Biotechnology of Microbial Enzymes. Production, Biocatalysis and Industrial Applications

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Alvarez-Macarie and Baratti 20 reported the production of a novel thermostable esterase from the highly thermotolerant Bacillus licheniformis heterologously expressed in E. Feruloyl esterase is a key enzyme in the biosynthesis of ferulic acid, which is the precursor for vanillin, an aroma compound used in foods and beverages Several researchers have reported the microbial production of ferulyl esterase 76 , Lipoxygenases LOX are involved in the dioxygenation of polyunsaturated fatty acids in lipids containing a cis -1,4-pentadiene.

BASF Enzyme Production

LOX enzymes are non-haem iron-containing enzymes. The LOX-catalyzed reaction produces different precursors for the production of different volatile and aroma-producing chemical substances in plants. LOXs are used in aroma generation in food industry and also in bread making Soya bean lipoxygenases LOX is the most studied lipoxygenase enzyme.

Bacterial LOXs possess different specificity towards fatty acids. LOX from Nostoc sp. The main applications of LOXs in dough are based on their ability to bleach the flour pigment carotenoid, by co-oxidation of the pigment with fatty acids 83 , Lipoxygenases are also employed to improve tolerance to mixing and different handling properties of dough This effect is due to the oxidation of thiol group in gluten, which may lead to redistribution of different disulphide bonds, tyrosine cross-linking and subsequent strengthening of the gluten.

This also leads to the improvement in dough rheology. Recently Patel et al. The catalytic modules of cellulases belong to glycosyde hydrolase GH family and have been classified in different groups based on differences in amino acid sequences and three-dimensional structural features. GH family enzymes mainly use acid—base catalysis mechanism for cleaving glycoside bonds in cellulose.

The catalysis is achieved by two major residues a proton donor and a nucleophile of the enzyme in the active site region The hydrolysis occurs via retention or inversion mechanism depending on the spatial position of these catalytic residues in the enzyme. Endoglucanases from various sources belong to different glycoside hydrolase families, among which the major are 5—9, 12, 44, 45, 48, 51 and Most of the fungal endoglucanases contain a catalytic module with carbohydrate-binding module CBM , but catalytic module without CBM was also reported from fungal species Multiple catalytic modules and CBMs are present in bacterial endoglucanases.

Exoglucanases or cellobiohydrolases CBHs act on available reducing or non-reducing ends of cellulose polymer and liberate cellobiose. Fungal and bacterial CBHs show diversity in catalytic module and belong to glycoside hydrolase families 5, 6, 7, 9, 48 and 74 A tunnel-shaped catalytic module is observed in most of the CBHs. The cellulolytic machinery of microbes is mainly regulated through feedback inhibition of b-glucosidases by their reaction product glucose.

A large diversity of microorganisms is reported to produce cellulases during their growth on cellulosic materials. The industrial making of cellulases is mainly from microbial sources, bacteria and fungi, and these microorganisms can be diverse in their habitat. The aerobic bacteria show similar mechanism of cellulose degradation to that of aerobic fungi.

In anaerobic bacteria, cellulosomes are located on the cell surface and operate via a different system. Cellulases from fungi Aspergillus and Trichoderma and bacteria Bacillus and Paenibacillus are potentially used in the production of food. They are also widely used for various industries such as textile, paper, detergent and food industry In juice industry, cellulases are applied in combination with other macerating enzymes for increasing process performance and yield, improving the extraction methods, clarification and stabilization of juices They can also reduce the viscosity of nectar and puree from fruits such as apricot, mango, plum, papaya, pear and peach, and are used for the extraction of flavonoids from flowers and seeds.

Microbial enzymes: industrial progress in 21st century

The preferences of cellulase-mediated extraction over conventional methods are due to higher yield, less heat damage and short processing time. Cellulases are utilized for the extraction of phenolic compounds from grape pomace They are also reported to reduce bitterness of citrus fruit and improve aroma and taste Cellulases are used with other enzymes for efficient olive oil extraction In wine production, cellulases are used in combination with other enzymes to increase yield and quality The main advantages of using these enzymes are improved maceration, better colour development, must clarification and finally wine stability and quality Studies of Oksanen et al.

Xylanases are produced by microorganisms to cleave xylans, a major constituent of hemicellulose. Endoxylanases EC 3.

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Exoxylanases EC 3. The major functions of xylanases are performed by a catalytic module and few classes possess an additional CBM for binding to substrates. The two major catalytic modules of hemicellulases are glycoside hydrolases GHs and carbohydrate esterases CEs. Endoxylanase hydrolyses the xylan backbone and has catalytic cores belonging to GH families 8, 10, 11, 30 and 43 with the most common ones being GH 10 and 11 These differ in their substrate specificities and the GH10 is more active on substituted xylan.

Similar to cellulases, they may also contain CBMs The catalytic module of these enzymes belongs to the GH families 3, 30, 39, 43, 52 and These two enzymes are often collectively called xylanases. Xylanases are produced by microbes like actinomycetes, bacteria and fungi. The major actinomycete and bacterial species producing xylanase are Streptomyces sp.

Applications of Microbial Enzymes in Food Industry - Europe PMC Article - Europe PMC

Those produced by bacteria and actinomycetes are effective in a broader range of pH 5. Fungi are major sources of xylanase due to their high content and extracellular release of the enzyme The major fungal species producing xylanase are Aspergillus sp. Fungal xylanases have higher activity than bacteria or yeast Carbohydrate-hydrolyzing enzymes are usually used in bread making industry. Rheological properties of dough are improved through enzymatic hydrolysis of non-starch polysaccharides Xylanases are widely used in bread making industry with other enzymes.

The potential effectiveness of xylanolytic enzymes increases its use in bread making. They can increase the specific bread volume and this improves the quality of bread. The hemicellulose in wheat flour is broken down by xylanase, which increases the binding of water in the dough. The dough becomes softer and crumb formation is delayed, allowing the dough to grow Xylanase is used to improve texture, tastiness and palatability in biscuits. They also play an important role in juice production by improving extraction, clarification and stabilization In combination with other enzymes, xylanases lead to better yield of juice and increased recovery of aromas, essential oils, vitamins, mineral salts, pigments, etc.

In beer making industries, xylanases are used for hydrolysing the cellular wall of barley. Hydrolysis leads to release of arabinoxylans and lower oligosaccharides, which reduces the muddy appearance and viscosity of the beer Pectinases are enzymes which catalyze the hydrolysis of glycosidic bonds in pectic polymers. Pectic substances found in tomato, pineapple, orange, apple, lemon pulp, orange peel and other citrus fruits act as natural substrate for this enzyme.

Pectinases can be produced from natural as well as recombinant microbes with attempts made to increase their thermostability and yield Pectinases can also act either on smooth or hairy regions of pectin Based on pH, there are acidic and alkaline pectinases also grouped in endopectinases when enzyme cleaves randomly, and exopectinases when the terminal ends are targeted. Pectinases find a multitude of industrial applications such as in paper bleaching, food industry, remediation, etc.

Juices with added pectinase have a clearer appearance and filterability than enzyme-depleted counterparts Apart from reducing the turbidity and haze generation of naturally derived fruit juices such as apple and banana, pectinases also improve the colour and flavour of drinks , The addition of gelatin and pectin greatly increases the viscosity and turbidity of juices, and removal of the haze is the most costly part of juice production.

The use of biogenic enzymes such as pectinases in juices would act almost nine times better than mechanical maceration to get good results. Glucose oxidase EC 1. He stated that in the presence of dissolved oxygen the enzyme can convert glucose to gluconic acid. The glucanolactone is then spontaneously hydrolysed to gluconic acid The enzyme is homodimeric and contains two similar polypeptide chain subunits 80 kDa.

The subunits are covalently linked by disulphide bonds and one flavin adenine dinucleotide FAD molecule non-covalently bound to active site region of each subunit. The glucose oxidase production has been reported from various microorganisms and it was first discovered in Aspergillus niger and Penicillium glaucum. Aspergillus niger species is widely used for production of glucose oxidase and its strains can produce higher amount of glucose oxidase Penicillium adametzii is a widely used fungus for the production of extracellular glucose oxidase The different bacterial species are also reported to produce glucose oxidase.

Although many species of bacteria and fungi are reported to produce this enzyme, fungi are considered for the industrial production of glucose oxidase Glucose oxidase has its wide use in various industries like pharmaceutical and food industries, and in biofuel cells Its use is increasing in baking industry because its oxidizing effects make stronger dough In food industry, it enhances the flavour, aroma and stability of food products by removing glucose and oxygen from diabetic drinks and egg white Glucose oxidase improves the colour, texture, flavour and shelf life of food products and prevents rottening During food packaging glucose oxidase is used for increasing storage life by removing oxygen Laccases EC 1.

Commonly known as blue oxidases, they are used for studying their potential to oxidize phenolic compounds and therefore applied in several industrial sectors - These enzymes act as a potent biocatalyst for application in chemical synthesis, biobleaching of paper pulp, bioremediation, biosensing, wine stabilization and textile finishing. They have different specificity for substrate and a wide range of oxidizable substrates, which further depends on the type of microbial sources producing the enzyme Laccases catalyze the oxidation of a wide range of compounds such as phenolics, aromatic amines and ascorbate , These enzymes combine reducing substrate having four oxidized electrons with four reduced electrons for cleaving dioxygen bond in the presence of four copper atoms present in laccases The mechanism of catalytic activity of laccase is described in the report of Madhavi and Lele and Morozova et al.

Laccases are secreted extracellularly by several fungi as a product of their secondary metabolism during fermentation, but their production is limited to a few fungal species Well known producers of laccases belong to Deuteromycetes, Ascomycetes and Basidiomycetes , Funalia trogii is a white rot fungus capable of producing laccase through absorbent fermentation.

The maximum laccase production by F. Bacillus licheniformis produces recombinant laccases for industrial applications Recently, heterologous expressions have been used for laccase production. Bacillus vallismortis fmb genes were cloned and heterologously expressed in Escherichia coli BL21 DE3 cells Laccase is used for modification of colour appearance of food and beverage industries, or for wine stabilization as an alternative to physical and chemical adsorbents.

Removal of polyphenols from wine should be chosen to avoid adverse changes in wine organoleptic characteristics including stability in acidic medium and reversible inhibition due to the presence of sulphite Furthermore, this enzyme is used in cork stopper manufacturing industry Haze formation is one of the problems in brewing industry. To avoid it, laccases have been applied for polyphenol oxidation as substitute for traditional approach by different researchers - This enzyme is also used for oxygen removal in the final step of beer production which prolongs the storage life of beer.

Commercial laccase called Flavourstar, manufactured by Novozymes, is used for removing the off-flavour formation in brewing industry It is used in baking because it has the capability to cross-link with biopolymers. The application of laccase in baking enhances stability, strength and decreases stickiness which further increases machinability of bread batter. Moreover, it increases volume and enhances softness of the product as reported by Labat et al.

Catalase EC 1. It helps hydrogen peroxide decomposition. This enzyme can be produced from microbial sources such as Aspergillus niger and Micrococcus luteus and from bovine liver. Microorganisms are usually preferred as sources for enzyme production due to their advantages such as fast growth, easy handling and genetic tuning for obtaining a desired product , The anaerobic Bacteroides fragilis exhibited increased catalase levels in media with haem Frankenberg et al. A facultative anaerobic catalase-producing Bacillus maroccanus resistant to hydrogen peroxides was isolated from textile effluents A potent catalase-producing bacterium Pyrobaculum calidifontis was isolated from hot springs in Los Banos and Calamba, Laguna, Philippines A thermo-alkaliphilic catalase-positive strain of Bacillus halodurans LBK was isolated from alkaline hot-spring waters of Kenya A halo alkali tolerant catalase-producing Oceanobacillus oncorhynchi ssp.

A catalase-positive psychrophile Bacillus N2a was isolated from seawater Other catalase-positive bacteria such as Rhizobium radiobacter were isolated from industrial effluent from beverage industry , Comamonas testosteroni and C. Psychrobacter piscatorii T-3, a psychrotolerant bacterium isolated from bleach-rich runoff, has high catalase activity Fungi and yeast are able to produce catalase.

In fabric industry, catalase is used for removing excess hydrogen peroxide from fabric. This enzyme is mostly used along with other enzymes in food processing industry. Catalase is often used with glucose oxidases for food preservation. Results showed that colour and amount of acetaldehyde were stable if treated properly with enzymes Catalase is applied in milk processing industry to eliminate peroxide from milk 31 , to remove glucose from egg white in baking industry and in food wrappers to prevent oxidation and control perishability of food.

This enzyme has limited use in cheese production. Peroxidases EC 1. They catalyse the reduction of peroxides and oxidation of a wide range of inorganic and organic compounds. Their molecular mass ranges from 30 to Da, and they comprise a group of unique enzymes such as iodide peroxidase, NADH peroxidase and glutathione peroxidase as well as a group of other nonspecific enzymes Peroxidases are present in plants, microorganisms and animals.

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They are involved in lignification processes in plants and defence mechanisms against damaged or infectious tissues Among microorganisms, Phanerochaete chrysosporium is the best characterized peroxidase-secreting organism Industrial scale applications of fungal peroxidases are limited by challenges associated with post-translational modification of proteins However, bacterial peroxidases are easier to produce and have better stability and activity suitable for industrial applications. These enzymes are applied with bacterial laccases for dye decolourization Peroxidase activities are reported in bacterial taxa, such as Firmicutes, Proteobacteria, Actinobacteria and Acidobacteria , Moreover, actinomycetes, which are soil bacteria, are able to grow like fungi and have similar ecological niche, and can produce peroxidases for lignin degradation , The first secreted extracellular lignin peroxidase was produced by Streptomyces viridosporus T7A Peroxidase catalyzes a wide range of substrates using hydrogen peroxide or other peroxides This enzyme is used in food industry for producing flavour, colour and texture and improving nutritional quality of food.

Other applications include as biosensors, in polymer synthesis and in the management of pollutants in the environment It can be used for treating phenolic effluents from industries. Thermal inactivation of peroxidases is used in food industry to measure the efficiency of blanching treatment, which further enhances the shelf life of food The negative effect of peroxidases is that they cause undesirable browning of fruits and off-flavours of vegetables.

This enzyme can be produced from natural microbes such as Brevibacillus brevis or from recombinant Saccharomyces cerevisiae The enzyme catalytically converts acetolactate to acetoin via a two-step reaction involving direct decarboxylation of substrate to an enol derivative and its further protonation to final product Moreover, the off-taste due to the presence of diacetyl in beer is nullified by the action of this enzyme. Studies indicate that both free and encapsulated form of this enzyme work efficiently in the process, thus aiding the use of immobilized enzymes at reduced costs Of the various microbially derived enzymes, asparaginases form a major class of pharmaceutical, nutraceutical and industrially significant enzymes widely used by man Asparaginase, as the name implies, catalyses the breakdown of the asparagine to subsequent acid derivative aspartic acid and NH 3 and can be considered as the asparagine-depleting enzyme.

Asparagine is a nonessential amino acid to humans, whereas it is an essential amino acid for cancerous cells. Thus, the depletion of asparagine critically affects the growth of cancerous cells, which forms the basis of this enzyme as anticancer agent Various food processing methods such as frying in oil and baking cause the conversion of asparagine to acrylamide, a known carcinogen.

Biotechnology of microbial enzymes

Naringinase EC 3. Nariginase is produced mostly by fungal isolates, viz. Fungal sources of naringinase are found to be more predominantly used than the bacterial ones due to increased yield. Naringinase has a major role in food processing as a debittering enzyme supplemented to fruit juices. Both free 35 and immobilized forms of this enzyme are used to get better results.

Immobilization of this enzyme has been done in a variety of substrates such as polyvinyl alcohol cryogels , packaging films , cellulose triacetate nanofibre , graphene , etc. Various food additives such as biopolymers and sweeteners can be synthesized using rhamnosidase or naringinase. The use of naringinase is also noted in tomato pulp preparation, kinnow peel waste treatment and prunin preparation Enzymes find application in food, detergent, pharmaceutical and paper industries.

Biotechnology of Microbial Enzymes

Nowadays, the enzymatic hydrolysis and enzyme-based processes are preferred to the chemical ones due to the environmentally friendly nature, efficient process control, high yield, low refining costs and process safety. In comparison with plant and animal enzymes, microbial enzymes can be produced very effectively by different fermentation techniques like solid-state and submerged fermentations.

It is also easy to produce microbial enzymes on a large scale. The microbial enzymes can be easily modified through various molecular and biochemical approaches. Hyperproduction of microbial enzymes with high specific activity can be achieved by overexpression of their genes. Many of the enzymes of microbial origin are still unexplored and there are many opportunities for finding wider industrial application of microbial enzymes, especially in food sector.

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The snippet could not be located in the article text. This may be because the snippet appears in a figure legend, contains special characters or spans different sections of the article. Food Technol Biotechnol. PMID: Received Aug 29; Accepted Jan Copyright notice. This article has been cited by other articles in PMC. Summary The use of enzymes or microorganisms in food preparations is an age-old process.

Key words: enzymes, food industry, brewing, baking, juice clarification. Introduction Microorganisms have been used in food fermentation since ancient times and fermentation processes are still applied in the preparation of many of the food items 1. Table 1 Applications of microbial enzymes in food industry.

see url Open in a separate window. Glucoamylases Glucoamylases EC 3. Proteases Proteases are enzymes which catalyze the hydrolysis of peptide bonds present in proteins and polypeptides. Lipases Lipases are enzymes which catalyze the hydrolysis of long-chain triglycerides. Phospholipases Phospholipases selectively break down phospholipids into fatty acids and other lipophilic substances. Esterases In aqueous solution, esterases are able to facilitate the splitting of esters into acid and alcohol.

Lipoxygenases Lipoxygenases LOX are involved in the dioxygenation of polyunsaturated fatty acids in lipids containing a cis -1,4-pentadiene. Xylanases Xylanases are produced by microorganisms to cleave xylans, a major constituent of hemicellulose. Pectinases Pectinases are enzymes which catalyze the hydrolysis of glycosidic bonds in pectic polymers. Glucose oxidase Glucose oxidase EC 1. Laccase Laccases EC 1. Catalase Catalase EC 1. Peroxidase Peroxidases EC 1. Asparaginase Of the various microbially derived enzymes, asparaginases form a major class of pharmaceutical, nutraceutical and industrially significant enzymes widely used by man Conclusions and Future Perspectives Enzymes find application in food, detergent, pharmaceutical and paper industries.

References 1. Enzyme Technology. A broader view: Microbial enzymes and their relevance in industries, medicine, and beyond. BioMed Res Int. Advances in microbial amylases. Biotechnol Appl Biochem. Braz J Microbiol.

Application of solid-state fermentation to food industry - A review. J Food Eng. J Biotechnol. Therefore, a huge number and quantity of enzymes are now available in the market Reetz, Although isolated enzymes are mainly chosen for their simple implementation in biocatalyzed processes, whole-cell biotransformations are generally preferred for complex reactions involving more than one enzyme or cofactor, or for reactions where enzymes are not suitable for isolation. We herein provide examples of the application of isolated enzymes wild-type or mutants and whole cells, either in soluble or immobilized form, in the synthesis of some valuable APIs.

Biblioteca de la Universidad Complutense. Biocatalytic processes are more ecofriendly, sustainable, and profitable, and hence biocatalysis is proving to be key for the development of the so-called bioeconomy. In addition to enantioselectivity, regioselectivity on complex molecules is another inherent feature of chemoenzymatic processes, which avoids the need of protecting groups, and reduces the number of synthetic steps.

Finally, biocatalysis can also be used to produce achiral APIs where classical chemical methods are too complicated. Therefore, a huge number and quantity of enzymes are now available in the market Reetz,