires low feed concentrations, expensive heat-transfer equipment, a large gas-handling job, and the need for good control and the exothermicity provides an important energy (steam) source. A typical plant consists of a tubular reactor, a partial condenser, an absorber, a dehydration unit, a refining column, and may or may not have a benzene recovery unit (see Figure 2.1). REACTION STEP: Benzene is usually mixed with air to give concentrations from 1-1.4 mol% and is passed through the catalyst at 60 130 g benzene/L catalyst/h. The heat generated by the exothermic reaction of producing maleic anhydride is used to heat water to produce steam for use elsewhere. Pressure is 1-2 atm. Steam is also produced in heat exchangers that cool the effluent gas from the reactors. The benzene-to-maleic catalyst has been improved since its discovery. Average conversion of benzene in the U.S. plants is reported to be 94.5%. most, if not all commercial benzene oxidation catalysts are of the supported type. The support may be metal shapes or low surface area, e.g., ceramically bonded alumina, silica, or carborundum. The active catalyst, containing a preponderance of vanadium oxides, usually is mudded onto the carrier by concentration from a solution, resulting in a catalyst with 1-2 m2/g surface area. The major modifying element is molybdenum. Also, phosphorous, alkali and alkaline earth metals, tin, boron, silver, titanium, tungsten, nickel, cobalt, and many other elements have been added to vanadia catalyst. RECOVERY AND PURIFICATION: In most of the manufacturing processes, the reaction gas, which typically contains 1% maleic anhydride, is cooled to below 200C by being passed through heat exchangers; then t is cooled further to near the dew point of water (55-65C). forty to sixty percent of the maleic anhydride is condensed as a liquid which is separated from the gas stream. The amount that condenses is limited by condensation and absorption of water by the maleic anhydride, forming maleic acid. The remaining maleic anhydride in the gas is removed as the acid by absorption in water.
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The maleic acid solution formed from water absorption is concentrated and dehydrated directly to crude maleic anhydride or by the use of entraining agents, usually o-xylene. the adequacy of the equipment and process in the recovery step is critical to an economic operation. The Figure 2.1 shows the process studied. Vaporized benzene and air are mixed before entering the tubular reactor. This mixture has a concentration of 1 mole% benzene. A low benzene concentration is used in order that that flammability of mixture is not exceeded. The reaction gas mixture is then passed over the catalyst in a multitubular fixed-bed reactor at an optimum pressure range of 0.15 0.25 MPa. The desired reaction, as given below, C6H6 + 4.5 O2 C4H2O3 + 2 CO2 + 2H2O is highly exothermic, causing hot spots of 340-500C to occur in the catalyst. Approximately 27MJ of heat are generated per ton of benzene reacted. Water is heated to steam to disspiate the heat produced. Upon exiting the reactor, the vapor mixture is cooled to 150-160C by heat exchangers. Partial condensers further cool the gas to 55C, the condensation point of maleic anhydride, to recover 50% of the maleic anhydride as liquid. The condensed maleic anhydride must be removed as soon as possible to avoid prolonged contact with the water in the reactant gas. Exposure of maleic anhydride results in the undesired formation of maleic acid that further limits recovery if allowed to continue for an extended period of time. The maleic anhydride that cannot be recovered is eventually washed out with water as maleic acid. Water scrubbing and subsequent dehydration of the maleic acid, in an evaporator with 95% conversion, is required to purify and reform the remaining maleic anhydride. CATALYST As a rule, commercial catalysts for benzene oxidation are supported by an alumina or silica carrier and have a surface area of 1-2 m2/g. a typical catalyst may be modified with a promoter to increase the conversion, yield, and selectivity. Molybdenum is the most popular modifying element, but phosphorous, alkali, alkaline earth metals, tin, boron, and silver, among others, are also used. Benzene is passed through the catalyst at 60-130 g
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benzene/L catalyst/h. the lifetime of the catalyst can be as long as 4 years depending mainly on the reactor operating temperature and the purity of the starting materials.
C4H2O3 C6H6
Tubular O2 reactor
C4H2O3 CO2 H2O O2 Cooler H2O O2 N2 C6H6 C6H6 H2O CO2

condenser
C4H2O3
CO2 H2O
AIR O2 N2 C6H6
N2
N2
Vapor C6H6 CO2 Steam

E V A P O R T O R
C4H2O3 vapor CO2 C6H6 O2 N2
H2O
O2 N2
Separator
Product Recovery Absorber
Liquid C4H2O3 to storage Figure 2.1 Process Flow Sheet C4H4O4 to storage
Liquid C4H2O3
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2.2 MATERIAL BALANCE
TUBULAR REACTOR BASIS: 100 kg benzene 1.28205 kmole benzene Main reaction: C6H6 + 4.5 O2 Conversion is taken as 95%. ENTERING STREAM C4H2O3 + 2H2O + 2 CO2 Benzene-air mixture enters at a concentration of 1 mol% benzene.
Compone nts C6H6 O2 N2

kg C6H6 = 100 AIR O2 = 852.92 N2 = 2807.54
weight( kg) 100 852.922 2807.54
Molecul 78 32 28
No. of Moles(in kmole) 1.28205 26.6538 100.2691

kg C4H2O3 = 119.35 CO2 = 107.18 H2O = 43.85 C6H6 = 5 O2 = 677.54 N2 = 2807.54
Reactor Tubular Reactor
Figure 2.2 Material balance for reactor
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LEAVING STREAM
Component s C4H2O3 H2O CO2 C6H6 O2 N2
weight(k g) 119.35 43.85 107.18 5 677.54 2807.54
Molecular weight 98 18 44 78 32 28
No. of moles (in kmole) 1.2179 2.436 2.436 0.0641 21.173 100.2691
PRODUCT RECOVERY ABSORBER C4H2O3 + H2O C4H4O4
40% solution of maleic acid contains 70.64kg the maleic acid solution contains 105.957kg of water. Water required for absorption = water for the reaction + water to make 40% maleic acid solution water required = 0.6099kmol + 5.8865kmol = 116.935kg ENTERING STREAM = 6.4955kmol
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Components C4H2O3 CO2 C6H6 O2 N2 H2O (solvent)
weight(kg) 59.68 107.18 5 677.54 2807.54 116.935
Molecular weight 98 44 78 32 28 18
C6H6 = 5 kg CO2 = 107.18 kg N2 = 2807.54 kg
No. of moles(in kmol) 0.6099 2.436 0.0641 21.173 100.269 6.4955
H2O = 116.92 kg
O2 = 677.54 kg
Product Recovery Absorber
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C4H4O4 = 70.64kg C4H2O3 = 59.68kg H2O = 105.96kg Component C4H4O4 CO2 weight(kg) CO2 = 107.18kg O2 = 677.54kg N2 Molecular= 2807.54kg of moles No weight 5kg (kmole) CH =
6 6
70.64 116 Figure 2.3 Material Balance for absorber 107.18 44 78 32 28 18
0.6099 2.436 0.0641 21.173 100.269 5.8865
C6H6 5 LEAVING STREAM O2 N2 H2O 677.54 2807.54 105.96
EVAPORATOR
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Steam
Vapor H2O = 10.41kg
C4H4O4 = 70.64 kg
Evaporator
C4H2O3 = 59.68 kg H2O = 0.55kg
Figure 2.7 Material Balance for evaporator 2.3 ENERGY BALANCE
REACTOR Heat liberated in the exothermic reaction: HR = 2700KJ Therefore, Heat to be removed = -2700KJ m.Cp.t = 2700KJ m * 4.186 * 25 = 2700KJ
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Mass of cooling water required, m = 25.8003kg
COOLER (m.Cp.t)entering mixture = (m.Cp.t)cooling water [{(119.35*2.122) + (107.18*8.9846) + (43.85*12.4729) + (677.54*1.024) + (2807.54*0.9718) + (5*2.3037)} * 200] = m * 4.186 * 25 mass of cooling water required, m = 9931.78kg
CONDENSER (m.Cp.t) + (m)entering mixture = (m.Cp.t)water [{(119.35*2.122)+(107.18*6.3146)+(43.85*8.7663)+(677.54*0.6576) + (2807.54*0.6803) + (5*1.619)} * 200] + [3760.46 * 584.18] * 25 mass of water required for heat exchange, m =28021.03kg/h = m * 4.186
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EVAPORATOR q = mf Cp f (T-Tf) + (mf m) =70.64 * 1.68 * (135 60) + (70.64 59.68) * 1927.3 = 30023.85 kJ q = ms.s => 30023.85 = ms * 1848.5 Mass of steam required, ms = 16.24kg/hr
2.4 DESIGN
REACTOR = V/VO = V/1995.99 Let =0.2, => V = 399.198 m3 COOLER Calculation of number of tubes N: Tube side flow rate = (3760.46 / 1.8840) = 1996 m3/hr V.Ai.N = 1996 Let V = 6m/s = 360m/min 360 * [/4 * (2.12/100)2] * N = 1996 N = 237 per pass For two passes, Total number of tubes = 474 Inside film coefficient (hi): Jh = (hi.Di / k) (Cp./k)-1/3 = 19 (hi * 0.0212 / 0.027)(0.25 * 0.021 * 3.6 / 0.027)-1/3 = 19 hi = 21.48 kcal/hr m2 K Outside film coefficient (ho): 74
Jh = (ho.Do/k) (Cp./k)-1/3 = 8.8 (ho * 0.0254 / 0.027)(1.0 * 0.75 * 3.6 / 0.539)-1/3 = 8.8 ho = 319.5 kcal/hr m2 K Overall transfer coefficient: hio = hi * ID/OD = 21.48 * 2.12/2.54 = 17.93 Uo = (hio ho)/(ho + hio) = 16.98 kcal/hr m2 K Calculation of area and tube length: Q = (m Cp t)water = 188023kcal/hr Q = Uo Ao t Ao = 55.37m2 = DoLN L = 55.37 / ( * 0.0254 * 474) = 1.464m. Length of tube = 146.4cm Number of tubes = 474.
2.5 INSTRUMENTATION AND PROCESS CONTROL The primary objectives of the designer when specifying instrumentation and control schemes are: 1. Safe Plant Operation: a. To keep the process variables within known safe operating limits b. To detect dangerous situations as they develop and to provide alarms and automatic shut-down systems. c. To provide interlocks and alarms to prevent dangerous operation procedures. 2. Production rate: To achieve the desired output, at the rate that is most economical and that which corresponds to the market needs. 3. Product quality: a. To maintain the product composition within specified quality standards. b. To produce products with the characteristics desired and to make sustained efforts to improve quality and minimize lapses at every stage of production. 4. Cost:
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To operate at the lowest production cost, commensurate with the other objectives. INSTRUMENTATION TEMPERATURE MEASUREMENT The temperature measuring element in a control system for jacketed tank is generally a thermocouple. The five most commonly used thermocouples are copper constantan, iron constantan, chromel alumel, platinum platinum 13% rhodium, platinum platinum 10% rhodium. LEVEL MESUREMENT The float- shaft type is employed either in open vessels. This method is suitable for a wide range of liquids and semi-liquids. Difficulties are sometimes encountered when the liquid deposits on the float and when the liquid level is foaming or turbulent. FLOW RATE MEASUREMENT The industrial devices for flow rate estimation are common orifice meter, venturimeter, pilot tube and the Rota meter. The piping system must be made of special corrosion resistant material when corrosive fluids are used. pH MEASUREMENT In this process we use the digital pH meters, these pH meters can measure the pH of the solution accurately for two decimal places. These pH meters can be used over wide range temperatures. These pH meters dont require additional current for the working once they are dipped in the solution they measure the pH of the solution on the display. FEED FORWARD CONTROL Feed forward control is becoming widely used. Process disturbances are measured and compensated for without waiting for change in the controlled variable to indicate that a disturbance has occurred. Feed forward control is also controlled when the final controlled variable cannot be measured. The equation solved by the controller relating input-liquid heat content, steam flow and output-liquid temperature is usually designated as the process model. Perfect models and controllers are rare, so a combination of feedback and feed forward control is desirable. The
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arrangement of a controller supplying the set point for another controller is known as cascade control and it is commonly called Feed back control. CONTROL VALVE The control valve is a pneumatic device that moves the valve stem as the pressure on a spring-loaded diaphragm changes. The stem positions a plug I the orifice of the valve body. As the pressure increases, the plug moves downward and restricts the flow of fluid through the valve. The action is referred to as air-to-close. The valve may also be constructed to have airto-open action. Valve motors are often constructed so that valve stem is proportional to the valve-top pressure. Most commercial valves move from fully open to fully close as the valvetop pressure changes from 3 to 15 psi. CONTROLLER The controller hardware is required to control the temperature of a stream leaving at refrigeration. This hardware, available from manufacturers of such equipment, consists of the following components listed here along with their respective conversions: 1. Transducer (temperature to current) 2. Controller-recorder (current to current) 3. Converter (current to pressure) 4. Control valve (pressure to flow rate) Thermocouple is used to measure the temperature, the signal from the thermocouple is sent to the transducer, which produces an output range of 4-20mA, which is a linear function of the input. The output of the transducers enters the controller where it is compared to a set point to produce an error signal. The controller converts the error to an output in the range of 4-20mA according to the control law we have considered so far has been proportional. The output of the controller enters the converter, which produces an output the range of 3-15 psig, which is a linear function of the input. Finally, the output of the converter is sent to the top of the control valve, which adjusts the flow of the cooling water to the heat exchanger. We shall assume that the valve is linear and the pressure-to-open type. Electricity is needed for transducer, controller and converter. A source of 20 psig air is needed for the converter.
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This concludes our brief introduction to valves and controllers. We know present transfer function for such devices. These transfer functions, especially for controllers are based on ideal devices that can be only approximated in practice. The degree of approximation is sufficiently good to warrant use of these transfer functions to describe the dynamic behavior of controller mechanism for ordinary design purpose.
2.6 PLANT LAYOUT Introduction The economic construction and efficient operation of a process unit will depend upon how well the plant and equipment laid on the process flowsheet is laid out and on the profitability of the project with its scope for future expansion. Plant location and site selection should be made before the plant layout. Plant layout and site selection: The location of the plant has a crucial effect on the profitability of the project. The important factors that are to be considered while selecting a site are: 1. Marketing area For materials that are produced in bulk quantities, such as cement, mineral acids, and fertilizers where the cost of product per ton is relatively low and the cost of transport a significant fraction of the sales price, the plant should be located close to the primary product. 2. Raw materials The availability and price of suitable raw materials will often determine the site location. Plants producing bulk chemicals are best located close to the source of major raw material
3. Transport Transport of raw materials and products is an important factor to be considered.
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4. Availability of labour Labour will be needed for construction of the plant and its operation. There should be an adequate pool of unskilled labours available locally. Skilled tradesmen will be needed for plant maintenance. Local trade union customs and restrictive practices will have to be considered when assessing the availability and suitability of the local labour for recruitment and training. 5. Environmental impact and effluent treatment All industrial processes produce waste products, and full consideration must be given to the difficulties and cost of their disposal. The disposal of toxic and harmful effluents will be covered by the local regulations and appropriate authorities must be consulted during the initial survey to determine the standards that must be met. 6. Local community consideration The proposed plant must fit in with and be acceptable to the local community. Full consideration must e given to the safe location of the plant so that it does not impose a significant additional risk to the community on a new site, the local community must be able to provide adequate facilities for the plant personnel. 7. Land Sufficient land must be available for the proposed plant and for the future expansion. The land should ideally flat, well drained and have suitable load-bearing characteristics. Full site evaluation must be made to determine the need for piling or other special foundations. 8. Climate Adverse climatic condition at a site will increase costs. Abnormally low temperatures will require the provision of additional insulation and special heating for equipment and pipe runs. 9. Political and strategic considerations Capital grants, tax concessions and other inducements are often given by governments to direct new investment to preferred locations; such as areas of high unemployment. The availability of such grants can be the overriding consideration in the site selection.
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After considering the location of the site the plant layout is completed. It involves placing of equipment so that the following are minimized: I. Damage to persons and property in case of fire explosion or toxic release. II. Maintenance costs III. Number of people required to operate the plant IV. Construction costs V. Cost of planned expansion. SITE LAYOUT The process units and ancillary building should be laid out to give the most economical flow of materials and personal around site. Hazardous process must be located at a safe distance from other buildings. Considerations must also be given to future expansion of the site. The ancillary buildings and services required on site, in addition to the main processing units(buildings),will include : 1. Storages for raw material and products: Tank farms and warehouse 2. Maintenance workshops. 3. Store for maintenance and operating supplies. 4. Laboratories for process control. 5. Fire stations and other emergency services. 6. Utilities: steam boilers, compressed air, power generation, Transformer station. 7. Effluent disposal plant. 8. Offices for general administration. 9. Canteens and other amenity buildings, such as medical centers. 10. Car parks. In preparing the plant layout one has to satisfy various regulations acts and considerations are: 1. Factories act 2. The explosives act 3. Excise rules 80
4. Health rules 5. The boilers act 6. Electricity regulations It is also advisable to check up the insurance regulations from the view of getting the best coverage at minimum cost for plant building and inventory. RAW MATERIALS SOURCES Careful considerations should be given to the sources of raw materials to be used, method of delivery and storage facilities of raw materials. WASTE PRODUCT DISPOSAL Another aspect, which is gaining importance these days, is the environmental considerations. Careful attention should be given to the nature of products to be wasted, their quantity, available methods of disposal and the legislations governing the disposal. Location can be an important factor for cost. If the bearing quality of the land is low, considerable amount may be spent in piling support for heavy equipments or multi-storied buildings. If the land is uneven and the site needs even level, the cost of leveling may be considerable. Sometimes advantage can be taken of uneven levels so as to use gravity as a means of transportation of materials. When planning the preliminary site layout, the process units will normally be sited first and arranged to give a smooth flow of materials through various processing steps, from raw material to final product storage. Process units are normally spaced at least 30 meters apart. Administration offices and laboratories, in which a relatively large number people will be working, should be located well away from potentially hazardous process control rooms. The siting of the main process units will determine the layout of the plant roads, pipes, alleys and drains. Access roads will also be constructed for operation and maintenance purpose. The main storage areas should be placed between the loading and unloading facilities and the process units they serve. MAINTENANCE Maintenance costs are high in the maleic anhydride industry. In some case, the cost of maintenance exceeds the companys profit. The engineer must design to reduce these costs.
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The easiest way to reduce maintenance costs is to allow lots of extra space and to construct everything at ground level for easy access. However, this may increase construction and operating expenses and decrease the ease of operatibility. Adequate space must be left around all equipment so that it can be easily serviced and operated. STORE AND WAREHOUSES The engineer must decide whether the warehouses must be at ground level or dock level. The latter facilitates loading trains and trucks, but costs 15-20% more than one placed on the ground. It is usually difficult to justify the added expenses of a dock-high warehouse To size the amount of space needed, it must be determined how much is to be stored in what size containers. The container sizes that will be used are obtained from the scope. Liquids are generally stored in bulk containers. No more than a weeks supply of liquid stored in drums should be planned. Solids, on the other hand, are frequently stored in smaller containers or in a pile on the ground.
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RAW MATERIAL STORAGE
TUBULAR REACTOR
HEAT EXCHANGE RS AND SEPARATOR
ABSORBER AND DEHYDRAT OR
WORKSHOP
WORK SHOP
PRODUCT
FIRE STATION HOSPITAL
TESTING AND LAB AREA
CANTEEN
ADMINISTRATIVE SECTION
QUALITY CONTROL
HEALTH CLUB
PARKING AREA
PACKAGING SECTION
SECURITY OFFICE
TRAINING
ENTRANCE
EXIT
STORE HOUSE
Figure 2.5 Plant layout
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2.7 COST ESTIMATION ESTIMATION OF THE TOTAL CAPITAL INVESTMENT The total capital investment I involves the following: A. The fixed capital investment in the process area, IF. B. The capital investment in the auxiliary services, IA. C. The capital investment as working capital, IW. i.e., I = IF + IA + IW
A. FIXED CAPITAL INVESTMENT IN THE PROCESS AREA, IF. This is the investment in all processing equipment within the processing area. Fixed capital investment in the process area, IF = Direct plant cost + Indirect Anhydride manufacturing plant are furnished below: S.No. 1 2 3 4 5 6 TOTAL Table 2.1 Cost of equipments 84 Equipment Tubular Reactor Heat exchanger Condenser Absorption column Storage tank sealed Miscellaneous Units 1 1 1 1 2 Cost in lakhs/unit 800 100 150 340 50 Cost lakhs 800 100 150 340 100 1000 2490 lakhs in plant cost The approximate delivered cost of major equipments used in the proposed Maleic
Direct cost factor S.No 1 2 3 4 5 6 7 8 9 Items Delivered equipments Equipment installation Insulation Instrumentation Piping Land & building Foundation Electrical Clean up Total direct cost factor Table 2.2 Direct cost factor Direct plant cost = (Delivered cost of major equipments) * (Total direct cost factor) / 100 Direct plant cost = (2490 * 280) / 100 = 6972 lakhs Indirect cost factor S.No. 1 2 3 Item Overhead contractor etc. Engineering fee Contingency Total indirect cost factor Table 2.3 Indirect cost factor Indirect plant cost = (Direct plant cost)( Total indirect cost factor) / 100 = (6972 * 56) / 100 = 3904.32 lakhs Indirect cost factor 30 13 13 56 15 15 15 75 30 10 15 5 280 cost of major Direct cost factor 100
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Fixed capital investment in the process area, IF = Direct plant cost + Indirect plant cost = 6972 + 3904.32 = 10876.32 lakhs
B. THE CAPITAL INVESTMENT IN THE AUXILLARY SERVICES, IA. Such items as steam generators, fuel stations and fire protection facilities are commonly stationed outside the process area and serve the system under consideration. S.No. 1 2 3 4 5 6 7 8 9 10 Items Auxiliary buildings Water supply Electric Main Sub station Process waste system Raw material storage Fire protection system Roads Sanitary and waste disposal Communication Yard and fence lighting Total Capital investment in the auxillary services = (Fixed capital investment in the process area)*(Auxillary services cost factor) / 100 = (10876.32* 12.3) / 100 = 1337.7874 lakhs Auxiliary factor 5 2 1.5 1 1 0.7 0.5 0.2 0.2 0.2 12.3 services cost
Table 2.4 Auxillary services cost factor
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Installed cost = Fixed capital investment in the process area + Capital investment in the auxiliary services = 10876.32+ 1337.7874 = 12214.1074 lakhs
C.
THE
CAPITAL
INVESTMENT
AS
WORKING
CAPITAL,
IW.
This is the capital invested in the form of cash to meet day-to-day operational expenses, inventories of raw materials and products. The working capital may be assumed as 15% of the total capital investment made in the plant ( I ). Capital investment as working capital, IW = ((10876.32 + 1337.7874)* 15) / 85 = (12214.1074 * 15) / 85 = 2155.4307 lakhs Total capital investment, I = IF+ IA+ IW = 10876.32 + 1337.7874 + 2155.4307 = 14369.5381 lakhs ESTIMATION OF MANUFACTURING COST The manufacturing cost may be divided into three items, as follows: A. Cost Proportional to total investment B. Cost proportional to production rate C. Cost proportional to labour requirement
A. COST PROPORTIONAL TO TOTAL INVESTMENT This includes the factors, which are independent of production rate and proportional to the fixed investment such as Maintenance-labour and material
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Property taxes Insurance Safety expenses Protection, security and first aid General services, laboratory, roads, etc. Administrative services For this purpose we shall charge 15% of the installed cost of the plant = (Installed cost * 15) / 100 = (12214.1074 * 15) / 100 = 1832.12 lakhs B. COST PROPORTIONAL TO PRODUCTION RATE The factors proportional to production rate are Raw material costs Utilities cost power, fuel, water. Steam, etc. Maintenance cost Chemical, warehouse, shipping expenses Assuming that the cost proportional to production rate is nearly 60% of total capital investment, Cost proportional to production rate = (Total capital investment*60) / 100 = (14369.5381* .6) = 8621.7228 lakhs C. COST PROPORTIONAL TO LABOUR REQUIREMENT The cost proportional to labour requirement might amount to 10% of total manufacturing cost. Cost proportional to labour requirement = (1832.116 + 8621.7228)(0.1) / (0.9) = 1161.5323 lakhs Therefore, manufacturing cost = (1832.116 + 8621.7228 + 1161.5323) = 11615.3233 lakhs
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SALES PRICE OF PRODUCT Market price of = Rs.65/kg Production rate =25000 TPA Total sales income = 65 * 25000 * 1000 = 16250 lakhs PROFITABILITY ANALYSIS A. DEPRECIATION According to sinking fund method: R = (V-VS) I / (1+ I)n -1 R = Uniform annual payments made at the end of each year V = Installed cost of the plant VS = Salvage value of the plant after n years N = life period (assumed to be 15 years) I R = Annual interest rate (taken as 15%) = (12213.7874 * .15) / (1+0.15)15-1 = 256.69 lakhs B. GROSS PROFIT Gross profit = Total sales income - manufacturing cost = 16250 11615.3233 = 4634.6767 lakhs C. NET PROFIT It is defined as the annual return on the investment made after deducting depreciation and taxes. Tax rate is assumed to be 40%. Net profit = Gross profit-Depreciation-(Gross profit*Tax rate) = 4634.6767-256.69-(4634.6767*0.4) = 2524.1160 lakhs D. ANNUAL RATE OF RETURN
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Rate of return = (100*Net profit/Installed cost) = (100*2524.1160) / 12213.7874 = 20.66% E. PAYOUT PERIOD Payout period = Depreciable fixed investment ((profit)+(depreciation)) = 12213.7874/ (2524.1160 + 256.69) = 4.39 years 2.8 HEALTH AND SAFETY Maleic anhydride is a strong eye irritant and can cause painful conjunctivitis and possible corneal dullness if not immediately removed. Eye contact with dusts or fumes of maleic anhydride also can cause temporary double vision and the impression of halos around lights. Painful irritation can result from prolonged exposure to the skin, sometimes with the formation of small blisters. Moist skin is especially susceptible because of the rapid exothermic hydrolysis of maleic anhydride in the presence of water. Sub-acute inhalation may cause headache, nosebleed, nausea, or vision disturbances. Prolonged inhalation has caused pulmonary edema. Rubber gloves and chemical safety goggles should be worn when handling maleic anhydride and dust respirators must be worn when maleic anhydride dust is present. To protect against fumes, organic-vapor-cartridge respirators or full-face gas masks are required. Respirators are mandatory if the material cannot be handled in a well-ventilated area. Although maleic anhydride does not present a severe fire hazard, it can burn if ignited. Fires can be extinguished with carbon dioxide. Dry chemicals are not recommended, especially if they contain sodium; nor is water or foam because frothing may result. EMISSIONS Nearly all emissions from maleic anhydride production are from the main process vent of the product recovery absorber, the largest vent in the process. The predominant pollutant is unreacted benzene, ranging from 3 to 10 percent of the total benzene feed. Fugitive emissions of benzene, maleic anhydride, and maleic acid also arise
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from the storage and handling of benzene and maleic anhydride. Dust from the briquetting operations can contain maleic anhydride. Potential sources of secondary emissions are spent reactor catalyst and excess water from the dehydration column. Benzene oxidation process emissions can be controlled at the main vent by means of carbon adsorption, thermal incineration, or catalytic incineration. Benzene emissions can be eliminated by conversion to the n-butane process. The vent from the refining vacuum system is combined with that of the main process as a control for refining vacuum system emissions. A carbon adsorption system or an incineration system can be designed and operated at a 99.5 percent removal efficiency for benzene and volatile organic compounds. PROCESS SAFETY Introduction: In recent years there has been an increased emphasis on process safety as a result of number of serious accidents. This is due in part to the worldwide attention to issues in the chemical industry brought on by several dramatic accidents involving gas releases, major explosions and several environmental accidents. Public awareness of these and other accidents has provided a driving force for industry to improve its safety record. Local and national governments are taking a hard look at safety in the industry as a whole and the chemical industry in particular. For many reasons, the public often associates chemical industry with environmental and safety problems. It is vital for the future of the chemical industry that process safety has a higher priority in the design and operation of chemical process facilities.
Industrial accidents: An accident has been defined as an unplanned or unexpected event, which causes or is likely to cause an injury. An accident occurs as a result of unsafe action or exposure to an unsafe environment. Reasons for accidents: Improper attitude Lack of knowledge or skill
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Physical unsuitability Improper mechanical or physical environment Accident prevention: From the foregoing, it will be seen that the occurrence of an injury is the culmination of a series of events circumstances that invariably occur in a fused and logical order. Knowledge of the factors in the accident sequence guides and assists in selecting the point of attack in prevention work. It permits simplification without sacrifice of effectiveness. The four factors that converge to cause accidents are: Personal factor Hazard factor Unsafe factor Proximate casual factor The solution under the four factors would also lead to the steps. These are planning and organizing to 1. Prevent unsafe mechanical or physical conditions. 2. Prevent unsafe action being committed. Unsafe condition examples : Operating or working at unsafe speed. Making safety devices inoperative. Unsafe loading, placing, mixing etc. Inadequately guarded. Unsafe design or construction. Hazardous arrangement or process. Unsafe dress or apparel. Unsafe method, process, planning etc. The most important means of accident prevention are: Engineering revision. Instruction.
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Personal adjustment. Discipline. PERSONAL PROTECTIVE DEVICES: Protective devices are required by regulation; the employers are required to provide it free of cost and also should be responsible to ensure its usage maintenance and renewal. Once it is decided to use personal protective devices, we must select the proper type of devices. For selection of device, two criteria should be used: 1. The degree of protection. 2. The ease with which it may be used. Protective devices are divided into two groups: a. Respiratory devices b. Non-respiratory devices
Safety appliances: S Helmets: Every employee inside the factory should always wear the safety helmet to avoid head injuries. S Safety shoes: All the employees working inside a factory should wear safety shoes and gumboots should be used while handling acids and alkalis. S Hand gloves: While operating any valve or equipment and also while executing any maintenance work including electrical maintenance work, the employees should wear appropriate type of safety gloves. S Dust mask: While working in a dusty atmosphere, the employees must wear dust masks to prevent dust and fumes entering the sensitive respiratory organs. S Plastic aprons:
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This along with the hood gives protection to the operation and maintenance staff while handling dangerous acids and other hazardous chemicals particularly when there is possible leakage. In spite of safety appliances, the companys medical center should be equipped to meet any emergency and any employee coming in contact with acids or any hazardous chemicals must be treated at the medical center immediately.
CHAPTER 3 CONCLUSION The manufacture of maleic anhydride by oxidation of benzene was studied as it was simple in concept. Maleic anhydride is an important product with a multitude of applications and its manufacture must be made most economical and pollution control should be given priority. In this project we dealt with the cost estimation and considerations of plant layout.
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CHAPTER 4 BIBLIOGRAPHY BOOKS 1) Charles E. Dryden, Outlines of Chemical Technology for the 21st century, Third edition, NEW YORK PRESS, 1997 2) Kirk and Othmer - Encyclopedia of Chemical Technology, Third edition Maleic Anhydride, Maleic Acid, and Fumaric Acid. Vol 14, 1981:780-788 3) Perry, R.H., and D.W. Green, Perrys Chemical Engineers Handbook, Seventh Edition, McGraw-Hill, 1997 4) McCabe, Smith and Harriot, Unit Operations of Chemical Engineering REFERENCES 1. Arqumedes Cruz-Lpez, Nolven Guilhaume, Sylvain Miachon and Jean-Alain Dalmon, (2005), Selective oxidation of butane to maleic anhydride in a catalytic membrane reactor adapted to rich butane feed, Catalysis Today Volumes 107-108, 30 October 2005, Pages 949-956 2. Beatriz T. Pierini and Eduardo A. Lombardo, (2005), Cr, Mo and W used as VPO promoters in the partial oxidation of n-butane to maleic anhydride, Catalysis Today Volumes 107-108 , 30 October 2005, Pages 323-329 3. G. Bignardi, F. Cavani, , C. Cortelli, T. De Lucia, F. Pierelli, F. Trifir, G. Mazzoni, C. Fumagalli and T. Monti, Influence of the oxidation state of vanadium on the reactivity of V/P/O, catalyst for the oxidation of n-pentane to maleic and phthalic anhydrides, WEBSITES Journal of Molecular Catalysis A: Chemical Volume 244, Issues 1-2 , 1 February 2006, Pages 244-251
http://www.che.Isu.edu/COURSES/4205/2000/Lee/paper.htm www.epa.gov
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KINETICS OF ENZYMATIC DEHAIRING USING PROTEASE ENZYME & MANUFACTURE OF MALEIC ANHYDRIDE

EXPANSION Michaelis-Menten constant Velocity Substrate concentration Temperature

Figure 1.1 Tanning Process

These enzymes catalyse intramolecular rearrangements and are subdivided into:

isomerisation of glucose to fructose.

Optical Density at 750 nm

Figure 2.2 Effect of time on protease activity at different protease concentrations

Enzyme conc 0.0025 0.005 0.0075 0.01

Od 0.178 0.432 0.782 1.15

Amino lib 0.612 1.484 2.687 3.951

acid Activity 2446.391 2968.654 3582.542 3951.333

Velocity 163.093 197.91 238.836 263.422

Effect of time for 15 minutes time interval

activity vs enzyme conc 15min

Figure 2.3 Effect on activity for 15 minutes

Table 2.2 Effect of time for 30 minutes time interval

activity vs enzyme conc 30min

Figure 2.5 Effect on activity for 30 minutes

Table 2.3 Effect of time for 45 minutes time interval

Figure 2.7 Effect on activity for 45 minutes

Table 2.4 Effect of time for 60 minutes time interval

Figure 2.9 Effect on activity for 60 minutes

Table 2.5 Effect of time for 120 minutes time interval

Figure 2.11 Effect on activity for 120 minutes

Table 2.6 Effect of time for 180 minutes time interval

Figure 2.13 Effect on activity for 180 minutes

Table 2.7 Effect of time for 240 minutes time interval

Figure 2.15 Effect on activity for 240 minutes

Table 2.8 Effect of time for 300 minutes time interval

Figure 2.17 Effect on activity for 300 minutes

Table 2.9 Effect of time for 360 minutes time interval

Figure 2.19 Effect on activity for 360 minutes

600 500 activity 400 300 200 100 0 0 50 time

0.2% subs 0.4% subs 0.6% subs 1% subs

Table 2.10 Effect of pH on protease activity

Figure 2.23 Effect of pH on velocity

Table 2.11 Effect of temperature on protease activity

effect of temperature on activity

8000 6000 4000 2000 0 0 10 20 30 40 50 60

temperature Figure 2.24 Effect of temperature on protease activity

Substrate conc % 0.2 0.4 0.6 1.0

Od 0.012 0.016 0.02 0.044

Amino acid lib 0.041 0.055 0.069 0.151

Activity 41.231 54.975 68.719 151.181

Velocity 2.749 3.665 4.581 10.079

1/[S] 5 2.5 1.667 1

1/V 0.364 0.273 0.218 0.099

Table 2.12 Effect of substrate concentration for 15 min time interval

velocity vs subs conc15 min

Figure 2.26 Effect on velocity for 15 min time interval

Substrate conc % 0.2 0.4 0.6 1.0

Od 0.014 0.021 0.033 0.055

Amino acid lib 0.048 0.072 0.113 0.189

Activity 48.103 72.155 113.386 188.977

Velocity 1.603 2.405 3.780 6.299

1/[S] 5 2.5 1.667 1

1/V 0.624 0.416 0.265 0.159

Table 2.13 Effect of substrate concentration for 30 min time interval

velocity vs subs conc 30min

Figure 2.28 Effect on velocity for 30 min time interval

Substrate conc % 0.2 0.4 0.6 1.0

Od 0.017 0.027 0.051 0.079

Amino acid lib 0.058 0.093 0.175 0.271

Activity 58.411 92.770 175.233 271.439