Production of Recombinant Enzymes

Production of Recombinant Enzymes Arnold L Demain, Research Institute for Scientists Emeriti (R.I.S.E.), Drew University, Madison, NJ, USA Preeti Vaishnav, Rustomjee Elanza CHS, Mind Space, Mumbai, India Ó 2016 Elsevier Inc. All rights reserved.

Industrial Importance of Enzymes Impact of Biotechnology and Genetic Techniques Production of Recombinant Enzymes Systems Used Escherichia coli and Other Nonfilamentous Bacteria Filamentous Bacteria Yeasts Filamentous Fungi Insect Cells Transgenic Plants Applications of Recombinant Enzymes Detergent Industry Food and Beverage Industries Feed Industry Textile Industry Pulp and Paper Industry Medicine Other Uses References

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Industrial Importance of Enzymes Enzymes have been predominantly used since ancient times in a wide range of industries such as food, textile, leather, indigo, medicine, and dairy. Initially, enzymes were obtained from natural sources such as papaya or even extracted from the rumen of calves. In the 1970s, most of the enzymes used were traditionally derived from plant and animal sources, which resulted in a low level of availability, high prices, and stunted growth of the enzyme industry. The industries flourished in the 1980s and 1990s when microbial enzymes came onto the scene. As times advanced, fermentation processes were used extensively to produce enzymes from selected strains to obtain purified and well-characterized enzymes on a large scale for industrial purposes. Microbial enzymes proved economically favorable since cultivation of microbes was much simpler and faster than that of plants and animals and the producing organisms could be more easily manipulated genetically to produce the desired qualities and quantities of enzymes. Production of over 500 industrial products involves enzymes (Kumar and Singh, 2013). The global market for industrial enzymes reached 2 billion dollars in the year 2000, was estimated at 3.3 billion dollars in 2010, and is probably more than 4 billion dollars today. Enzymes play key roles in numerous processes that are commonly encountered in the production of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, and pharmaceuticals. 65% of the enzymes are used to prepare detergents, starch, leather, textiles, personal care products, pulp, and paper. 25% are employed in processing of food and 10% are used in animal feed supplements. The majority of currently used industrial enzymes are hydrolytic in action, being employed mainly for degradation of various natural substances. The most important hydrolases are the proteases which account for 57% of the market, mainly for the detergent and dairy industries. The second largest group is the carbohydrases, primarily amylases and cellulases, used in industries such as starch, textile, detergents, and baking. Other useful enzymes include lipase, glucose isomerase, xylose isomerase, lactase, pullulanase, and xylanase. Also important are invertases from Kluyveromyces fragilis, Saccharomyces carlsbergensis, and Saccharomyces cerevisiae for candy and jam manufacture; b-galactosidase (lactase) from Kluyveromyces lactis, K. fragilis, or Candida pseudotropicalis for hydrolysis of lactose from milk or whey; and galactosidase from S. carlsbergensis for crystallization of beet sugar. ‘Fructose syrup,’ produced by xylose isomerase on glucose, reached a production level of 15 million tons per year over 10 years ago (Vandamme et al., 2005). Some of the major industrial uses of enzymes in manufacturing include Escherichia coli amidase to produce 6-aminopenicillanic acid (6-APA) at 40 000 tons/year, Streptomyces xylose (glucose) isomerase to isomerize D-glucose to D-fructose at 100 000 tons/year, and Pseudomonas chloraphis nitrile hydratase to produce acrylamide from acrylonitrile at 30 000 tons/year (Jaeger et al., 2002). Amylases are produced at an annual rate of 95 000 tons/year. Over half of the industrial enzymes are made by yeasts and molds, with bacteria producing about 30%.

Reference Module in Food Sciences

http://dx.doi.org/10.1016/B978-0-08-100596-5.03023-7

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Production of Recombinant Enzymes

Enzymes play an integral role in the food and feed industries. Biotechnology has been used to manufacture food products for more than 8000 years, offering ways to improve the processing of raw materials into final products. Bread, alcoholic beverages, vinegar, cheese and yogurt, and many other foods have been made using enzymes found in various microorganisms. Today, biotechnology is still affecting the food industry by providing new products, lowering costs, and improving processes on which food producers have long relied. Without a doubt, this will continue into the future. Using biotechnology, improvements in functionality, nutritional value, and sensoric properties (e.g., flavor and texture) have been achieved, as well as improvements in the processing itself, using new tools, such as enzymes, emulsifiers, and improved starter cultures. Biotechnology also offers improved ways to deal with waste problems, food safety problems, packaging issues, etc. Enzymes used predominantly in the food industry are (1) glucose oxidase for dough strengthening, (2) laccase for clarification of juices and as a flavor enhancer (beer), (3) lipoxygenase for dough strengthening and bread whitening, (4) cyclodextrin transferases for cyclodextrin production, (5) glycosyltransferase and fructosyltransferase for synthesis of fructose oligomers, (6) transglutaminase for modification of viscoelastic properties, dough processing, and meat processing, and (7) amylases for starch liquefaction and saccharification, increasing shelf life and improving quality by retaining a moist, elastic and soft nature, bread softness and volume, and for flour adjustment, ensuring uniform yeast fermentation, juice treatment, and to produce low-calorie beer. (8) Galactosidase is important for viscosity reduction in lupins and grain legumes used in animal feed and enhanced digestibility. Also important is (9) glucanase for viscosity reduction in barley and oats used in animal feeds, and enhanced digestibility. (10) Glucoamylase is employed for saccharification, (11) invertase for sucrose hydrolysis and production of invert sugar syrup, and (12) lactase for lactose hydrolysis and whey hydrolysis. (13) Lipase is used for cheese flavoring, in situ emulsification for dough conditioning, support for lipid digestion in young animals, and synthesis of aromatic molecules. (14) Proteases (namely, chymosin, and papain) are used for protein hydrolysis, milk clotting, low-allergenic infant-food formulation, enhanced digestibility and utilization, flavor improvement in milk and cheese, as a meat tenderizer, and for prevention of chill haze formation in brewing. (15) Pectinase is employed for mash treatment and juice clarification. (16) Hydrolysis of proteins (namely, soy, gluten) by peptidase is used to create savory flavors and for cheese ripening. (17) Phospholipase is employed for in situ emulsification for dough conditioning. (18) Phytases are used for release of phosphate from phytate and enhanced digestibility. Other applications include (19) pullulanase for saccharification, (20) xylanases for viscosity reduction, enhanced digestibility, and dough conditioning, (21) acetolactate decarboxylase for beer maturation, and (22) xylose (glucose) isomerase for conversion of glucose to fructose. Certain recombinant enzymes were approved by the FDA in 2003 for use in medicine. They included Fabrazyme (agalsidase beta), an enzyme replacement for Fabry disease caused by deficiency of alpha-glucosidase, and Aldurazyme (laronidase) for Hurler and Hurler-Scheie forms of mucopolysaccharidosis I in which patients lack L-iduronidase.

Impact of Biotechnology and Genetic Techniques Biotechnology has played a major role in furthering the application of enzymes. By 1994, over 50% of the industrial enzyme market involved recombinant processes (Cherry and Fidasstef, 2003) and by 2003, approximately, 90% of all industrial enzymes were recombinant forms. Recombinant DNA techniques have opened the door to the application of enzymes from so-called extremophiles, such as thermophiles, psychrophiles, barophiles, halophiles, alkaliphiles, and acidophiles. Furthermore, developments in biotechnology, such as protein engineering and directed evolution, had a major effect on the enzyme industry. These advances in biotechnology provide different kinds of enzymes displaying new activities and adaptability to new conditions, leading to increased use for industrial purposes. Recombinant DNA technology acted as a boon for the enzyme industry in the following ways (Falch, 1991): (1) plant and animal enzymes such as chymosin could be made by microbial fermentation; (2) enzymes from organisms difficult to grow or handle genetically were able to be produced by industrial organisms such as species of Aspergillus and Trichoderma, and K. lactis, S. cerevisiae, Yarrowia lipolytica, and Bacillus licheniformis; (3) enzyme productivity was increased by the use of multiple gene copies, strong promoters, and efficient signal sequences; (4) production of a useful enzyme from a pathogenic or toxin-producing species could now be done in a safe host; and (5) protein engineering was employed to improve the stability, activity, and/or specificity of an enzyme. By the 1990s, many enzymes were produced by recombinant techniques. In 1993, over 50% of the industrial enzyme market was provided by recombinant processes (Hodgson, 1994) and sales were $140 million (Stroh, 1994). Plant phytase, produced by recombinant Aspergillus niger, has been used as a feed for 50% of all pigs in Holland. A 1000-fold increase in phytase production was achieved in A. niger by the use of recombinant technology (Van Hartingsveldt et al., 1993). Industrial lipases were cloned in Humicola and industrially produced by Aspergillus oryzae. They are used for laundry cleaning, interesterification of lipids, and esterification of glucosides, producing glycolipids which have applications as biodegradable nonionic surfactants for detergents, skin care products, contact lenses, and as food emulsifiers. Mammalian chymosin was cloned and produced by A. niger or E. coli and recombinant chymosin was approved in the United States; its price was half that of natural calf chymosin. Over 60% of the enzymes used in the detergent, food, and starch processing industries were recombinant products as far back as the mid-1990s (Cowan, 1996). Enzymes of superior quality have been obtained by protein engineering, specifically by site-directed mutagenesis. Single changes in amino acid sequences have yielded changes in pH optimum, thermostability, feedback inhibition, carbon source inhibition, substrate specificity, Vmax, Km, and Ki. An important method for improving enzymes is directed evolution (also known as applied molecular evolution or directed molecular evolution) (Kuchner and Arnold, 1997; Arnold, 1998; Johannes and Zhao, 2006; Kumar and Singh, 2013). Improved properties include activity, stability, solubility, and specificity (Johannes and Zhao, 2006). It increased the activity of

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glyphosate-N-acetyltransferase by 10 000-fold as well as its thermostability by 5-fold (Siehl et al., 2005). Unlike site-directed mutagenesis, this method of pooling and recombining parts of similar genes from different species or strains yielded remarkable improvements in enzymes in a very short amount of time. The procedure actually mimics nature in that mutation, selection, and recombination are used to evolve highly adapted proteins, but it is much faster than nature. The technique has been used to improve enzymes and to change substrate specificity. It also has been employed to improve protein pharmaceuticals, small molecule pharmaceuticals, gene therapy, DNA vaccines, recombinant protein vaccines, viral vaccines and to evolve viruses. It involves iterative mutagenesis, screening, and selection, i.e., repeated rounds of generating random gene libraries, expressing the genes in a suitable host, and screening the libraries for the desired property; i.e., improved enantioselectivity, catalytic efficiency, catalytic rate, and enzyme stability. Proteins from directed evolution work were already on the market in 2000 (Tobin et al., 2000). Many of these enzymes are used as therapeutic agents to treat gastrointestinal and rheumatic diseases, thromboses, cystic fibrosis, metabolic disease, and cancer. Sales of therapeutic enzymes were $2.3 billion in 1996, while in 1998, markets for therapeutic enzymes were as follows (Stroh, 1994): Pulmozyme (DNase) for cystic fibrosis, acute myocardial infarction, and ischemic stroke, $350 million; CeredaseÒ and CerezymeÒ (r-DNA version) for Gaucher’s disease, $387 million. By 2007, the market for CerezymeÒ reached $1.1 billion.

Production of Recombinant Enzymes An enzyme is labeled as ‘recombinant’ based on the following guidelines: (1) the enzyme’s amino acid sequence is the same as can be found in nature, but the organism used to produce it is nonnative (Mule et al., 2007); (2) the organism used to produce it has been genetically modified (e.g., a stronger promoter in front of the gene (Valle and Ferrari, 2005); and/or (3) the enzyme itself (i.e., its amino acid sequence) has been engineered (Kragh et al., 1999). To keep with the idea of minimizing the impact of genetic engineering on the environment, a development in strain construction has been to remove the antibiotic marker which often accompanies the gene coding for the enzyme of interest (Widner et al., 2000). Recombinant DNA technologies involve isolating a target gene, connecting it with a carrier, transforming it into another organism, and using that organism to propagate the gene product. This process is called ‘cloning.’ There are a few elemental factors that need to be considered in order to succeed in cloning a gene (Whitehurst and van Oort, 2009). They are as follows: 1. A donor organism providing the gene of interest. It can be a mammalian source, plant, or, in most cases, a microorganism. The choice of a proper donor depends on the specific requirements for the property of the gene product. The donor can be found through either a screening process designed for locating the gene of interest, or through a DNA matching process performed on genomic DNA with known partial gene sequence information. 2. A host organism. This is a host in which the target gene can be replicated, transcribed, and further translated into products of interest. In most cases, a host microorganism is chosen because of its easy reproduction. It can also be a mammalian, plant, or an insect system. Choosing the correct host organism is very important for successful expression of a target gene. Although, unfortunately, there are no definite rules in this respect, it has been generally established that homologous gene expression, i.e., genes of one origin expressed in the same or a closely related organism, is the better choice. Nevertheless, the vast number of heterologous expressions that have succeeded in the bacterium E. coli support the conclusion that it is a rather universal host. Choosing a host cannot be separated from choosing a vector. The choice of a host–vector system as a set should be the consideration for the purpose of each target expression. Some commonly used host–vector systems are E. coli (pUC18/19), Bacillus subtilis, (pUB110), and Streptomyces lividans (pIJ702). 3. A vector functioning as the carrier. The vector should be capable of carrying the target gene, replicating it in the corresponding host cells, and integrating it into the chromosome of the host. The most commonly used vector is closed double-helix circular plasmid DNA. It usually contains an ‘ori’ region for replication and typically some antibiotic-resistance genes as selection markers and multiple restriction sites for splicing and joining it with target genes. The vector can also be single-stranded phage DNA or cosmid DNA. A shuttle vector is a vector with the characteristic of functioning in multiple hosts of a different genus. 4. A target gene. This is a gene which can be isolated from the donor organism by ‘shotgun cloning’ or by using primers designed from known sequences of the target gene. These sequences can be deduced from N-terminal or internal amino acid sequences of the corresponding protein. A target gene can also be made synthetically on the basis of knowledge of the known sequences. If a target gene is from a eukaryotic organism such as mammalian cells, the gene should be obtained from a cDNA library in order to remove the introns that are processed in mammalian cells but not in the prokaryotic host. 5. Tool enzymes. Restriction enzymes are necessary tools because they can cleave DNA molecules at specific sites, producing ‘sticky ends’ or ‘blunt ends’ on the action pattern of the enzyme. DNA molecules can be rejoined by a ligase, which is able to connect different gene segments with complementary sequences together, for example, to ligate the isolated target gene to plasmid carriers. Another important enzyme used in the manipulation of DNA is a polymerase, which is used in the amplification of DNA molecules by the ‘polymerase chain reaction.’ 6. Introduction of recombinant DNA molecules into host cells. Three methods are currently used to introduce foreign DNAs into host cells. The first is to use competent E. coli cells. Another method involves exposing the cells and DNA molecules to an electrical field (electroporation). This method is used in both transforming DNA into bacteria and eukaryotic cells with a typical frequency of 109–1010 colonies per gram of DNA. The third method involves using protoplast cells generated by treating cells with polyethylene glycol, or sucrose and lysozyme, and allowing cells to take up foreign DNA at a lower frequency of 106–107

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colonies per gram of supercoiled plasmid DNA. The frequency of transformation depends on the state of the cells and treatment conditions. 7. Isolation of the recombinant clone. Once a recombinant DNA (target gene in a plasmid) is introduced into the host cell, cells are cultivated and placed on agar plates, often containing chemicals such as antibiotics for easy selection of recombinant clones. Colonies depending on the required gene can be screened, selected, or confirmed by assaying enzyme activities, detecting proteins with antibodies or by in situ hybridization with probes derived from known DNA sequences. 8. Fermentation or cultivation technologies for expression. Once a recombinant DNA (target gene in a plasmid) is introduced into the host cell and the recombinant organism isolated, fermentation or cell cultivation technologies are necessary for optimal expression of the cloned gene. Understanding the metabolism of the recombinant organism or cell system and the characteristics of regulating elements, such as gene promoters and regulators, is essential in this respect. Traditional techniques for growing an organism under optimum production conditions are also applicable. In this article, we shall be mainly discussing and evaluating the systems used for producing recombinant enzymes.

Systems Used Recombinant enzymes represent about 90% of industrial enzymes. It is fortunate that efficient and scaled-up systems are available for producing enzymes from rare organisms, i.e., those that have not been used in industry, and in organisms that are known in industry. The latter include E. coli, B. subtilis, other species of Bacillus, Pseudomonas fluorescens, Ralstonia eutropha, Pichia pastoris, Hansenula polymorpha, S. cerevisiae, and species of Trichoderma and Aspergillus (Demain and Vaishnav, 2009). In addition to microbes, expression of the recombinant enzyme can be carried out in mammals, plants, or insects. To choose the best system for expression of the recombinant enzyme, the following factors should be considered: enzyme quality, functionality, production speed, and yield (Adrio and Demain, 2014). Nonglycosylated proteins are usually made in E. coli or yeasts and they constitute 40% of the therapeutic protein market. N-glycosylated proteins are usually made in mammalian cells which mimic human glycosylation. Chinese hamster ovary (CHO) cells provide about 50% of the therapeutic protein market, but the process is very expensive and the glycoproteins made are not exactly the human type, and in some cases, they must be modified. Yeasts, molds, and insect cells are generally unable to provide mammalian glycosylation. However, the popular methylotrophic yeast, P. pastoris, has been genetically engineered to produce a human type of glycosylation. Development of efficient and scale-up expression systems enables the production of enzymes from industrially unknown microorganisms and other living organisms using the industrial microbes mentioned above.

Escherichia coli and Other Nonfilamentous Bacteria Already in 1993, recombinant processes in E. coli were responsible for almost $5 billion worth of products (Demain and Vaishnav, 2009). Escherichia coli has certain advantages for use as a recombinant host including the following: (1) genome modification is easy, quick, and precise; (2) growth to high cell densities is rapid; (3) inexpensive media can be used; and (4) protease activity is easily reduced. The organism can accumulate heterologous proteins up to 50% of its dry cell weight (Swartz, 1996). Research has led to increased yields, secretion, posttranslational modifications, and reduction in production of acetate (Waegeman and Soetaert, 2011). Titers reached 14 g l
1 of recombinant protein by 1998 (Kim et al., 1998). With regard to recombinant enzymes, levan fructotransferase (LFT) was secreted at 4 g l
1 (Lee et al., 2001) and asparaginase at 5 g l
1 (Khushoo et al., 2005). However, E. coli does have some drawbacks, e.g., inability to carry out posttranslational modifications, presence of toxic cell wall pyrogens, and occasionally, formation of inclusion bodies which contain insoluble, inactive, and aggregated heterologous proteins. Some of these problems have been eliminated by use of different promoters to regulate expression, use of different host strains, coexpression of chaperones, lowering temperature, and achieving secretion of the proteins into the periplasmic space or into the medium (Choi and Lee, 2004). As a result, good production and excretion has been obtained with some heterologous enzymes, including alkaline phosphatase at 5.2 g l
1 into the periplasm and LFT at 4 g l
1 into the medium. Lipoxygenase from Pseudomonas aeruginosa, an enzyme that is widely used in food industry to improve aroma, rheological or baking properties of foods, was successfully characterized, expressed, and secreted by E. coli (Lu et al., 2013). Recombinant E. coli containing the isoeugenol monooxygenase gene from Pseudomonas putida converted isoeugenol to vanillin with a conversion yield of 81% and a titer of 28 g l
1 in a 6-h bioconversion (Yamada et al., 2008). Other nonfilamentous bacteria have also shown good production. For example, organophosphohydrolase has been produced at 10 g l
1 by R. eutropha (Barnard et al., 2004). Also, a level of 20 g l
1 of recombinant protein has been produced by the P. fluorescens culture of the Pfenex company (Squires and Lucy, 2008; DePalma, 2013).

Filamentous Bacteria Streptomycetes are used as producers of industrial enzymes. Fermentation technologies have developed rapidly, and together with the discovery of numerous unique enzymes, streptomycetes are attracting more and more attention in industry. Streptomycetes have been called an enzyme treasure trove by Professor Horinouchi of Tokyo University (Horinouchi, 2007). Genetic aspects of

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Streptomyces were intensively studied in the 1960s and 1970s by the Hopwood group (Chater and Hopwood, 1984). Besides cell morphology, genetic phenomena such as chromosomal recombination through transformation, conjugation, and transduction within Streptomyces species and the presence of plasmids were discovered and investigated thoroughly. Streptomycetes have a very large genome size among bacteria and the number of genes encoding proteins exceeds some of the eukaryotic microorganisms such as S. cerevisiae. Gene clusters for the biosynthesis of antibiotics and other functional products are abundant. Hence, streptomycetes have great potential to be harnessed in the recombinant enzyme industry. Commercial glucose isomerases that are used in production of high-fructose corn syrup are those of Streptomyces olivaceus, Streptomyces rubiginosus, and Streptomyces murinus. An enzyme of great economical impact, a trans-glutaminase that is used in the quality improvement of processed meat and fish products and for textural improvement of noodles, comes from Streptomyces mobaraensis (Enzyme Technical Association). However, there are some negative effects of using filamentous bacteria for industrial production. The mycelial lifestyle of streptomycetes often results in viscous broths and unfavorable pellet formation during deep liquid cultivation processes. For this reason, the application of Streptomyces as a general producer of industrial enzymes is, so far, still limited.

Yeasts Yeasts are more suited than more complex eukaryotes to make recombinant enzymes (Celik and Calik, 2012). These include rapid growth, high levels of production, lack of pyrogens, pathogens, and viral inclusions. There are some advantages in using S. cerevisiae as a cloning host, as compared to bacteria (Romanos et al., 1992). These include rapid growth in simple media, high cell density, and secretion of heterologous enzymes into the extracellular broth. Furthermore, its genetics are more advanced than any other eukaryote. 9 g l
1 of glucose oxidase from A. niger has been produced by S. cerevisiae (Demain and Vaishnav, 2009). Transgenic wine yeast, genetically engineered to contain six copies of the bovine L-lactate dehydrogenase gene, produced 122 g l
1 of L-(þ)-lactate from cane sugar with optical purity of 99% or higher (Saitoh et al., 2005). However, S. cerevisiae does have problems, such as plasmid instability, hyperglycosylation of the recombinant enzymes, lack of tightly regulated and strong promoters, and addition of an alpha-1,3-linked mannose residue which could cause immunogenic response in patients if the recombinant product is to be used medically. One of the most popular expression systems for the production of microbial enzymes is the methylotrophic yeast P. pastoris (Higgins and Cregg, 1998; Demain and Vaishnav, 2009; Celik and Calik, 2012). Recombinant intracellular proteins have been made with this yeast at 22 g l
1 (Hasslacher et al., 1997) and recombinant secreted proteins reached 14.8 g l
1 (Werton et al., 1999). It has been stated that P. pastoris can produce up to 30 g l
1 of recombinant proteins (Morrow, 2007). Furthermore, it can excrete proteins, as compared to retaining them in the periplasm, as is the case with S. cerevisiae. A recombinant lipase from Rhizomucor miehei (RML) has been used to convert oil from microalgae to biodiesel (Huang et al., 2014). This 1,3-specific lipase is a highly versatile biocatalyst used in laboratory and industrial applications. When cloned from R. miehei and expressed in P. pastoris strain GS115, it was able to catalyze methanolysis of soybean oil and showed strong position specificity. One advantage of the P. pastoris expression system is its ability to perform eukaryotic posttranslational modifications of heterologous proteins, such as glycosylation. N-linked glycosylation is very common when heterologous proteins are expressed in P. pastoris. It was found that N-linked glycosylation also plays an essential role in protein secretion. Pichia pastoris has produced over 700 proteins (Zhang et al., 2009). Of these, over 350 recombinant proteins were expressed, with production of more than 10 g l
1 of protein in some cases. High-level expression in P. pastoris can be affected by factors such as nucleotide sequence properties, gene copy number, mRNA transcription, promoter choice, secretion signals, and protein folding in the endoplasmic reticulum; these factors cannot be regulated solely through control of the fermentation process. Pichia pastoris has several more advantages over S. cerevisiae. They include (1) a methanol promoter (AOX1) which is efficient and tightly regulated, and yields alcohol oxidase at 30% of soluble protein. (2) It also has less-extensive glycosylation, which is due to shorter chain lengths of N-linked high-mannose oligosaccharides. These are usually up to 20 residues lacking the terminal a-1,3-mannose linkages (Bretthauer and Castellino, 1999). Additional advantages include (3) integration of multicopies of foreign DNA into chromosomal DNA yielding stable transformants; (4) ability to secrete high levels of foreign proteins; and (5) high-density growth and straightforward scale-up (Romanos, 1995). Heterologous gene expression in another methylotrophic yeast, i.e., H. polymorpha, is similar to that of P. pastoris. Expression of foreign genes uses the promoter of the methanol oxidase gene. Similar to the AOX1 gene in P. pastoris, the MOX gene of H. polymorpha is highly expressed and tightly regulated. It can yield enzyme levels up to 37% of total cell protein (Giuseppin et al., 1988). It has produced lipase, hexose oxidase, catalase, glucoamylase, phytase, and other recombinant proteins. Titers include 1.4 g l
1 of secreted glucoamylase and 13.5 g l
1 of phytase. Other useful yeasts include (1) K. lactis making bovine chymosin (rennin) and glucoamylase, (2) Y. lipolytica, and (3) Schizosaccharomyces pombe.

Filamentous Fungi Compared to the successful use of yeasts for production of recombinant enzymes, the use of filamentous fungi (molds) has been much more difficult. It is still difficult to introduce or delete genes, but some advances in transformation have been achieved, e.g., promoter-tagged restriction enzyme-mediated insertion mutagenesis in A. niger (Shuster and Connelley, 1999) and transformation of Aspergillus awamori by Agrobacterium tumefaciens-Ti plasmid–mediated homologous recombination (Gouka et al., 1999). Aspergillus niger strains are attractive hosts for recombinant DNA technology because of their ability to secrete high levels of bioactive proteins with posttranslational processing, such as glycosylation. Aspergillus niger excretes 25 g l
1 of glucoamylase

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(Ward et al., 2006). Foreign genes can be incorporated via plasmids into chromosomes of these filamentous fungi where they integrate stably into the chromosome as tandem repeats providing superior long-term genetic stability. As many as 100 copies of a gene have been observed. The titer of a genetically engineered bovine chymosin–producing strain of A. awamori was improved by 500% via conventional mutagenesis and screening (Lamsa and Bloebaum, 1990). It was then increased from 250 mg l
1 to 1.1 g l
1 by nitrosoguanidine mutagenesis and selection for 2-deoxyglucose resistance (Dunn-Coleman et al., 1991, 1993). Transformants contained 5–10 integrated copies of the chymosin gene. Aspergillus awamori produced 4.6 g l
1 of glucoamylase from A. niger. Mucor rennin was produced by A. oryzae at 3.3 g l
1. Fusarium alkaline protease was made by Acremonium chrysogenum at 4 g l
1. 35 g l
1 of recombinant proteins have been made by Trichoderma reesei. Chrysosporium lucknowense can produce 50–80 g l
1 of proteins, and mutants have been obtained that produce low viscosity and low levels of protease (Gusakov et al., 2007; Verdoes et al., 2007). Production of nonfungal proteins has been low, compared to that of homologous proteins. To overcome such problems, certain steps have been taken, including construction of protease-deficient strains (Van der Hombergh et al., 1997), introduction of a large number of gene copies (Gouka et al., 1997), employment of strong fungal promoters and efficient secretion signals, and gene fusions with a gene encoding part of, or an entire, well-secreted protein (Morelejo et al., 1999). With respect to glycosylation in molds, hyperglycosylation does not seem to occur and low-mannose side chains are formed. Trichoderma reesei has been shown to glycosylate in a manner similar to that in mammalian cells (Salovouri et al., 1987). Trichoderma reesei can produce 35 g l
1 of recombinant enzymes (Durand et al., 1988). Despite the above successes, secreted yields of some heterologous proteins have been comparatively low in some cases. The strategies for yield improvement have included the use of strong homologous promoters, increased gene copy number, gene fusions with a gene encoding a naturally well-secreted protein, protease-deficient host strains, and screening for high titers following random mutagenesis. Such approaches have been effective with some target heterologous proteins, but not with others. Hence, although there has been an improvement in the production of fungal proteins by recombinant DNA methods, there are usually transcription limitations (Verdoes et al., 1995). Although an increase in gene copies of up to about five usually results in an equivalent increase in protein production, higher numbers of gene copies do not give equivalently high levels of protein. Since the level of mRNA correlates with the level of protein produced, transcription is the main problem. Studies on overproduction of glucoamylase in A. niger indicate the problem in transcription to be due to (1) the site of integration of the introduced gene copies and (2) the available amount of trans-acting regulatory proteins. Also, heterologous protein production by filamentous fungi is sometimes severely hampered by fungal proteases. Aspergillus nidulans contains about 80 protease genes (Machida, 2002).

Insect Cells Insect cells are able to carry out more complex posttranslational modifications than can be accomplished with fungi. They also have the best machinery for the folding of mammalian proteins and are therefore quite suitable for making soluble protein of mammalian origin (Agathos, 1991). The most commonly used vector system for recombinant protein expression in insects is the baculovirus. The most widely used baculovirus is the nuclear polyhedrosis virus (Autographa californica) which contains circular double-stranded DNA, is naturally pathogenic for lepidopteran cells, and can be grown easily in vitro. The usual host is the fall armyworm (Spodoptera frugiperda) in suspension culture. A larval culture can be used which is much cheaper than a mammalian cell culture. Recombinant insect cell cultures have yielded over 200 proteins encoded by genes from viruses, bacteria, fungi, plants, and animals (Knight, 1991). Human beta-secretase (BACE-1) is a type I integral membrane aspartic protease that catalyzes the internal cleavage of the amyloid precursor protein, generating the N-terminus of the Abeta peptide. The generation and subsequent extracellular deposition of Abeta (1-42) peptide into amyloid plaques in the brain constitute one of the hallmarks of Alzheimer’s disease (AD), a common debilitating neurodegenerative disorder. Inhibition of BACE-1 is considered an excellent therapeutic strategy against AD. To generate a pure enzyme for protein crystallography and subsequent structure-based drug design, a soluble, unglycosylated, 6xHis-tagged form of proBACE-1 was expressed in insect cells using baculovirus infection (Bruinzeel et al., 2002). Human 15-lipoxygenases are also expressed in insect cells using baculovirus infection. The baculovirus-assisted insect cell expression offers many advantages, as follows: (1) eukaryotic posttranslational modifications without complication, including phosphorylation, N- and O-glycosylation, correct signal peptide cleavage, proper proteolytic processing, acylation, palmitoylation, myristoylation, amidation, carboxymethylation, and prenylation (Luckow and Summers, 1988; Miller, 1988); (2) proper protein folding and S–S bond formation, unlike the reducing environment of E. coli cytoplasm; (3) high expression levels. The virus contains a gene encoding the protein polyhedron which is made at very high levels normally and is not necessary for virus replication. The gene to be cloned is placed under the strong control of the viral polyhedrin promoter, allowing expression of heterologous protein of up to 30% of cell protein. Production of recombinant proteins using the baculovirus expression vector system in insect cells reached 600 mg l
1 back in 1988 (Maiorella and Harano, 1988). The baculovirus insect cell system can produce 11 g l
1 of recombinant protein (Morrow, 2007). Also, (4) easy scale-up with high-density suspension culture; (5) safety: expression vectors are prepared from the baculovirus which can attack invertebrates but not vertebrates or plants, thus ensuring safety; (6) lack of limit on protein size; (7) efficient cleavage of signal peptides; and (8) simultaneous expression of multiple genes (Wilkinson and Cox, 1998).

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Insect cell systems, however, do have some shortcomings, some of which can be overcome. They are as follows. (1) Particular patterns of posttranslational processing and expression must be empirically determined for each construct. (2) Differences exist in proteins expressed by mammalian and baculovirus-infected insect cells. For example, inefficient secretion from insect cells may be circumvented by the addition of insect secretion signals (e.g., honeybee melittin sequence). (3) Improperly folded proteins and proteins that occur as intracellular aggregates are sometimes formed, possibly due to expression late in the infection cycle. In such cases, harvesting cells at earlier times after infection may help. (4) Low levels of expression are sometimes encountered. This can often be increased with optimization of time of expression and multiplicity of infection. (5) Incorrect glycosylation has been a problem with insect cells as hosts (Bisbee, 1993). The complete analysis of carbohydrate structures has been reported for a limited number of glycoproteins. Potential N-linked glycosylation sites are often either fully glycosylated or not glycosylated at all, as opposed to expression of various glycoforms that may occur in mammalian cells. Species-specific or tissue-specific modifications are unlikely to occur.

Transgenic Plants For recombinant protein production, use of plants, as compared to live animals and animal cell cultures, is much safer and less expensive, requires less time, and is superior in terms of storage and distribution issues. In fact, plant expression systems are believed to be even better than microbes in terms of cost, protein complexity, storage, and distribution. The use of plants offers a number of advantages over other expression systems. The low risk of contamination with animal pathogens includes viruses since no plant viruses have been found to be pathogenic to humans. Another advantage is that growth on an agricultural scale requires only water, minerals, and sunlight, unlike mammalian cell cultivation which is an extremely delicate process, very expensive, requiring bioreactors that cost several hundred million dollars when production is scaled up to commercial levels. Some added advantages of plant systems are glycosylation and targeting, compartmentalization, and natural storage stability in certain organs. Transgenic plants have been used to produce valuable enzymes such as D-glucuronidase (GUS), laccase, and trypsin (Hood, 2002). However, production of recombinant proteins in plant cell culture has a low titer, i.e., 200 mg l
1 (Weathers et al., 2010). Transgenic plants can be produced in two ways. One way is to insert the desired gene into a virus that is normally found in plants, such as the tobacco mosaic virus in the tobacco plant. The other way is to insert the desired gene directly into the plant DNA. Products with titers as high as 0.02–0.2% of dry cell weight have been achieved. Recombinant enzymes have been produced in transgenic plants at levels as high as 14% of total tobacco soluble protein, e.g., phytase from A. niger (Kusnadi et al., 1997). By 1997, GUS was already on the market. GUS from E. coli was produced in corn at 0.7% of soluble seed protein. Potential disadvantages of transgenic plants include possible contamination with pesticides, herbicides, and toxic plant metabolites (Fitzgerald, 2003).

Applications of Recombinant Enzymes Enzymes have great significance for industrial, pharmaceutical, and biotechnological processes (Sanchez and Demain, 2011). Industrial enzymes reached a market of $3.3 billion in 2010 (BBC Research, 2011) and recently, a value of $4.4 billion appears to have been reached. Technical enzymes are typically used as bulk enzymes in the detergent, textile, pulp, paper, and biofuel industries. Leather and bioethanol are responsible for the highest sales figures. Revenues for such enzymes amounted to nearly $1.2 billion in 2011 and $1.5 billion in 2015 and are predicted to rise to $1.7 billion by 2016. The biofuel (bioethanol) industry is expected to reach high sales figures in the future (World Enzymes, 2011). Enzymes for foods and beverages are expected to reach a market of $1.3 billion by the end of 2015. Over 60% of the global enzyme market is represented by proteases which are used to produce detergents, foods, pharmaceuticals, leather, agrochemical products, and silk.

Detergent Industry The major application of industrial enzymes is that of detergent additives. Enzymes such as proteases, lipases, amylases, oxidases, peroxidases, and cellulases are added to detergents to catalyze the breakdown of chemical bonds upon the addition of water. A requirement is that they must be active under thermophilic (60  C) and alkalophilic (pH 9–11) conditions. They must also be active in the presence of the various components of washing powders. Enzymes in detergents enhance the detergent’s ability to remove tough stains and also make the detergent eco-friendly. Approximately 25% of the total worldwide sales of enzymes involve the use of proteases in laundry detergents. Novo Industry A/S (Novozymes) introduced the first detergent containing a bacterial protease (‘Biotex’) in 1956, i.e., an alcalase produced by B. licheniformis. In 1994, Novo Nordisk introduced LipolaseÔ, the first commercial recombinant lipase for use in a detergent, by cloning the Humicola lanuginosa lipase into the A. oryzae genome. Genencor International introduced two bacterial lipases in 1995: one from Pseudomonas mendocina (LumafastÔ) and another from Pseudomonas alcaligenes (LipomaxÔ). Another enzyme added recently to detergents is MannawayÔ, a Bacillus mannanase which removes food stains containing guar gum (Kirk et al., 2002). Amylases are another type of enzymes used in detergent formulations, and 90% of all liquid detergents contain such enzymes. The enzymes are used for laundry and automatic dishwashing to clean up residues of starchy foods, such as custard, gravies, potato, and chocolate, and other smaller oligosaccharides. The hydrolytic lipases are commercially very important, and their addition to detergents is mainly for use in laundries and household dishwashers. Enzymes reduce the environmental load of detergent

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products, as they save energy by enabling a lower wash temperature to be used, and use of chemicals in detergents is reduced. They are mostly biodegradable, leaving no harmful residues, having no negative impact on sewage treatment processes, and not possessing any kind of risk to aquatic life.

Food and Beverage Industries Enzymes for manufacture of foods and beverages are the largest segment of industrial enzyme use. Revenues in 2011 reached $1.2 billion and have been projected to reach $1.8 billion by 2016 at an annual growth rate of 10.4% (World Enzymes, 2011). There is an extensive use of amylase in the processed food industry for baking, brewing, production of cakes, preparation of digestive aids, fruit juices, and starch syrups. The a-amylases have been widely used in the baking industry. These enzymes are generally added to the dough of bread in order to degrade the starch into smaller dextrins, which are further fermented by yeast. The a-amylase enhances the fermentation rate and the reduction of the viscosity of dough, which results in improvements in the volume and texture of the product. To modify food flavor and fragrance by synthesis of esters of short-chain fatty acids and alcohols, lipases have been frequently used. Lipases are used in the production of fruit juices, baked foods, vegetable fermentations, and for dairy enrichment. Fats, oils, and related compounds are their main targets. They play a major role in the fermentative steps during manufacturing of sausage and also to measure changes in long-chain fatty acids liberated during ripening. Lipases of different microbial sources have been used for refining rice flavor, modifying soybean milk, and for enhancing the aroma and speeding up the fermentation of apple wine. Three recombinant fungal lipases made in A. oryzae are used in the food industry, i.e., one from R. miehei, one from Thermomyces lanuginosus, and another from Fusarium oxysporum. By adding lipases, fat is removed during processing of meat and fish, and this process is called biolipolysis. Lipase mediation of carbohydrate esters of fatty acids provides a potential market for use as emulsifiers in foods (and also in the manufacture of pharmaceuticals and cosmetics). Protease use in the dairy industry mainly involves cheese manufacture. Calf rennin was originally used for cheese making because of its high specificity, but it is being replaced by proteases from GRAS organisms such as B. subtilis, Mucor miehei, Mucor pusillus Lindt, and Endothia parasitica. Their primary function in cheese making is hydrolysis of the specific peptide bond (Phe105-Met106) generating para-k-casein and macropeptides (Rao et al., 1998). Production of calf rennin (chymosin) by recombinant A. niger var. awamori reached 1 g l
1 via nitrosoguanidine mutagenesis and selection for 2-deoxyglucose resistance (Dunn-Coleman et al., 1991). Further application of parasexual recombination with parental cultures making 1.2 g l
1 yielded a strain making 1.5 g l
1 (Bodie et al., 1994). The U.S. Food and Drug Administration (FDA) approved four recombinant proteases for production of cheese (Pariza and Johnson, 2001). Production of ‘high-fructose corn syrup’ from starch by bacterial glucose isomerase, fungal a-amylase, and glucoamylase is over a $1-billion business. Other enzymes important in the food industry are invertase from K. fragilis, S. carlsbergensis, and S. cerevisiae for production of candy and jam; b-galactosidase from K. lactis, K. fragilis, or C. pseudotropicalis for hydrolysis of lactose in milk and whey; and galactosidase from S. carlsbergensis for beet sugar crystallization. Production of ‘fructose syrup’ by action of xylose isomerase on glucose reached 15 million tons per year by 2005 (Vandamme et al., 2005).

Feed Industry Feed enzymes are a promising segment of the enzyme industry. Their global market was predicted to reach about $730 million in 2015 (Feed Enzymes, 2007). Feed enzymes lead to greater efficiency in feed utilization by increasing the digestibility of nutrients. They also degrade unacceptable components in feed, which are otherwise harmful or of little or no value (Choct, 2006). Commercially available feed enzymes are phytases, proteases, a-galactosidases, glucanases, xylanases, a-amylases, and polygalacturonases, mainly used for swine and poultry (Selle and Ravindran, 2007). Recent developments include the development of heat-stable enzymes, improved specific activity, new nonstarch polysaccharide-degrading enzymes, and rapid, economical, and reliable assays for measuring enzyme activity. Applications vary from country to country since the use of enzymes as feed additives is restricted in many countries by local regulatory authorities (Pariza and Cook, 2010).

Textile Industry For textile manufacture, enzymes are also increasingly being used to develop cleaner processes and reduce the use of raw materials and production of waste. The most recent commercial advances are (1) the application of cellulases for denim finishing and laccases for decolorization of textile effluents and (2) textile bleaching (Araujo et al., 2008). For cotton manufacture, an alternative enzymatic process was developed based on a pectate lyase (Tzanov et al., 2001). Much lower temperatures are used in this process and it uses less water than the classical method. Amylases are utilized for the desizing process in the textile industry. Sizing agents such as starch are added to yarn before fabric production for a fast and secure weaving process. Desizing is the process where removal of starch from the fabric takes place and acts as the strengthening agent to prevent breaking of the warp thread during the weaving process. The a-amylases selectively remove the size and do not affect the fibers. For many years, amylase from a Bacillus strain has been employed. In this industry, lipases are used for the removal of size lubricants, which increases the fabric’s absorbance ability for improved levelness in dyeing. In denim

Production of Recombinant Enzymes

9

abrasion systems, they are used to lessen the frequency of cracks and streaks. Commercial preparations used for the desizing of cotton fabrics and denim contain both a-amylases and lipase enzymes.

Pulp and Paper Industry The use of lipases, xylanases, and laccases in removing pitch (hydrophobic components of wood, mainly triglycerides and waxes) in the pulp industry is another application of increasing importance (Gutierrez et al., 2009). Candida rugosa lipase is used by Nippon Paper Industries to remove up to 90% of these compounds (Farrell et al., 1997). The main use of a-amylases in the pulp and paper industry is the modification of starch of coated paper, i.e., for the production of low-viscosity, high-molecular-weight starch. The coating treatment makes the surface of the paper smooth and strong to improve the writing quality of the paper.

Medicine Lipases isolated from Galleria mellonella (wax moth) were found to have a bactericidal action on Mycobacterium tuberculosis H37Rv. Lipases can be used as digestive aids. They also can be of use in the treatment of malignant tumors, since they are the activators of tumor necrosis factor. Human gastric lipase is the most stable acid lipase and is considered to be a good tool for enzyme substitution therapy. Lipases have been used in the treatment of gastrointestinal disturbances, dyspepsias, cutaneous manifestations of digestive allergies, and so forth. The asymmetric hydrolysis of 3-phenylglycidic acid ester, which is a key intermediate in the synthesis of diltiazem hydrochloride, a widely used coronary vasodilator, is carried out using Serratia marcescens lipase. Therapeutic enzymes have a wide variety of specific uses such as oncolytics, thrombolytics, or anticoagulants and as replacements for metabolic deficiencies. Proteolytic enzymes serve as good anti-inflammatory agents. Some enzymes are being produced in increasing amounts for therapeutic purposes; this applies especially to recombinant enzymes such as factor VIII, tPA, and urokinase that cannot be produced in sufficient amounts from natural sources (blood serum or urine). Another advantage of the recombinant production of these enzymes is that possible contamination with pathogenic human viruses (HIV, herpes) can be avoided. Enzymes are now orally administrated to treat several diseases (e.g., pancreatic insufficiency and lactose intolerance). Since enzymes are proteins themselves, they are potentially subject to inactivation and digestion in the gastrointestinal environment. Therefore, a noninvasive imaging assay had been developed to monitor gastrointestinal activity of exogenous enzymes like prolyl endopeptidase as potential adjuvant therapy for celiac disease (Tufvesson et al., 2011).

Other Uses Retinoids (vitamin A and derivatives) are commercially very important in cosmetics and pharmaceuticals, such as skin care products. Immobilized lipases are used for the preparation of water-soluble retinol derivatives. Lipases are used in hair waving preparations and have also been used as an ingredient of topical antiobese creams or for oral administration (Ness et al., 1999). The enzyme-catalyzed dissolution of biodegradable polymer films based on biosensors has been developed. The polymer system, poly(trimethylene) succinate, which was investigated, is degraded by a lipase and can be used as a biosensor. Within the last few years, different processes have been designed using enzyme-labeled probes in order to avoid unstable and harmful isotopes. While screening various hydrolytic enzymes to fulfill the special demands, fungal lipases turned out to be the most relevant ones. Immobilization of lipases can be done on pH/oxygen electrodes along with glucose oxidase, and these serve as lipid biosensors and can be used in triglycerides and blood cholesterol determinations (Jaeger and Reetz, 2000). It is thought that soil microbial lipase activity can be an important indicator of diesel oil biodegradation in freshly contaminated, unfertilized, and fertilized soils. In coastal environments, fungal strains are used to degrade oil spills, which in turn increases ecorestoration and enzymatic oil processing in industries. Lipases produced by B. subtilis, B. licheniformis, Bacillus amyloliquefaciens, S. marcescens, P. aeruginosa, and Staphylococcus aureus were reported to degrade palm oil mill, from dairy, slaughter houses, the soap industry, and domestic wastewater. Pseudomonas aeruginosa lipases were recommended for castor oil degradation. 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 alkaline or neutral proteases, are currently being used in this industry. Laccases oxidize phenolic and nonphenolic lignin-related compounds as well as environmental pollutants (Rodriguez-Couto and Toca-Herrera, 2006). They are used to detoxify industrial effluents from the paper and pulp, textile, and petrochemical industries; as medical diagnostic tools, for bioremediation of herbicides, pesticides, and explosives in soil; as cleaning agents for water purification systems; as catalysts in drug manufacture; and as cosmetic ingredients. Cellulases have been widely used in textile applications for many years, and these enzymes are gaining additional consideration in the enzyme market, owing to their ability in the breakdown of lignocellulosic feedstocks. The cost of cellulases is a key issue in achieving a low-price conversion of lignocellulosic biomass into biofuels and other products (Rubin, 2008; Wilson, 2009; Zhang, 2011). Filamentous fungi can produce native cellulases at levels greater than 100 g l
1 (Demain and Vaishnav, 2009). Enzymes needed to hydrolyze cellulose include (1) endoglucanases, which break down cellulose chains in a random manner; (2) cellobiohydrolases, which liberate glucose dimers from both ends of cellulose chains; and (3) beta-glucosidases, which produce glucose from oligomer chains. Hypocrea jecorina (T. reesei) is the main industrial source of cellulases and hemicellulases used to depolymerize plant biomass to simple sugars (Zhang et al., 2006;

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Kumar and Singh, 2013; Singh and Singh, 2008; Kubicek et al., 2009). The overall action of T. reesei on cellulosic biomass is limited by its low content of beta-glucosidase. The result is the accumulation of cellobiose which limits further breakdown. The expression of the beta-glucosidase gene from Pericona sp. in T. reesei resulted in an increased level of beta-glucosidase, thus increasing the overall cellulase activity and action on biomass residues (Dashban and Qin, 2012). Cellulases are formed adaptively, and several positive (XYR1, ACE2, HAP2/3/5) and negative (ACE1, CRE1) components involved in this regulation are now known (Kubicek et al., 2009). In addition, the complete genome sequence of T. reesei has been published (Martinez et al., 2008), thus making the organism susceptible to targeted improvement by metabolic engineering. It has been reported that the extreme thermophilic bacterium Caldicellulosiruptor bescii produces a cellulase/hemicellulase system that is twice as active as that from T. reesei (Kanafusa-Shinkai et al., 2013).

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