Immunosensor

An amperometric enzyme immunosensor for the determination of antigen (Ag) of pathogenic fungi Trichophyton
rubrum (TR) was developed. The immunosensor is based on a specific immunoreaction of Ag with antibodies which
were immobilized together with cholinesterase into a nitrocellulose membrane (biosensitive part of immunosensor).
The biosensitive part could be regenerated and is stable for at least 35 days. The developed immunosensor allows
the determination of down to 1×10−15 mg ml−1 of Ag TR. The total time of immunoassay is less than 20 min. The
method was applied for the determination of Ag TR in human blood serum.

An enzyme immunosensor has been developed for assaying human immunoglobulin G (IgG). The
sensor is composed of an oxygen sensoring system and an antibody-binding membrane. The assay
procedure involves the competitive immunochemical reaction of the membrane-bound antibody
with nonlabeled and catalase-labeled IgG and the electrochemical determination of membrane-
bound catalase activity. The analytical result is directly displayed by the output current of the
sensor. The sensor exhibited an excellent performance in monitoring specifically human IgG.

Types of Biosensors- As mentioned, the biosensors are of 5 types:

calorimetric,
potentiometric
amperometric,
opticalandacousticwavebiosensors.

The chief features of calorimetric biosensors are briefly described here.Many

enzyme catalyzed reactions produce heat (exothermic). Calorimetric biosensors
measure the change in temperature of the solution containing the analyte following
enzyme action and interpret it in terms of the analyte concentration in the solution.

The analyte solution is passed through a small (Ca. 1 ml) packed bed column
containing immobilized enzyme; the temperature of the solution is determined just
before entry of the solution into the column and just as it is leaving the column
using separate thermistors.

This is the most generally applicable type of biosensor, and it can be used for
turbid and strongly coloured solutions. The greatest disadvantage is to maintain
the temperature of the sample stream at a constant, say+- 0.01˚C, temperature.
The sensitivity (10-4 M) and tbe range (10-4 - 10-2 M) of such biosensors is quite
low for most applications.

The sensitivity can be increased by using two or more enzymes of the pathway in
the biosensor to link several reactions to increase the heat output. Alternatively,
multifunctional enzymes may be used .An example is the use of glucose oxidase
for determination of glucose.
Calorimetric Biosensor
When the physical change is heat, released or absorbed by the reaction it is
calorimetric biosensor. It measures the change in temperature in the solution
containing analyte and interprets it in terms of analyte concentration.

1
Separate thermistors measure the temperature of the solution before entry into the
small packed bed column containing immobilized enzyme and also at the time of
leaving the column.
Calorimetric biosensors are most widely applicable and can be used to measure
turbid and strongly coloured solutions. Maintenance of constant sample
temperature is the disadvantage of this type.

At the transducer surface, an electrical potential is produced due to changed

distribution of electrons and this type of biosensors are called potentiometric
biosensors. They use ion sensitive electrodes, commonly pH meter glass electrodes
for cations, glass pH electrodes coated with a gas-selective membrane for CO2,
NH3 or H2S or solid-state electrodes. These electrodes convert the biological
reaction into electric signal.
Potentiometric Biosensors
At the transducer surface, an electrical potential is produced due to changed
distribution of electrons and this type of biosensors are called potentiometric
biosensors. They use ion sensitive electrodes, commonly pH meter glass electrodes
for cations, glass pH electrodes coated with a gas-selective membrane for CO2,
NH3 or H2S or solid-state electrodes. These electrodes convert the biological
reaction into electric signal.

Optical biosensor
A most promising optical biosensor utilizes luminescence due to firefly luciferase for
detection of bacteria in food or clinical samples. The bacteria are specifically lysed
to release ATP. This ATP is used by luciferase in the presence of O2 to produce
light, which is measured by the biosensor.
Acoustic wave biosensors sense the change in mass of the biological
components as a result of the reaction.

They are also called piezoelectric devices. The surface of the transducer is usually
coated with antibodies which bind to the complementary antigen present in the
sample solution. The resulting increase in mass reduces their frequency of
vibration. This change in frequency is measured in terms of antigen present in the
sample solution.

An artificial enzyme is a synthetic, organic molecule prepared to recreate the active site of
an enzyme.

Enzyme catalysis of chemical reactions occur with high selectivity and rate in a small part of the
enzyme macromolecule known as the active site. There, the binding of a substrate close to
functional groups in the enzyme causes catalysis by so-called proximity effects. It is therefore
2
possible to create similar catalysts from small molecule mimics of enzyme active sites by
combining, in a small molecule, the ability to bind substrate with catalytic functional groups.
Since the artificial enzymes need to bind molecules, they are made based on a host-molecule
such as a cyclodextrin, crown ethers or calixarene etc.

A number of artificial enzymes have been reported catalysing various reactions with rate
increases up to 103; this is nevertheless substantially lower than natural enzymes that typically
causes rate increases above 106. One of the pioneers in artificial enzyme research is chemist
Ronald Breslow.

New approaches based on amino acids or peptides as characteristic molecular moieties have led
to a significant expansion of the field of artificial enzymes or enzyme mimics. For instance,
recent results by the group of Rob Liskamp[1] have shown that scaffolded histidine residues can
be used as mimics of certain metalloproteins and -enzymes. Especially the structural mimicry of
certain copper proteins (e.g. hemocyanin, tyrosinase and catechol oxidase), containing so-called
type-3 copper binding sites, has been shown. This is a significant improvement since the use of
scaffolded histidine residues is one step closer to the mimicry of enzymes by biological relevant
species such as amino acids and peptides[2].

For the first time ever, a completely artificial chemical enzyme has been
successfully used to neutralise a toxin found naturally in fruits and vegetables.

 First, water miscible solvents like ethanol and acetone were added. If the water
concentration was high enough, activity remained.
 Biphasic mixtures were made in which an aqueous solution of an enzyme was
emulsified in a water immiscible solvent like chloroform or ethylacetate. The substrate
would partition into both phases, while the product hopefully would end up into the
organic phase.
 Nearly nonaqueous solvents were used, with a few % water at less than the solubility
limits of water.
 Finally, anhydrous organic solvents (0.01% water) were used. It is this later case that is
most astonishing, since at first glance it is hard to believe that enzymatic activity was
retained.

It is important to realize that in this last case, the ENZYME IS NOT IN SOLUTION. It is
rather in suspension and acts as a heterogeneous catalyst, much like palladium acts as
a heterogeneous catalyst in the hydrogenation of alkenes. The suspension must be
mixed vigorously and then sonicated to produce small suspended particles, so diffusion
of reactants into the enzyme and out is not rate limiting. Let's explore the activity of
chymotrypsin in a nonpolar solvent. Consider the following questions.

 Why aren't the enzymes inactive? Surely it must seem ridiculous that they aren't, since as we
learned earlier, proteins are not that stable. A 100 amino acid protein on average is stabilized
only about 10 kcal/mol over the denatured state, or the equivalent of a few H bonds. Surely the
3
 hydrophobic effect, one of the dominant contributors to protein folding and stability, would not
stabilize the native structure of enzymes in nonpolar organic solvents, and the protein would
denature. It doesn't however! Maybe the real question should be not whether water is
necessary, but rather how much water is necessary. The enzyme can't "see" more than a
monolayer or so of water around it. The data suggests that the nature of the organic solvent is
very important. The most hydrophobic solvents are best in terms of their ability to maintain
active enzymes! Chymotrypsin retains 104 more activity in octane than pyridine (see kcat/Km
below), which is more hydrophilic than octane. The more polar the solvent, the more it can strip
bound water away from the protein. If you add 1.5% water to acetone, the bound water increases
from 1.2 to 2.4%, and the activity of chymotrypsin increases 1000 fold.

Chymotrypsin Activity in Organic Solvents

relative ratio H2O bound to

Solvent Structure kcat/Km (M-1min-1)
kcat/Km enzyme (%, w/w)

Octane 63 15000x 2.5

Toluene 4.4 1000x 2.3

Tetrahydrofuran 0.27 175x 1.6

Acetone 0.022 5.5x 1.2

Pyridine <0.004 1x (.004) 1.0

 How active are enzymes in nonpolar solvents? Enzymes are often studied in model
transesterification reactions. Typical reaction conditions are enzyme at 1 mg/ml, with one
substrate, an ester such as N-acteyl-L-Phe-ethyl ester,at 2-12 mM, and the other substrate an
alcohol, such as n-propanol (instead of being water as in a typical hydrolysis reaction) at 0.25-1.5
M. The more concentrated alcohol replaces the alcohol (ethanol) esterified in the ester. Michaelis-
Menten kinetics are followed, with biomolecular rate constants of 1010 > than without the enzyme.
 How much water do the enzymes need? 1 molecule of chymotrypsin in octane has < 50
molecules of water associated and can demonstrate activity. To form a monolayer requires about

4
 500 water molecules. Water can be added which presumably leads to more bound water and
higher activity.
 How stable are the enzymes? Denaturation requires conformational flexibility, which apparently
requires water. The half-life of chymotrypsin in water at 60oC is minutes, but in octane at 100oC it
is hours. At 20oC, the half-life in water is a few days, but in octane it is > 6 months. Remember
two things contribute to stability. The protein can denature at high temperatures. Also since
chymotrypsin is a protease, it can cleave itself in a autoproteolytic reaction.

Half-Life of Chymotrypsin Activity in Water and Octane

Solvent 60oC 100oC 20oC

water minutes - few days

octane - hours > 6 months

 Is the enzyme specificity changed? The net binding energy is a function of the binding energy
of the substrate - the binding energy of the water, since water must be displaced from the active
site on binding. In an anhydrous solvent, specificity changes must be expected. For
chymotyrpsin, the driving force for binding of substrates in water is mostly hydrophobic. In water,
the kcat/Km for the reaction of N-acteyl-L-Ser-esters is reduced 50,000x compared to the Phe
ester. However, in octane, chymotrypsin is three times more active toward Ser esters than Phe
esters.

Chymotrypsin Specificity Changes in Water and Octane

kcat/Km
Substrate
solvent: H2O solvent: Octane

N-acetyl-L-Ser-ester 1x 3x

N-acetyl-L-Phe-ester 50,000x 1x

Now consider competitive inhibitors. Napthalene binds 18 times more tightly than 1-
napthoic acid, but in octane, the chymotrypsin binds napthoic acid 310 times as tightly.
Likewise the ratio of [kcat/Km (L isomer)]/[kcat/Km (D isomer)] of N-acetyl-D- or N-acetyl-
L-Ala-chloroethyl esters is 1000-10,000 in water, but less than 10 in octane.

Chymotrypsin Inhibition Constants in Water and Octane

Inhibition Constant Ki (nM)

Inhibitor In water In Octane

Benzene 21 1000

5
Benzoic acid 140 40

Toluene 12 1200

Phenylacetic acid 160 25

Naphthalene 0.4 1100

1-Naphthoic acid 7.2 3

 Can new reactions be carried out in nonpolar solvents? The quick answers is yes, since
reactions in aqueous solutions can be unfavorable due to low Keq's, side reactions, or insolubility
of reactants. Consider lipases which cleave fatty acid esters by hydrolysis in aqueous solutions.
In nonaqueous solutions, reactions such as transesterification or ammonolysis can be performed.

Enzymes are clearly active in organic solvents which appears to contradict our central
concepts of protein stability. Two reasons could could explain this stability.

1. It is possible that from a thermodynamic view, the enzyme is stable in organic solvents. However,
as was discussed above, this is inconceivable given the delicate balance of noncovalent and
hydrophobic interactions required for protein stability.
2. The second reason must win the day: the protein is unable to unfold from a kinetic point of view.
Conformational flexibility is required for denaturation. This must require water as the solvent.

A specific example helps illustrate the effects of different solvents on chymotrypsin

activity. Dry chymotrypsin can be dissolved in DMSO, a water miscible solvent. In this
solvent it is completely and irreversibly denatured. If it is now diluted 50X with acetone
with 3% water, no activity is observed. (In the final dilution, the concentrations of
solvents are 98% acetone, 2.9% water, and 2% DMSO.) However, if dry chymotrypsin
was added to a mixture of 98% acetone, 2.9% water, and 2% DMSO, the enzyme is
very active. We end up with the same final solvent state, but in the first case the
enzyme has no activity while in the second case it retains activity.

6
Dry enzymes added to a concentrated water-miscible organic solvent (like DMSO)
will dissolve and surely denature, but will retain activity when added to a
concentrated water-immiscible solvent (like octane), in which the enzyme will not
dissolve but stay in suspension.

It appears the enzymes have very restricted conformational mobility in nonpolar

solvents. By lyophilizing (freeze-drying) the enzyme against a specific ligand, a given
conformation of a protein can be trapped or literally imprinted onto the enzyme. For
example, if the enzyme is dialyzed against a competitive inhibitor (which can be
extracted by the organic solvent), freeze-dried to remove water, and then added to a
nonpolar solvent, the enzyme activity of the "imprinted" enzyme in nonpolar solvents is
as much as 100x as great as when no inhibitor was present during the dialysis. If
chymotrypsin is lyophilized from solutions of different pHs, the resulting curve of V/Km
for ester hydrolysis in octane is bell-shaped with the initial rise in activity reaching half-
maximum activity at a pH of around 6.0 and a fall in activity reaching half-maximum at
pH of approximately 9.

Enzymes in organic solvent allow new routes to organic solvents. Enzymes, which are
so useful in synthetic reactions, are:

 stereoselective - can differentiate between enantiomers and between prochiral substrates

 regioselective - can differentiate between identical functional groups in a single substrate

7
  chemoselective - can differentiate between different functional groups in a substrate (such as
between a hydroxyl group and an amine for an acylation reaction)

Enzyme in anhydrous organic solvents are so useful (from a synthetic point) not only since new types of
reactions can be catalyzed (such as transesterification, ammonolysis, thiolysis) but also because the
stereoselectivity, regioselectivity, and chemoselectivity of the enzyme often changes from activities of the
enzyme in water.

Organic Reactions in Water?

Organic reactions are usually conducted in organic solvents, since many organic molecules react with water, and the
reagents and products are usually not soluble in water. In a manner analogous to using an enzyme as a
heterogeneous catalyst in nonpolar solvent, Sharpless is pioneering a technique to conduct organic reactions in
water. They (Narayan et al.) have shown that many unimolecular and bimolecular reactions occur faster in water
than in organic solvents. As in enzyme catalysis in nonpolar solvent, the reactions must be mixed vigorously to
disperse reactants in micro-drops (a suspension) in water, greatly increasing the surface area that might allow water
to act on transition states or intermediates to stabilize them through hydrogen bonding. They called these reactions
"on water" reactions since reactants usually float on water. They have performed cycloadditions, alkene reactions,
Claisen rearrangments, and nucleophilic substitution reactions using this process. One cycloaddition reaction went
to completion in ten minutes at room temperature, compared to 18 hours in methanol and 120 in toluene. Adding
nonpolar solvent at certain times greatly increased the rate of the reaction.

Enzyme-catalyzed processes in organic solvents

Three different lipases (porcine pancreatic, yeast, and mold) can vigorously act as catalysts
in a number of nearly anhydrous organic solvents. Various transesterification reactions
catalyzed by porcine pancreatic lipase in hexane obey Michaelis-Menten kinetics. The
dependence of the catalytic activity of the enzyme in organic media on the pH of the
aqueous solution from which it was recovered is bell-shaped, with the maximum coinciding
with the pH optimum of the enzymatic activity in water. The catalytic power exhibited by the
lipases in organic solvents is comparable to that displayed in water. In addition to
transesterification, lipases can catalyze several other processes in organic media including
esterification, aminolysis, acyl exchange, thiotransesterification, and oximolysis; some of
these reactions proceed to an appreciable extent only in nonaqueous solvents

Amylase enzymes find use in bread making and to break down complex sugars such as
starch (found in flour) into simple sugars. Yeast then feeds on these simple sugars and
converts it into the waste products of alcohol and CO2. This imparts flavour and causes the
bread to rise. While Amylase enzymes are found naturally in yeast cells, it takes time for the
yeast to produce enough of these enzymes to break down significant quantities of starch in
the bread. This is the reason for long fermented doughs such as sour dough. Modern bread
making techniques have included amylase enzymes (often in the form of malted barley)
into bread improver thereby making the bread making process faster and more practical for
commercial use.[3]
When used as a food additive Amylase has E number E1100, and may be derived from swine
pancreas or mould mushroom.
Bacilliary amylase is also used in clothing and dishwasher detergents to dissolve starches
from fabrics and dishes.
Workers in factories that work with amylase for any of the above uses are at increased risk
of occupational asthma. 5-9% of bakers have a positive skin test, and a fourth to a third of
bakers with breathing problems are hypersensitive to amylase. [4]
An inhibitor of alpha-amylase called phaseolamin has been tested as a potential diet aid. [5]

8
Blood serum amylase may be measured for purposes of medical diagnosis. A normal
concentration is in the range 21-101 U/L. A higher than normal concentration may reflect
one of several medical conditions, including acute inflammation of the pancreas (concurrently
with the more specific lipase)[6], but also perforated peptic ulcer, torsion of an ovarian
cyst,strangulation ileus, macroamylasemia and mumps. Amylase may be measured in other
body fluids, including urine and peritoneal fluid.
In molecular baiology, the presence of amylase can serve as an additional method of
selecting for successful integration of a reporter construct in addition to antibiotic
resistance. As reporter genes are flanked by homologous regions of the structural gene for
amylase, successful integration will disrupt the amylase gene and prevent starch
degradation, which is easily detectible through iodine staining.

ProductionofProteases.
Proteases are the second most important group ofindustrial enzymes after alpha amylases.
They are used primarily in the detergent and dairy industry. In the market, they are
available as alkaline, neutral and acid proteases.
(a) Alkaline Proteases. They are produced and secreted out by many bacteria and fungi.
The most important producers are species of Bacillus (e.g. licheniformis, B.
amyloliquefaciens, B. firmus, B. megaterium, and B pumilus), species of Streptomyces (e.g.
S. fradiae, S. griseus, and S. rectus) and some fungi (e.g. Aspergillus niger, A. sojae, A
oryzae and A. flavus).

The cultures are maintained at 30-37°C in fermenters or flasks, where production of a

protease is chiefly regulated by the composition of culture medium. High partial pressure of
oxygen is generally necessary for optimal protease production. In most cases, the enzymes
are collected, purified and immobilized before their use inthedetergentindustry.

(b)NeutralProteases.
They are also produced and excreted out by both bacteria and fungi including Bacillus
subtilis, B. cereus, B. megaterium, Pseudomonas deruginosa, Streptomyces griseus,
Aspergillus oryzae, A sojae and Percularia oryzae. Neutral proteases are used in leather
industry and food industry. They are relatively unstable.

(c)AcidProteases.
They include rennin, which is an important enzyme used in cheese production. Many other
acid proteases are used in medicines. The important fungal genera producing rennin are
Aspergillus, Candida, Coriolus, Endothia and Mucor. Rennin produced by Endothia parasitica
was the first rennin marketed commercially in 1967. The enzyme is excreted out by the
fungus in the medium. It is concentrated and precipitated out in an evaporation process.

Applications of proteases in the food industry

Certain proteases have been used in food processing for centuries and any record of the discovery of
their activity has been lost in the mists of time. Rennet (mainly chymosin), obtained from the fourth
stomach (abomasum) of unweaned calves has been used traditionally in the production of cheese.
Similarly, papain from the leaves and unripe fruit of the pawpaw (Carica papaya) has been used to
tenderise meats. These ancient discoveries have led to the development of various food applications for a
wide range of available proteases from many sources, usually microbial. Proteases may be used at
9
various pH values, and they may be highly specific in their choice of cleavable peptide links or quite non-
specific. Proteolysis generally increases the solubility of proteins at their isoelectric points.

The action of rennet in cheese making is an example of the hydrolysis of a specific peptide linkage,
between phenylalanine and methionine residues (-Phe105-Met106-) in the κ -casein protein present in milk
(see reaction scheme [1.3]). The κ -casein acts by stabilising the colloidal nature of the milk, its
hydrophobic N-terminal region associating with the lipophilic regions of the otherwise insoluble α - and β -
casein molecules, whilst its negatively charged C-terminal region associates with the water and prevents
the casein micelles from growing too large. Hydrolysis of the labile peptide linkage between these two
domains, resulting in the release of a hydrophilic glycosylated and phosphorylated oligopeptide (caseino
macropeptide) and the hydrophobic para-κ -casein, removes this protective effect, allowing coagulation of
the milk to form curds, which are then compressed and turned into cheese (Figure 4.1). The coagulation
process depends upon the presence of Ca2+ and is very temperature dependent (Q10 = 11) and so can be
controlled easily. Calf rennet, consisting of mainly chymosin with a small but variable proportion of pepsin,
is a relatively expensive enzyme and various attempts have been made to find cheaper alternatives from
microbial sources These have ultimately proved to be successful and microbial rennets are used for about
70% of US cheese and 33% of cheese production world-wide.

Figure 4.1. Outline

method for the preparation of cheese.

The major problem that had to be overcome in the development of the microbial rennets was temperature
lability. Chymosin is a relatively unstable enzyme and once it has done its major job, little activity remains.
However, the enzyme from Mucor miehei retains activity during the maturation stages of cheese-making
and produces bitter off-flavours. Treatment of the enzyme with oxidising agents (e.g. H2O2, peracids),
which convert methionine residues to their sulfoxides, reduces its thermostability by about 10�C and
renders it more comparable with calf rennet. This is a rare example of enzyme technology being used to
destabilise an enzyme Attempts have been made to clone chymosin into Escherichia
coli and Saccharomyces cerevisiae but, so far, the enzyme has been secreted in an active form only from
the latter.

The development of unwanted bitterness in ripening cheese is an example of the role of proteases in
flavour production in foodstuffs. The action of endogenous proteases in meat after slaughter is complex
but 'hanging' meat allows flavour to develop, in addition to tenderising it. It has been found that peptides
10
with terminal acidic amino acid residues give meaty, appetising flavours akin to that of monosodium
glutamate. Non-terminal hydrophobic amino acid residues in medium-sized oligopeptides give bitter
flavours, the bitterness being less intense with smaller peptides and disappearing altogether with larger
peptides. Application of this knowledge allows the tailoring of the flavour of protein hydrolysates. The
presence of proteases during the ripening of cheeses is not totally undesirable and a protease
from Bacillus amyloliquefaciens may be used to promote flavour production in cheddar cheese. Lipases
from Mucor miehei or Aspergillus niger are sometimes used to give stronger flavours in Italian cheeses by
a modest lipolysis, increasing the amount of free butyric acid. They are added to the milk (30 U l-1) before
the addition of the rennet.

When proteases are used to depolymerise proteins, usually non-specifically, the extent of hydrolysis
(degree of hydrolysis) is described in DH units where:

(4.1)

Commercially, using enzymes such as subtilisin, DH values of up to 30 are produced using protein
preparations of 8-12% (w/w). The enzymes are formulated so that the value of the enzyme : substrate
ratio used is 2-4% (w/w). At the high pH needed for effective use of subtilisin, protons are released during
the proteolysis and must be neutralised:

subtilisin (pH 8.5)

H2N-aa-aa-aa-aa-aa-COO- H2N-aa-aa-aa-COO- + H2N-aa-aa-COO- + H+ [4.1]

where aa is an amino acid residue.

Correctly applied proteolysis of inexpensive materials such as soya protein can increase the range and
value of their usage, as indeed occurs naturally in the production of soy sauce. Partial hydrolysis of soya
protein, to around 3.5 DH greatly increases its 'whipping expansion', further hydrolysis, to around 6 DH
improves its emulsifying capacity. If their flavours are correct, soya protein hydrolysates may be added to
cured meats. Hydrolysed proteins may develop properties that contribute to the elusive, but valuable,
phenomenon of 'mouth feel' in soft drinks.

Proteases are used to recover protein from parts of animals (and fish) would otherwise go to waste after
butchering. About 5% of the meat can be removed mechanically from bone. To recover this, bones are
mashed incubated at 60�C with neutral or alkaline proteases for up to 4 h. The meat slurry produced is
used in canned meats and soups.

Large quantities of blood are available but, except in products such black puddings, they are not generally
acceptable in foodstuffs because of their colour. The protein is of a high quality nutritionally and is de-
haemed using subtilisin. Red cells are collected and haemolysed in water. Subtilisin is added and
hydrolysis is allowed to proceed batchwise, with neutralisation of the released protons, to around 18 DH,
when the hydrophobic haem molecules precipitate. Excessive degradation is avoided to prevent the
formation of bitter peptides. The enzyme is inactivated by a brief heat treatment at 85�C and the product
is centrifuged; no residual activity allowed into meat products. The haem-containing precipitate is recycled
and the light-brown supernatant is processed through activated carbon beads to remove any residual
haem. The purified hydrolysate, obtained in 60% yield, may be spray-dried and is used in cured meats,
sausages and luncheon meats.

Meat tenderisation by the endogenous proteases in the muscle after slaughter is a complex process
which varies with the nutritional, physiological and even psychological (i.e. frightened or not) state of the
animal at the time of slaughter. Meat of older animals remains tough but can be tenderised by injecting

11
inactive papain into the jugular vein of the live animals shortly before slaughter. Injection of the active
enzyme would rapidly kill the animal in an unacceptably painful manner so the inactive oxidised disulfide
form of the enzyme is used. On slaughter, the resultant reducing conditions cause free thiols to
accumulate in the muscle, activating the papain and so tenderising the meat. This is a very effective
process as only 2 - 5 ppm of the inactive enzyme needs to be injected. Recently, however, it has found
disfavour as it destroys the animals heart, liver and kidneys that otherwise could be sold and, being
reasonably heat stable, its action is difficult to control and persists into the cooking process.

Proteases are also used in the baking industry. Where appropriate, dough may be prepared more quickly
if its gluten is partially hydrolysed. A heat-labile fungal protease is used so that it is inactivated early in the
subsequent baking. Weak-gluten flour is required for biscuits in order that the dough can be spread thinly
and retain decorative impressions. In the past this has been obtained from European domestic wheat but
this is being replaced by high-gluten varieties of wheat. The gluten in the flour derived from these must be
extensively degraded if such flour is to be used efficiently for making biscuits or for preventing shrinkage
of commercial pie pastry away from their aluminium dishes.

Pectinases are one of the upcoming enzymes of fruit and textile industries. These enzymes break down
complex polysaccharides of plant tissues into simpler molecules like galacturonic acids. The role of acidic
pectinases in bringing down the cloudiness and bitterness of fruit juices is well established. Recently,
there has been a good number of reports on the application of alkaline pectinases in the textile industry
for the retting and degumming of fiber crops, production of good quality paper, fermentation of coffee and
tea, oil extractions and treatment of pectic waste water. This review discusses various types of pectinases
and their applications in the commercial sector.

Over the years, pectinases have been used in several conventional industrial processes,
such as textile, plant fiber processing, tea, coffee, oil extraction, treatment of industrial
wastewater, containing pectinacious material, etc. They have also been reported to work on
purification of viruses and in making of paper. They are yet to be commercialized.
Fruit juice extraction The largest industrial application of pectinases is in fruit juice
extraction and clarification. Pectins contribute to fruit juice viscosity and turbidity. A mixture
of pectinases and amylases is used to clarify fruit juices. It decreases filtration time up to
50%. Treatment of fruit pulps with pectinases also showed an increase in fruit juice volume
from banana, grapes and apples. Pectinases in combination with other enzymes, viz.,
cellulases, arabinases and xylanases, have been used to increase the pressing efficiency of
the fruits for juice extraction. Vacuum infusion of pectinases has a commercial application to
soften the peel of citrus fruits for removal. This technique may expand in future to replace
hand cutting for the production of canned segments. Infusion of free stone peaches with
pectinmethylesterase and calcium results in four times firmer fruits. This may be applied to
pickle processing where excessive softening may occur during fermentation and storage.

Textile processing and bioscouring of cotton fibers

Pectinases have been used in conjunction with amylases, lipases, cellulases and
hemicellulases to remove sizing agents from cotton in a safe and ecofriendly manner,
replacing toxic caustic soda used for the purpose earlier. Bioscouring is a novel process for
removal of noncellulosic impurities from the fiber with specific enzymes. Pectinases have
been used for this purpose without any negative side effect on cellulose degradation.
Degumming of plant bast fibers
Bast fibers are the soft fibers formed in groups outside the xylem, phloem or pericycle,
e.g. Ramie and sunn hemp. The fibers contain gum, which must be removed before its use
for textile making. The chemical degumming treatment is polluting, toxic and non-
biodegradable. Biotechnological degumming using pectinases in combination with xylanases
presents an ecofriendly and economic alternative to the above problem.
Retting of plant fibers
Pectinases have been used in retting of flax to separate the fibers and eliminate pectins.

12
Waste water treatment Vegetable food processing industries release pectin,
containing wastewaters as by-product. Pretreatment of these wastewaters with pectinolytic
enzymes facilitates removal of pectinaceous material and renders it suitable for
decomposition by activated sludge treatment.
Coffee and tea fermentation
Pectinase treatment accelerates tea fermentation and also destroys the foam forming
property of instant tea powders by destroying pectins. They are also used in coffee
fermentation to remove mucilaginous coat from coffee beans.

Paper and pulp industry During papermaking, pectinase can depolymerise pectins
and subsequently lower the cationic demand of pectin solutions and the filtrate from
peroxide bleaching.
Animal feed
Pectinases are used in the enzyme cocktail, used for the production of animal feeds.
This reduces the feed viscosity, which increases absorption of nutrients, liberates nutrients,
either by hydrolysis of non-biodegradable fibers or by liberating nutrients blocked by these
fibers, and reduces the amount of faeces.

Purification of plant viruses In cases where the virus particle is restricted to phloem,
alkaline pectinases and cellulases can be used to liberate the virus from the tissues to give
very pure preparations of the virus.
Oil extraction
Citrus oils such as lemon oil can be extracted with pectinases. They destroy the
emulsifying properties of pectin, which interferes with the collection of oils from citrus peel
extracts.
Improvement of chromaticity and stability of red wines
Pectinolytic enzymes added to macerated fruits before the addition of wine yeast in
the process of producing red wine resulted in improved visual characteristics (colour and
turbidity) as compared to the untreated wines. Enzymatically treated red wines presented
chromatic characteristics, which are considered better than the control wines. These wines
also showed greater stability as compared to the control.

Cellulases

Microbial cellulases find applications in various industries and constitute a major group of the industrial
enzymes. Recently, there is resurgence in utilization of biomass for fuel production employing
cellulases and hence forth in obtaining better yields and novel activities. Improving the economics of
such processes will involve cost reduction in cellulase production which may be achieved by better
bioprocesses and genetic improvement of cellulase producers to yield more of the enzyme. The review
discusses the current knowledge on cellulase production by microorganisms and the genetic controls
exercised on it. It discusses the industrial applications of cellulases and the challenges in cellulase
research especially in the direction of improving the process economics of enzyme production.

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