Biotechnology of Microbial Enzymes: Production, Biocatalysis and Industrial Applications

Brazil

Preface

Goutam Brahmachari

Department of Chemistry, Visva-Bharati (a Central University), Santiniketan, West Bengal, India

Arnold L. Demain

Research Institute for Scientists Emeriti (RISE), Drew University, Madison, NJ, United States

Jose L. Adrio

NEOL Biosolutions, S.A., Granada, Spain

This single volume, titled Biotechnology of Microbial Enzymes: Production, Biocatalysis, and Industrial Applications, is an endeavor focusing on the recent cutting-edge research advances in the field of microbial enzymes, very particularly emphasizing their production, modifications, and industrial applications. This book, comprising a total of 20 chapters written by active researchers and leading experts in microbial enzymes from several countries, in response to our personal invitation, brings together an overview of current discoveries and trends in this remarkable field. We are most grateful to the contributors for their generous and timely response in spite of their busy and tight schedules with academics, research, and other responsibilities.

Enzymes are potential biocatalysts produced by living cells to bring about specific biochemical reactions, linked with the metabolic processes of the cells. Due to the unique biochemical properties of enzymes, the demand for industrial enzymes is continuously rising driven by a growing need for sustainable solutions. Microorganisms have provided, and continue to provide, an impressive number of such biocatalysts with a wide range of applications across several industries such as food, animal feed, household care, technical industries, biofuels, fine chemicals, and pharmaceuticals. As mentioned, the unique properties of enzymes, such as high specificity, fast action, and biodegradability, allow enzyme-assisted processes in industry to run under mild reaction conditions, with improved yields and reduced waste generation. However, despite these advantages, natural enzymes do not often fulfill all process requirements and need further tailoring or redesign in order to fine-tune key catalytic properties.

Recent advances in omics technologies (eg, genomics, metagenomics, proteomics), efficient expression systems, and emerging recombinant DNA techniques, facilitate the discovery of new microbial enzymes from nature, or by creating (or evolving) enzymes with improved catalytic properties. Combinations of engineered and de novo designed enzymes, coupled with chemistry, have been successful in generating more (and even new) chemicals and materials from cheaper and renewable resources, thereby opening a new window to establishing a bio-based economy and achieving low carbon green growth. The ongoing progress and interest in enzymes provide further success in many areas of industrial biocatalysis. In addition, the application of one-pot multistep reactions using multifunctional catalysts, or new and improved enzyme immobilization techniques, is also receiving growing interest in biocatalysis.

Many of the technologies and strategies mentioned above are gathered together in this book. In the introductory chapter (see chapter: Useful Microbial Enzymes—An Introduction), Sanchez and Demain have offered an overview on the importance and significance of useful microbial enzymes in both academia and industry. Production, Purification, and Application of Microbial Enzymes by Pandey and his coauthors deals with the production, purification, and applications of microbial enzymes focusing on their industrial benefits. Solid State Fermentation for Production of Microbial Cellulases by Ray and Behera is devoted to solid state fermentation techniques for production of microbial cellulases from various lignocellulosic substrates. The authors have critically analyzed the pros and cons of this viable technique along with its future openings and prospects. Koga and his group in "Hyperthermophilic Subtilisin-Like Proteases From Thermococcus kodakarensis" have described in detail the unique maturation and stabilization mechanisms of two subtilisin-like proteases, Tk-subtilisin and Tk-SP, isolated from Thermococcus kodakarensis, highlighting the prospect of their applications in novel medical and industrial fields. In Enzymes from Basidiomycetes—Peculiar and Efficient Tools for Biotechnology, Peralta and coauthors have offered an excellent overview on the potential biotechnological applications of basidiomycete-produced enzymes in the degradation of the complex structure of lignocelluloses, thereby opening a useful window towards the exploration of such lignocellulosic biomass for the production of fuel ethanol and other value-added commodity chemicals. Liu and his group have overviewed, in Microbial Production and Molecular Engineering of Industrial Enzymes: Challenges and Strategies, the commonly used strategies for microbial enzyme production and molecular engineering, emphasizing their opportunities and challenges in this remarkable field. Metagenomics and the Search for Industrial Enzymes by Ferrer and coauthors highlights the metagenomic techniques and possible applications in achieving collections of sequences encoding enzymes applicable in industrial sectors. Alcalde and his group have elaborated in detail (see chapter: The Pocket Manual of Directed Evolution: Tips and Tricks) the recent research advances in the field of directed evolution, a robust approach to tailor enzymes with improved attributes. Useful applications, opportunities, challenges, and future scopes of such Tips and Tricks are examined thoroughly by the authors in their discussion. Insights into the Structure and Molecular Mechanisms of β-Lactam Synthesizing Enzymes in Fungi by Martín and Liras deals with the structure and molecular mechanisms of β-lactam synthesizing enzymes in fungi. They have offered insights into the molecular characterization of the enzymes involved in penicillin biosynthesis and the early steps of cephalosporin formation, so as to have an idea of their molecular mechanisms. Recent studies on the cellulosome, a supramolecular assembly of microbial biomass-degrading enzymes, have provided researchers with an insight into how microbial enzymes can exist in the form of a mysterious extracellular organelle and how abundant water-insoluble plant biomass is degraded in nature by such a well-organized protein complex. Chow and Wu have outlined these aspects in The Cellulosome: A Supramolecular Assembly of Microbial Biomass-Degrading Enzymes, highlighting the composition, structure, and assembly of the cellulosome of Clostridium thermocellum, a thermophilic and anaerobic bacterium.

Liu and Kokare have overviewed the classification, microbial resources, microbial production, and industrial applications of a group of industrially-used microbial enzymes, such as carbohydrases, proteases, and lipases, in Microbial Enzymes of Use in Industry. This chapter also focuses on the challenging potential of enzyme technology required to redesign naturally occurring enzymes in order to fine-tune their substrate specificity and thermostability for proper utilization on the industrial scale. Glucokinases (Glks) are responsible for glucose phosphorylation utilizing diverse phosphoryl donors such as ATP, ADP, and/or polyphosphate. Sanchez and coauthors have highlighted the significance of such microbial glucokinases in Significance of Microbial Glucokinases, emphasizing their physicochemical and biochemical characteristics, as well as their potential applications. Microbial enzymes, today, find useful applications as biocatalysts in a wide array of organic transformations with a handful of advantages; Brahmachari has offered a recent update on lipase-catalyzed organic transformations focusing on the biocatalytic efficacy of the enzyme in carrying out various types of organic reactions, including esterification, transesterification, additions, ring-closing, oxidation, reduction, amidation, and many others, in Lipase-Catalyzed Organic Transformations: A Recent Update. Enzymatic Biocatalysis in Chemical Transformations: A Promising and Emerging Field in Green Chemistry Practice by Blamey et al. elaborates the chemo-enzymatic uses of a variety of green biocatalysts in chemical transformations, yielding fine chemicals with potential applications, both in academia and industry. Microbial enzymes carry out many targeted chemical reactions with ease and specificity satisfying many aspects of green chemistry as well, and the authors have offered a useful discussion herein. Desmet and his group have outlined one such important chemical transformation, that is, glycosylation of useful chemical entries, with the help of microbial enzyme-catalysis, in Microbial Enzymes for Glycoside Synthesis: Development of Sucrose Phosphorylase as a Test Case, focusing on recent research outcomes in optimizing the glycosylation potential of sucrose phosphorylase as a test case. In Industrial Applications of Multistep Enzyme Reactions, Honda overviews the current status of commercially exploited multistep enzymatic reactions and ongoing challenges for expanding the industrial applicability of multienzyme systems. Advancing gene- and enzyme-engineering technologies have provided more versatile and less expensive approaches to prepare a variety of enzymes, leading to more flexible design of multistep enzyme cascades; the chapter focuses on such developments with representative examples of such reactions and the current state-of-the-art for developing further advanced multienzyme systems in detail. Biocatalysis for Industrial Production of Active Pharmaceutical Ingredients (APIs), by Barredo and coauthors, presents an excellent overview on biocatalysis for industrial production of active pharmaceutical ingredients, with examples of the application of isolated enzymes (wild-type or mutant) and whole cells, either in soluble or immobilized form, in the synthesis of such chemical entities, at both the industrial and laboratory scales. The authors also discuss the biocatalytic production of enzyme inhibitors with therapeutic effects, and drugs that modify the transport across membranes, along with those that interact with membrane and intracellular receptors as well. Agro-Industrial Residues and Microbial Enzymes: An Overview on the Eco-Friendly Bioconversion into High Value-Added Products by Valdo Madeira Jr et al. provides an updated and succinct overview of agro-industrial residues, applications of enzymes in lignocellulosic materials, and production of chemical compounds with potential applications from such biomass. Fernandes and Carvalho have underlined the fruitful applications of microbial enzymes for the food industry in Microbial Enzymes for the Food Industry. Productive Chain of Biofuels and Industrial Biocatalysis: Two Important Opportunities for Brazilian Sustainable Development by Ferreira-Leitão and her collaborators offers insight into the current situation of the two main biofuels in Brazil, that is, ethanol and biodiesel, and introduces a discussion of opportunities and bottlenecks in the exploitation of lignocellulosic and oleaginous materials, focusing on the important role of enzymatic and microbial processes to support a sustainable industry.

Representation of facts and their discussions in each chapter are extensive, authoritative, and deeply informative; hence, the book serves as a key reference for recent developments in the frontier research on the biotechnological developments of microbial enzymes and their prospective industrial applications. The broad interdisciplinary approach of this book will surely make the work very interesting to scientists deeply engaged in the research and/or use of microbial enzymes. We would like to express our sincere thanks to all the contributors for their excellent reviews in this remarkable area. It is their participation that made our effort to organize such a book possible. Their masterly accounts will surely provide the readers with a strong awareness of current cutting-edge approaches in this remarkable field of research.

We would also like to express our deep sense of appreciation to all the editorial and publishing staff members associated with Elsevier Inc. for their keen interest in publishing this book, as well as their all-around help to ensure that the highest standards have been maintained in publishing this book.

Chapter 1

Useful Microbial Enzymes—An Introduction

Sergio Sanchez¹ and Arnold L. Demain²,    ¹Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México (UNAM), Mexico City, Mexico,    ²Research Institute for Scientists Emeriti (RISE), Drew University, Madison, NJ, United States

Abstract

Enzymes are important due to their many useful properties. Their development, to a great extent, has been possible due to the availability of microbial sources. Microorganisms are of much attention because they can be produced economically and are amenable to genetic improvement. Microbial enzymes have replaced many plant and animal enzymes. They have found application in many industries including foods, beverages, pharmaceuticals, detergents, textiles, leather, chemicals, biofuels, animal feed, personal care, pulp and paper, diagnostics, and therapy. New molecular methods, including genomics and metagenomics, are being employed for the discovery of new enzymes from microbes. The development of recombinant DNA technology has had a major effect on production levels of enzymes and represent a way to overproduce industrially important microbial, plant, and animal enzymes. It has been estimated that between 50–60% of the world enzyme market is supplied by recombinant enzymes. In addition, directed evolution techniques have allowed design of enzyme specificities and better performance.

Keywords

Microbial enzymes; improvement; discovery; industry

1.1 The Enzymes: A Class of Useful Biochemicals

According to the International Union of Biochemistry (IUB), and based on their nature of reaction, enzymes are divided into six classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. The use of enzymes in industrial processes has been of crucial importance since they can eliminate the use of high temperatures, extreme pH values, organic solvents, and at the same time, offer high substrate specificity, low toxicity, product purity, reduced environmental impact, and ease of termination of activity. Microorganisms constitute the major source of enzymes as they produce high concentrations of extracellular enzymes. Screening for the best enzymes is simple, allowing the examination of thousands of cultures in a short period of time. Microorganisms used for enzyme production include around 50 GRAS bacteria and fungi. Bacteria are mainly represented by Bacillus subtilis, Bacillus licheniformis, and various Streptomyces species. Fungi are usually represented by Aspergillus, Mucor, and Rhizopus. Microorganisms can be cultured in large quantities in a relatively short period by established methods of fermentation. Microbial enzyme production is economical on a large scale due to inexpensive culture media and short fermentation cycles.

There are more than 3000 different enzymes known but only 5% are commercially used (Binod et al., 2013). Over 500 commercial products are made using enzymes (Johannes and Zhao, 2006). In regard to the total enzyme market, its global figures depend on the consulted source. In one case, the market reached $5.1 billion in 2009 and is predicted to rise 6.45 per annum to grasp $6.9 billion in 2017 (The Freedonia Group, Inc., 2014). In a second report, it was estimated to be $3.3 billion in 2010 and to reach $4.4 billion by 2015 (BBC Research, 2011; Binod et al., 2013). The major technical enzymes are used in bulk form for manufacture of detergents, textiles, leather, pulp, paper, biofuels, and the market for these enzymes reached $1.2 billion in revenues in 2011 and is still on the rise. Other applications include household care, foods, animal feed, fine chemicals, and pharmaceuticals. Enzymes have unique properties such as rapid action, high specificity, biodegradability, high yields, ability to act under mild conditions, and reduction in generation of waste materials. These properties offer flexibility with respect to operating conditions in the reactor.

Sales of feed enzymes are expected to reach $730 million in 2015. They are used to increase nutrient digestibility, and to degrade unacceptable components of feed. Included, mainly for poultry and swine, are proteases, phytases, glucanases, alpha-galactosidases, alpha-amylases, and polygalacturonases. Recent emphasis has been on development of heat-stable enzymes, economical and rapid assays that are more reliable, improvement of activity, and discovery of new nonstarch polysaccharide-degrading enzymes.

Enzymes for food and beverage manufacture are a major part of the industrial enzyme market, reaching sales of almost $1.2 billion in 2011. Lipases constitute a major portion of the usage, targeting fats and oils. In order to maximize flavor and fragrance, control of lipase concentration, pH, temperature, and emulsion content is necessary. Lipases are potentially useful as emulsifiers for foods, pharmaceuticals, and cosmetics. Aspergillus oryzae is used as a cloning host to produce fungal lipases, such as those from Rhizomucor miehi, Thermomyces lanuginosus, and Fusarium oxysporum.

Important detergent additives include proteases, lipases, oxidases, amylases, peroxidases, and cellulases which catalyze the breakdown of chemical bonds upon the addition of water. The useful ones are active at thermophilic temperatures (c. 60οC) and alkalophilic pH (9–11), and in the presence of components of washing powders.

Over 60% of the worldwide enzyme market is devoted to proteases. These enzymes are involved in the manufacture of foods, pharmaceuticals, leather, detergents, silk, and agrochemicals. Their use in laundry detergents constitute 25% of global enzyme sales. They include (1) the B. licheniformis alkalase Biotex, (2) the first recombinant detergent lipase called Lipolase, made by cloning the lipase from Humicola lanuginose into A. oryzae, (3) the Pseudomonas mendocina lipase (Lumafast), and (4) the Pseudomonas alcaligenes lipase (Lipomax).

Natural enzymes are often unsuitable for use as industrial biocatalysis and need modifications for industrial use. The production strains are usually modified by genetic manipulation to gain improved properties, including high production levels. With the introduction of recombinant DNA technology, it has been possible to clone genes encoding enzymes from microbes and expressing them at levels tens and hundreds times higher than those produced by unmodified microorganisms. Because of this, the enzyme industry rapidly accepted the technology and moved enzyme production from strains not suited for industry into industrial strains (Galante and Formantici, 2003). Genomics, metagenomics, proteomics, and recombinant DNA technology are employed to facilitate the discovery of new enzymes from microbes in nature, and to create or evolve improved enzymes. A number of new and useful enzymes have been obtained by metagenomics (Ferrer et al., 2007).

Directed evolution of proteins includes DNA shuffling, whole genome shuffling, heteroduplex, random chimeragenesis of transient templates, assembly of designed oligonucleotides, mutagenic and unidirectional reassembly, exon shuffling, Y-ligation-based block shuffling, nonhomologous recombination, and the combination of rational design with directed evolution (Yuan et al., 2005; Siehl et al., 2005; Bershstein and Tewfic, 2008; Reetz, 2009). Directed evolution has yielded increased activity, stability, solubility, and specificity of enzymes. For example, it increased the activity of glyphosate-N-acetyltransferase 10,000-fold and, at the same time, its thermostability by 5-fold.

1.2 Microbial Enzymes for Industry

According to their applications, microbial enzymes have been applied to make numerous biotechnology products and in processes commonly encountered in the production of laundry, food and beverages, paper and textile industries, clothing, etc. The use of enzymes as detergent additives represents a major application of industrial enzymes. The detergent market for enzymes has grown strongly in the last 25 years. In the year 2003, it was around $0.79 billion, with proteases as the major detergent enzyme product. The detergent industry uses more than 25% of the total enzyme production.

Proteases, lipases, amylases, oxidases, peroxidases, and cellulases are added to the detergents where they catalyze the breakdown of chemical bonds on the addition of water. For this purpose, they must be active under thermophilic (60oC) and alkalophilic (pH 9–11) conditions, as well as in the presence of various components of washing powders (Stoner et al., 2005). The market share of detergent proteases is estimated to be at 72% of the global detergent enzyme market (Maurer, 2015). The first detergent containing a bacterial protease was introduced in 1956, and in 1960, Novo Industry A/S introduced alcalase produced by B. licheniformis (Biotex). Cellulase from Bacillus sp. KSM-635 has been used in detergents because of its alkaline pH optimum and insensitivity to components in laundry detergents (Ozaki et al., 1990). Later, Novozymes launched a detergent using a cellulase complex isolated from Humicolla insolence (Celluzyme). Certain microorganisms called extremophiles grow under extreme conditions such as 100oC, 4oC, 250 atm., pH 10, or 5% NaCl. Their enzymes, which act under such extreme conditions, are known as extremozymes. One such enzyme, called Cellulase 103, was isolated from an alkaliphile and commercialized because of its ability to break down microscopic fuzz of cellulose fibers which trapped dirt on the surface of cotton textiles. It has been used for over 10 years in detergents to return the newness of cotton clothes, even after many washings. As early as the mid-1990s, virtually all laundry detergents contained genetically-engineered enzymes (Stoner et al., 2005). Over 31% of the enzymes used in detergents are recombinant products (McAuliffe et al., 2007).

The major application of proteases in the dairy industry is for the manufacturing of cheese. Four recombinant proteases have been approved by FDA for cheese production. Calf rennin had been preferred in cheese-making due to its high specificity, but microbial proteases produced by GRAS microorganisms, such as Mucor miehei, Mucor pusilis, B. subtilis, and Endothia parasitica, are gradually replacing it. The primary function of these enzymes in cheese-making is to hydrolyze the specific peptide bond (Phe105-Met106) that generates para-k-casein and macropeptides. Nearly 40,000 U/g bran of milk-clotting activity are produced by A. oryzae at 120 h by solid state fermentation (Vishwanatha et al., 2009). For many years, proteases have also been used for production of low allergenic milk proteins used as ingredients in baby milk formulas (Gupta et al., 2002).

Proteases can also be used for synthesis of peptides in organic solvents. Thermolysin is used in this way to make aspartame (Oyama et al., 1981). Aspartame sales reached $1.5 billion in 2003 (Baez-Viveros et al., 2004). In 2004, the production of aspartame amounted to 14,000 metric tons. The global sugar substitute market is the fastest growing sector of the sweetener market.

Fungal alpha-amylase, glucoamylase, and bacterial glucose isomerase are used to produce high fructose corn syrup from starch in a business amounting to $1 billion per year. Fructose syrups are also made from glucose by glucose isomerase (actually xylose isomerase) at a level of 15 million tons per year. The food industry also uses invertase from Kluyveromyces fragilis, Saccharomyces cerevisiae, and Saccharomyces carlsbergensis for manufacture of candy and jam. Beta-galactosidase (lactase), produced by Kluyveromyces lactis, K. fragilis, or Candida pseudotropicalis, is used to hydrolyze lactose in milk or whey and alpha-galactosidase from S. carlsbergensis is employed in the crystallization of beet sugar.

Microbial lipases catalyze the hydrolysis of triaclyglycerol to glycerol and fatty acids. They are commonly used in the production of a variety of products ranging from fruit juices, baked foods, pharmaceuticals, and vegetable fermentations to dairy enrichment. Fats, oils, and related compounds are the main targets of lipases in food technology. Accurate control of lipase concentration, pH, temperature, and emulsion content is required to maximize the production of flavor and fragrance. The lipase mediation of carbohydrate esters of fatty acids offers a potential market for use as emulsifiers in foods, pharmaceuticals, and cosmetics. Another application of increasing importance is the use of lipases in removing pitch (hydrophobic components of wood, mainly triglycerides and waxes). A lipase from Candida rugosa is used by Nippon Paper Industries to remove up to 90% of these compounds (Jaeger and Reetz, 1998). The use of enzymes as alternatives to chemicals in leather processing has proved successful in improving leather quality and in reducing environmental pollution. Alkaline lipases from Bacillus strains, which grow under highly alkaline conditions in combination with other alkaline or neutral proteases, are currently being used in this industry. Lipases are also used in detergent formulations for removal of lipid stains, fatty food stains, and sebum from fabrics (Hasan et al., 2010). Alkaline yeast lipases are preferred because they can work at lower temperatures, as compared to bacterial and fungal lipases. Cold-active lipase detergent formulation is used for cold washing which reduces energy consumption and wear and tear of textile fibers. It is estimated that every year, about 1000 tons of lipases are added to approximately $13 billion tons of detergents (http://www.aukbc.org/beta/bioproj2/introduction.html).

In other enzyme applications, laccases oxidize phenolic and nonphenolic lignin-related compounds as well as environmental pollutants (Rodríguez-Couto and Toca-Herrera, 2006). They are used to detoxify industrial effluents of the paper and pulp, textile, and petrochemical industries, as a medical diagnostic tool, for bioremediation of herbicides, pesticides, and explosives in soil, cleaning agent for water purification systems, catalyst in drug manufacture, and as ingredients in cosmetics.

Enzymes are also used in a wide range of agro-biotechnological processes, and the major application is the production of feed supplements to improve feed efficiency. A recent advance in feed enzymes involves the application of phytases in agriculture as an animal feed ingredient and also in foods to improve plant phosphorous uptake by monogastric animals (Vohra and Satyanarayana, 2003). Phytate phosphorus is often unavailable to farm animals and chelates valuable minerals. Phytase allows liberation of phosphorus from plant feedstuffs, which contain about 2/3 of their phosphorus as phytate. Hydrolysis of phytate prevents its passage via manure into the soil where it would be hydrolyzed by soil and water microbes causing eutrophication. Therefore, the use of phytase in the food industry involves removal of phytic acid which acts as an antinutritional factor. The annual market of phytase is about $500 million. The enzyme is made by many bacteria, yeasts, and filamentous fungi. Production is controlled by phosphate. Cloning of the phytase-encoding gene phyA from Aspergillus niger var. awamori and reintroduction at a higher dosage increased phytase production by sevenfold (Piddington et al., 1993). Recombinant Hansenula polymorpha produced 13 g/L of phytase (Mayer et al., 1999). New fungal phytases with higher specific activities or improved thermostability have been recently identified (Haefner et al., 2005).

In the paper and textile industries, enzymes are being increasingly used to develop cleaner processes and reduce the use of raw materials and production of waste. An alternative enzymatic process in the manufacture of cotton was developed based on a pectate lyase. The process for removing pectin and other hydrophobic materials from cotton fabrics is performed at much lower temperatures and uses less water than the classical method. Applying Gene Site Saturation Mutagenesis™ technology on DNA encoding for pectinolytic enzymes (selected from more than 100 environmental DNA libraries), single site mutants, exhibiting improved thermotolerance, were produced. In addition, variants with improved thermotolerance were produced by Gene Reassembly™ technology (Solvak et al., 2005). The best performing variant (CO14) contained eight mutations and had a melting temperature 16οC higher than the wild-type enzyme while retaining the same specific activity at 50οC. Optimal temperature of the evolved enzyme was 70οC, which is 20οC higher than the wild-type. Scouring results obtained with the evolved enzyme were significantly better than the results obtained with chemical scouring, making it possible to replace the conventional and environmentally harmful chemical scouring process (Solvak et al., 2005). Furthermore, alkaline pectinases are used for treatment of pectic wastewaters, degumming of plant bast fibers, paper making, and coffee and tea fermentations.

In the chemical industry, enzymes are used to replace chemical processes if they successfully compete on a cost basis. They sometimes require less energy, yield a higher titer with enhanced catalytic efficiency, produce less catalyst waste and by-products, and lower volumes of wastewater streams. They often involve hydrolases and ketoreductases that are stable in organic solvents. They also can be used to produce valuable compounds such as L-amino acids. For example, L-tyrosine has been made from phenol, pyruvate, pyridoxal phosphate, and ammonium chloride with a thermostable and chemostable tyrosine phenol lyase from Symbiobacterium toebii. The amino acid was produced at 130 g/L in 30 h with continuous substrate feeding. About 150 biocatalytic processes are used in the chemical industry and this number will increase with the application of genomics and protein engineering.

Enzymes are also important in the pharmaceutical industry. They are used in the preparation of beta-lactam antibiotics such as semisynthetic penicillins and cepalosporins. This antibiotic group is extremely important, making up 60–65% of the total antibiotic market. Enzymes are also involved in the preparation of chiral medicines, that is, complex chiral pharmaceutical intermediates. For example, esterases, proteases, lipases, and ketoreductases are used to prepare chiral alcohols, carboxylic acids, amines, and epoxides.

1.3 Improvement of Enzymes

Certain enzymes face problems such as poor stability, substrate/product inhibition, narrow substrate specificity, or enantioselectivity. To solve these problems, genetic modification is often carried out using recombinant DNA techniques. In some cases, this has improved activity by 100-fold. Modification of the enzyme is carried out by (1) rational redesign of the biocatalyst and/or by (2) combinatorial methods in which the desired functionality is searched for in randomly generated libraries. The rational design approach is carried out by site-directed mutagenesis to target amino acid substitutions. It requires knowledge about the three-dimensional structure of the enzyme and the chemical mechanism of the reaction. It often fails but successes have been achieved (Beppu, 1990; Van den Burg et al., 1998). Combinatorial methods include directed evolution which does not require extensive knowledge about the enzyme. Here, a large number of variants are created for screening for catalytic efficiency, enantioselectivity, solubility, catalytic rate, specificity, and enzyme stability. It is rapid and inexpensive. It includes a range of molecular biological methods which allow the achievement of genetic diversity, mimicking mechanisms of evolution in nature. Random mutagenesis of the protein-encoding gene is carried out by various techniques such as (1) error-prone polymerase chain reaction (PCR), (2) repeated oligonucleotide directed mutagenesis, or (3) action of chemical agents. Error-prone PCR introduces random point mutations in a population of enzymes. Molecular breeding techniques, such as DNA shuffling, allow in vitro random homologous recombination, usually between parental genes with homology above 70% (Ness et al., 2000). After cloning and expression, a large collection of enzyme variants, that is, about 10⁴ to 10⁶, is generated and subjected to screening or selection.

1.4 Discovery of New Enzymes

Screening of natural microbes for enzymes suffers from the fact that less than 1% of the microbes inhabiting the biosphere can be cultivated in the laboratory by standard techniques. New enzymes can be obtained from nature by three techniques, that is, metagenomic screening, genome mining, and taking advantage of the diversity of extremophiles.

Metagenomic screening involves preparation of a genomic library from environmental DNA and the systematic screening of the library for open reading frames potentially encoding novel enzymes (Uchiyama and Miyazaki, 2009; Gilbert and Dupont, 2011). Metagenomic screening of particular habitats (arctic tundra, cow rumen, volcanic vents, marine environments, and termite guts) has yielded enzymes such as lipase, oxidoreductase, amidase, amylase, nitrilase, decarboxylase, epoxide hydrolase, and beta-glucosidase. Although Escherichia coli has been the usual host for screening of foreign genes, the system has been improved by the use of alternative hosts and expression systems such as Streptomyces lividans, Pseudomonas putida, and Rhizobium leguminosarum.

Genome mining involves exploring genome sequence databases for genes encoding new enzymes. An example of a useful database is the NCBI database (NCBI Microbial Genomes, 2013) which includes more than 2000 genome sequences and draft assemblies. Two methods are used for the discovery of new enzymes. One of these, that is, genome hunting, involves the search for open reading frames in the genome of a particular microbe. Those sequences that are annotated as putative enzymes are subjected to subsequent cloning, overexpression, and activity screening. A second approach, called data mining, is based on homology alignment among all sequences deposited in databases. Using bioinformatic tools such as BLAST, the search for conserved regions between sequences yields homologous protein sequences that are then considered candidates for further study.

Extremophiles can survive under extreme conditions. These include temperature (−2οC to 12οC, 60οC to 110οC) pressure, radiation, salinity (2–5 M NaCl), and pH (<2, >9). They contain extremely stable enzymes. Genera such as Clostridium, Thermotoga, Thermus, and Bacillus contain extreme thermophiles growing at 60–80οC, whereas hyperthermophiles are members of Archea, for example, Pyrococcus, Methanopyrus, and Thermococcus. An example of an extremely useful enzyme is the Taq DNA polymerase from the thermophile Thermus aquaticus, which had sales of $500 million in 2009 (De Carvalho, 2011). Industry already uses thermophilic cellulases, amylases, and proteases.

Psychrophiles are already supplying cold-active enzymes, such as proteases, amylases, and lipases, for future development of detergents to reduce wear and tear of textile fibers. Cold-active cellulases and xylanases are of interest in the pulp and paper industry and for production of second generation biofuels via saccharification of pre-treated lignocellulosic biomass. They are also potentially useful for extraction and clarification of fruit juices, improvement of bakery products, bioremediation of waters contaminated with hydrocarbons or oils, and polishing and stone-washing of textiles. Halophilic xylanases, proteases, amylases, and lipases have been isolated from halophiles, such as species of Halobacillus, Halobacterium, and Halothermothrix (Van den Burg, 2003).

Also of interest are microbes surviving under extreme pH conditions that could be useful for isolation of thermoalkiphilic proteases and lipases as additives in laundry and dishwashing detergents (Shukla et al., 2009).

1.5 Concluding Remarks

Microbial enzymes have long been used by industrial product makers as major catalysts to transform raw materials into end products. Over 500 commercial products are made using enzymes. They are economically produced by different microorganisms and are quickly broken down when they have done their job. New technical tools to use enzymes as crystalline catalysts, for ability to recycle cofactors, and engineering enzymes to function in various solvents with multiple activities are important technological developments, which will steadily create new applications.

The industrial enzyme market will grow steadily mainly due to improved production efficiency resulting in cheaper enzymes, new application fields, new enzymes from screening programs, and by engineering properties of traditional enzymes. Tailoring enzymes for specific applications will be a future trend with continuously improving tools, further understanding of structure-function relationships, and increased searching for enzymes from exotic environments. New applications are to be expected in the field of textiles and new animal diets, such as ruminant and fish feed. It can be expected that breakthroughs in pulp and paper applications will materialize. The use of cellulases to convert waste cellulose into sugars and further to ethanol or butanol by fermentative organisms has been a major study topic for years. Increasing environmental pressures and energy prices will make this application a real possibility in the future.

Enzymes should never be considered alone but rather as a part of a biocatalyst technology. Recent developments in the fields of genetic engineering and protein chemistry are bringing ever more powerful means of analysis to bear on the study of enzyme structure and function, that will undoubtedly lead to the rational modification of enzymes to match specific requirements, and also the design of new enzymes with novel properties. Techniques such as protein engineering, gene shuffling, and directed evolution will enable the development of enzymes better suited to industrial environments. These tools will also allow the synthesis of new biocatalysts for completely novel applications, resulting in the production and commercialization of new enzymes, thus seeding a second explosive expansion to the current multibillion dollar enzyme industry.

Acknowledgements

We are indebted to Beatriz Ruiz and Marco Antonio Ortíz for helping in manuscript preparation.

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Chapter 2

Production, Purification, and Application of Microbial Enzymes

Anil Kumar Patel¹, Reeta Rani Singhania¹ and Ashok Pandey²,    ¹DBT-IOC Centre for Advanced Bio-Energy Research, Indian Oil Corporation, R&D Centre, Faridabad, Haryana, India,    ²Biotechnology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum, Kerala, India

Abstract

The present human era aspires to change from chemical domination to biological domination. Enzymes are the biocatalysts required by all living organisms for syntheses as well as breakdown reactions. Enzymes are dynamic molecules and many harmful chemical reactions have been replaced by eco-friendly biological processes due to these biocatalysts. Enzyme technology is evolving rapidly. Enzymes, being highly specific in nature, have the capacity to revolutionize the whole industrial sector. Industrial enzyme production technology has attained a huge success in recent years due to major advancements in bioprocess technology. Enzymes with desired properties and improved functionality could be developed with the advent of genetic engineering as well as protein engineering. This chapter deals with industrial enzyme production, purification, formulation, commercial application, and provides a short account of the market position of enzymes globally. Novel enzymes as well as novel applications are continuing to emerge providing new opportunities for enzyme technology to flourish.

Keywords

Enzyme; enzyme technology; solid state fermentation; submerged fermentation; downstream process

2.1 Introduction

Enzymes are the active proteins (except RNAse) that can catalyze biochemical reactions. These are biomolecules required for both syntheses as well as breakdown reactions by living organisms. All living organisms are built and maintained by these enzymes, which are truly termed as biological catalysts having the capability to convert a specific compound (as substrate) into products at higher reaction rates. Like chemical catalysts, enzymes increase the reaction rate by lowering its activation energy (Ea), hence, products are formed faster and reactions reach their equilibrium state more rapidly. The rates of most enzymatic reactions are millions of times faster than those of the uncatalyzed reactions. They can perform conversions in minutes or even in seconds which otherwise may take hundreds of years (Dalby, 2003; Otten and Quax, 2005). Enzymes are known to catalyze about 4000 biochemical reactions in living beings (Bairoch, 2000). For example, lactase is a glycoside hydrolase that is able to hydrolyze lactose (milk sugar) into its constituent galactose and glucose monomers. It is produced by various microorganisms and also in the small intestine of humans and other mammals helping to digest milk completely. Enzymes are also enantioselective catalysts, which can be used either in the separation of enantiomers from a racemic mixture or in the synthesis of chiral compounds.

Humans recognized the importance of enzymes thousands of years ago; clarification and filtration of wines and beer being the earliest examples of the application of industrial enzymes. Enzymes have been used in brewing, baking, and alcohol production since prehistoric times; however, they did not call them enzymes. One of the earliest written references to enzymes is found in Homer’s Greek epic poems dating from about 800 BC, where it was mentioned that enzymes were used in the production of cheese. The Japanese have also used naturally-occurring enzymes in the production of fermented products like sake, Japanese schnapps brewed from rice, for more than a thousand years. Some enzymes have been designed by nature to form complex molecules from simpler ones while others have been designed for breaking up complex molecules into simpler ones, also a few modify molecules. These reactions involve the making and breaking of the chemical bonds in the components. Owing to their specificity, a property of an enzyme that allows it to recognize a particular substrate that they are designed to target, they are useful for industrial processes and are capable of catalyzing the reaction between particular chemicals even if they are present in mixtures with many chemicals. These enzymes are environmentally safe, natural, and are applied very safely in food and even pharmaceutical industries. Still, enzymes are proteins, which like any protein can cause and have caused in the past allergic reactions, hence, protective measures are necessary in their production and applications.

Enzyme technology is an ever evolving branch of Science and Technology. With the intervention and influence of Biotechnology and Bioinformatics, continuously novel or improved applications of enzymes are emerging. With novel applications, the need for enzymes with improved properties are also emerging simultaneously. Development of commercial enzymes is a specialized business which is usually undertaken by companies possessing high skills in:

 Screening for new and improved enzymes

 Selection of microorganisms and strain improvement for qualitative and quantitative improvement

 Fermentation for enzyme production

 Large-scale enzyme purifications

 Formulation of enzymes for sale

Enzyme technology offers industries and consumers an opportunity to replace processes using aggressive chemicals with mild and environmentally friendly enzyme processes. About 3000 enzymes are known of which only 150–170 are being exploited industrially. At present only 5% of chemical products are produced through a biological route in this green era. However, economically feasible and eco-friendly enzymatic processes are emerging as alternatives to physico-chemical and mechanical processes. Based on the different application sectors, industrial enzymes can be classified as: (1) Enzymes in the food industry, (2) Enzymes for processing aids, (3) Enzymes as industrial biocatalyst, (4) Enzymes in genetic engineering, and (5) Enzymes in cosmetics.

Today, enzymes are envisaged as the bread and butter of biotechnology because they are the main tools for several biotechnological techniques (gene restriction, ligation, and cloning, etc.), bioprocesses (fermentation and cell culture), and in analytics in human and animal therapy as medicines or as drug targets. Furthermore they find applications in several other industries, such as food and feed, textiles, effluent and waste treatment, paper, tannery, baking, brewing, dairy, pharmaceuticals, confectionary, etc. (Pandey et al., 2006).

The enzymes utilized today are also found in animals (pepsin, trypsin, pancratin, and chimosin) and plants (papain, bromelain, and ficin), but most of them are of microbial origin, such as glucoamylase, α-amylase, pectinases, etc. The advantage of using microbes for enzyme production is their higher growing abilities, higher productivity, and their easier genetic manipulation for enhanced enzyme production, etc. Enzymes produced from microbial origins are termed as microbial enzymes. Microbes are mainly exploited in industries for enzyme production. Moreover, microbial enzymes are supplied, well standardized, and marketed by several competing companies worldwide. Depending on the type of process, enzymes can be used in soluble form (animal proteases and lipases in tannery) and in immobilized form (isomerization of glucose to fructose by glucose isomerase).

2.2 Production of Microbial Enzymes

Bacteria and fungi produce most industrial enzymes. Naturally occurring microorganisms are the most productive producers of enzymes. This knowledge has been exploited by industry for more than 50 years. Bacteria and fungi are the microorganisms best suited to the industrial production of enzymes. They are easy to handle, can be grown in huge tanks without light, and have a very high growth rate.

Bacterium Bacillus subtilis and the fungus Aspergillus oryzae are the most employed microorganisms for enzyme production by the global biotechnology company Novozymes. Both have immense capacity for producing enzymes and are considered completely harmless for humans (http://www.novozymes.com/en/about-us/our-business/what-are-enzymes/Pages/creating-the-perfect-enzyme.aspx).

The ideal microorganism grows quickly and produces lots of the desired enzyme at mild temperatures whilst consuming inexpensive nutrients. However, like most things in life, the ideal microorganism is hard to come by. Most microorganisms found in the wild are not well suited to domestication in large fermentation tanks. Some only produce tiny quantities of enzyme or take a long time to grow. Others can produce undesired by-products that would disturb industrial processes. So for industrial production a perfect microorganism is the foremost requirement. Table 2.1 shows the list of microorganisms involved for the production of enzymes along with their industrial applications.

Table 2.1

Industrial Application of Enzymes

SSF, solid-state fermentation; SmF, submerged fermentation.

2.2.1 Enzyme Production in Industries

Different microorganisms have been employed for industrial enzyme production, varying from eukaryotic systems, such as yeast and fungi, to prokaryotic system involving both Gram positive and Gram negatives bacteria.

The first enzyme industry emerged for producing subtilisin, an alkaline protease, naturally secreted by Bacillus licheniformis to break down proteainaceous substrate, and used in detergent. An industry for alpha amylase production was also based on Bacillus licheniformis that naturally secretes a highly thermostable α-amylase capable of breaking down starch to easily digestible oligosaccharides. Hence, strains of Bacillus were regarded as workhorses of enzyme production for decades because of their ability to overproduce substilisin and α-amylase. Amylase from Bacillus has long been used to liquefy starch. For the complete breakdown of starch into its monosaccharide other enzymes called glucoamylases are required. The most widely used glucoamylases for complete hydrolysis of starch into glucose are produced by a fungal strain of the genus Aspergillus (Sonenshein et al., 1993). Overproducing strains have been isolated over the years leading to high producing glucoamylases. Likewise, an acidic cellulase complex is secreted by a fungal strain of Trichoderma. Initially it was thought that this enzyme complex would convert cellulosic substrate to glucose in a similar way to the starch degrading enzymes but this application was not initially commercialized due to its slow action. Instead it has found an application in the treatment of textiles and as an additive in detergents. The potential of cellulases for biomass hydrolysis for bioethanol production was ignored for some years, but recently its potential for cellulose hydrolysis has regained attention from researchers all over the world, mainly for the application of bioethanol production using cellulosic biomass which is the most abundant material available for use to mankind. Several efforts were made to improve the enzyme complex and its expression. At present there are several enzyme companies preparing a cellulase cocktail for bioethanol application, for example, Genencor and Novozymes.

Glucose isomerase catalyzes the conversion of glucose into fructose; resulting in a product sweeter in taste. This particular enzyme was produced by a species of Streptomyces which led to the development of an industry based on the above application.

All of the strains described above that have been employed for industrial application are capable of differentiation. For example, Bacillus shows the tendency to survive adverse environmental conditions by forming spores which are dormant yet viable. The spore remains dormant until it reaches a favorable environment where it can germinate and multiply.

This differentiation is highly associated with the regulation of enzyme production by microorganisms. This type of highly complex behavior makes modeling studies difficult for process development. Protease production by Bacillus is highly regulated with differentiation and it is similarly the case for Aspergillus and Trichoderma. Streptomyces does not truly sporulate, although it differentiates by forming filaments unlike isolated single cells. This property exerts an effect on the production as well as the physical properties of fermentation broth. An organism can be considered as a metabolic system capable of utilizing substrates to produce cell mass and by-products. Enzymes catalyze the various reactions that are vital for an organism’s growth and metabolic activities. Each cell is equipped with a mechanism that regulates the synthesis of enzymes in an economical way, enabling the cell to respond adequately to environmental changes.

The basic mechanism of enzyme synthesis includes transcription, translation, and posttranslational processing which is highly conserved (Rhem and Reed, 1985). However, several differences exist between various classes of organisms, as well as some fundamental differences between prokaryotic and eukaryotic organisms. The enzymes themselves differ in their molecular structure, number of polypeptide chain, degree of glycosylation, and isoelectric point. Although all the differences influence the synthetic pattern, the basic enzyme synthesis mechanisms are similar enough to allow a general treatment of the microbiological production process. However, differences exist between the production kinetics of different enzymes by different microorganisms because of their varied physical characteristics and growth pattern, which necessitates the optimization of each production process separately.

2.2.2 Industrial Enzyme Production Technology

Fermentation technologies have been employed exclusively for the production of industrial enzymes, preferably by microorganisms such as bacteria or fungi under carefully controlled conditions due to their ease of multiplication and handling. Microorganisms employed are GRAS (Generally Recognized as Safe) strains due to their application in the food and feed industries (Pandey et al., 2008; Singhania et al., 2010). Currently, researchers are locating extremophile organisms from different parts of the world, ranging from the rain forest to arid regions to the bottom of the ocean, that may produce enzymes with a promising industrial nature. In practice, the great majority of microbial enzymes come from a very limited number of genera, of which Aspergillus species, Trichoderma species, Bacillus species, Streptomyces species, and Kluyveromyces species predominate. Most of the strains used have either been employed by the food industry for many years or have been derived from such strains by mutation and selection (Sarrouh et al., 2012).

Selection of the strain for industrial enzyme production is a very important factor for a successful industrial process. Ideally a strain producing an extracellular enzyme should be selected as it makes the purification and recovery far easier than when the enzyme is produced intracellularly. Different organisms may also differ in their suitability for fermentation; process characteristics, such as viscosity or recoverability, or legal clearance of the organism should also be considered before selection. The industrial strains typically produce up to 50 g/L of extracellular