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Enzymes are biocatalysts produced by living cells to bring about specific biochemical reactions generally forming parts of the metabolic processes of the cells. Enzymes are highly specific in their action on substrates and often many different enzymes are required to bring about, by concerted action, the sequence of metabolic reactions performed by the living cell. All enzymes which have been purified are protein in nature, and may or may not possess a nonprotein prosthetic group.
The practical application and industrial use of enzymes to accomplish certain reactions apart from the cell dates back many centuries and was practiced long before the nature or function of enzymes was understood. Use of barley malt for starch conversion inbrewing, and of dung for bating of hides in leather making, are examples of ancient use of enzymes. It was not until nearly the turn of this century that the causative agents or enzymes responsible for bringing about such biochemical reactions became known. Then crude preparations from certain animal tissues such as pancreas and stomach mucosa, or from plant tissues such as malt and papaya fruit, were prepared which found technical applications in the textile, leather,brewing, and other industries.
Dr. Jokichi Takamine (1894, 1914) was the first person to realize the technical possibility of cultivated enzymes and to introduce them to industry. He was mainly concerned with fungal enzymes, whereas Boidin and Effront (1917) in France pioneered in the production of bacterial enzymes about 20 years later.Technological progress in this field during the last decades has been so great that, for many uses, micro-bial cultivated enzymes have replaced the animal or plant enzymes. Once the favorable results of employing such enzyme preparations were established, a search began for better, less expensive, and more readily available sources of such enzymes.It was found that certain microorganisms produce enzymes similar in action to the amylases of malt and pancreas, or to the proteases of the pancreas and papaya fruit. This led to the development of processes for producing such microbial enzymes on a commercial scale Example, in textile desizing, bacterial amylase has largely replaced malt or pancreatin. At present, only a relatively small number of microbial enzymes have found commercial application, but the number is increasing, and the field will undoubtedly be much expanded in the future.
Enzyme classification:-Presently more than 3000 different enzymes have been isolated and classified. The enzymes are classified into six major categories based on the nature of the chemical reaction they catalyze:
1. Oxidoreductases :- Catalyze oxidation or reduction of their substrates.
2. Transferases :- Catalyze group transfer.
3. Hydrolases :- Catalyze bond breakage with the addition of water.
4. Lyases :- Remove groups from their substrates.
5. Isomerases :- Catalyze intramolecular rearrangements.
6. Ligases :- Catalyze the joining of two molecules at the expense of chemical energy.
Only a limited number of all the known enzymes are commercially available . More than 75 % of industrial enzymes are hydrolases. Protein-degrading enzymes constitute about 40 % of all enzyme sales. More than fifty commercial industrial enzymes are available and their number is increasing steadily
PRODUCTION OF MICROBIAL ENZYMES
Enzymes occur in every living cell, hence in all microorganisms. Each single strain of organism produces a large number of enzymes, hydrolyzing, oxidizing or reducing, and metabolic in nature. But the absolute and relative amounts of the various individual enzymes produced vary markedly between species and even between strains of the same species. Hence,
it is customary to select strains for the commercial production of specific enzymes which have the capacity for producing highest amounts of the particular enzymes desired. Commercial enzymes are produced from strains of molds, bacteria, and yeasts
Up until less than 10 years ago, commercial fungal and bacterial enzymes were produced by surface culture methods. Within the past few years, however, submerged culture methods have come into extensive use.
For fungal enzymes, the mold is cultivated on the surface of a solid substrate. Takamine used wheat bran and this has come to be recognized as the most satisfactory basic substrate although other fibrous materials can be employed.
Other ingredients may be added, such as nutrient salts, acid or buffer to regulate the pH, soy bean meal or beet cosettes to stimulate enzyme production. In one modification of the bran process, the bran is steamed for sterilization, cooled, inoculated with the mold spores and are then spreaded .Incubation takes place in chambers where the temperature and humidity are controlled within limits by circulated air. It may be stated that instead of trays for incubation, Takamine, as well as other producers, at one time used slowly rotating drums. Generally tray incubation gives more rapid growth and enzyme production. Bacterial enzymes have been and are also produced by the bran process. .Incubation takes place in chambers where the temperature and humidity are controlled within limits by circulated air However, until recently the process originally invented by Boidin and Effront was most extensively employed In this process, the bacteria are cultivated in special culture vessels as a pellicle on the surface of thin layers of liquid medium, the composition of which is adjusted for maximum production of the desired enzyme. Different strains of Bacillus subtilis and different media are employed, depending on whether bacterial amylase or protease is desired.
PRODUCTION PROCESS OF INDUSTRIAL ENZYMES USING MICROBES
Solid State Fermentation
Solid-state fermentation (SSF) is a method used for the production of enzymes. Solid-state fermentation involves the cultivation of microorganisms on a solid substrate, such as grains, rice and wheat bran. This method is an alternative to the production of enzymes in liquid by submerged fermentation. SSF has many advantages over submerged fermentation. These include, high volumetric productivity, relatively high concentration of product, less effluent generated and simple fermentation equipment.. SSF requires moisture to be present on the substrate, for the microorganisms to produce enzymes. As a consequence the water content of the substrate must also be optimized, as a higher or lower presence of water may adversely affect the microbial activity. Water also has implications for the physicochemical properties of the solid substrate. Enzymes of industrial importance have been produced by SSF. Some examples are, proteases, pectinases, glucoamylases andcellulases
Microorganisms used for the production of enzymes in S.S.F.
A large number of microorganisms, including bacteria, yeast and fungi produce different groups of enzymes.Selection of a particular strain, however, remains a tedious task, especially when commercially competent enzyme yields are to be achieved. The selection of a suitable strain for the required purpose depends upon a number of factors, in particular upon the nature of the substrate and environmental conditions. Generally, hydrolytic enzymes, e.g. cellulases, xylanases, pectinases, etc. are produced by fungal cultures, since such enzymes are used in nature by fungi for their growth. Trichoderma spp. and Aspergillus spp. have most widely been used for these enzymes. Amylolytic enzymes too are commonly produced by filamentous fungi and the preferred strains belong to the species of Aspergillus and Rhizopus. Although commercial production of amylases is carried out using both fungal and bacterial cultures, bacterial a -amylase is generally preferred for starch liquefaction due to its high temperature stability. In order to achieve high productivity with less production cost, apparently, genetically modified strains would hold the key to enzyme production.
Substrates used for the production of enzymes in SSF systems
Agro-industrial residues are generally considered the best substrates for the SSF processes, and use of SSF for the production of enzymes is no exception to that. A number of such substrates have been employed for the cultivation of microorganisms to produce host of enzymes .Some of the substrates that have been used included sugar cane bagasse, wheat bran, rice bran, maize bran, gram bran, wheat straw, rice straw, rice husk, soyhull, sago hampas, grapevine trimmings dust, saw dust, corncobs, coconut coir pith, banana waste, tea waste, cassava waste, palm oil mill waste, aspen pulp, sugar beet pulp, sweet sorghum pulp, apple pomace, peanut meal, rapeseed cake, coconut oil cake, mustard oil cake, cassava flour, wheat flour, corn flour, steamed rice, steam pre-treated willow, starch, etc.Wheat bran however holds the key, and has most commonly been used, in various processes.
The selection of a substrate for enzyme production in a SSF process depends upon several factors, mainly related with cost and availability of the substrate, and thus may involve screening of several agro-industrial residues. In a SSF process, the solid substrate not only supplies the nutrients to the microbial culture growing in it but also serves as an anchorage for the cells. The substrate that provides all the needed nutrients to the microorganisms growing in it should be considered as the ideal substrate. However, some of the nutrients may be available in sub-optimal concentrations, or even absent in the substrates. In such cases, it would become necessary to supplement them externally with these. It has also been a practice to pre-treat (chemically or mechanically) some of the substrates before using in SSF processes (e.g. ligno-cellulose), thereby making them more easily accessible for microbial growth.
Design of bioreactor in Solid State Fermentations
Over the last decade, there has been a significant improvement in understanding of how to design, operate and scale up SSF bioreactors. The key to these advances has been the application of mathematical modelling techniques to describe various physicochemical and biochemical phenomena within the system . The basic principle of SSF is the ââ‚¬Å“solid substrate bedââ‚¬Â. This bed contains the moist solids and an inter particle voids phase. SSF has been conventionally more applicable for filamentous fungi, which grow on the surface of the particle and penetrate through the inter particle spaces into the depth of the bed. The process in most of the cases is aerobic in nature. The suitable bioreactor design to overcome the heat and mass transfer effects, and easy diffusion and extraction of metabolites has become the topic of hot pursuit. While tray and drum type fermenters have been studied and used since long, much focus has been paid in last few years on developing packed bed fermenters as they could provide better process economics and a great deal of handling ease . A tray bioreactor could have unmixed beds without forced aeration of (manually) mixed bed without forced aeration. However, there has been no significant advances in tray design. Packed beds could be unmixed beds with forced aeration and rotating drums could have intermittent agitation without forced aeration, operating on continuous or semi-continuous mode. The bed could be agitated intermittently or continuously with forced aeration.
Factors affecting enzyme production in SSF
The major factors that affect microbial synthesis of enzymes in a SSF system include: selection of a suitable substrate and microorganism; pre-treatment of the substrate; particle size (inter-particle space and surface area) of the substrate; water content and aw of the substrate; relative humidity; type and size of the inoculum; control of temperature of fermenting matter/removal of metabolic heat; period of cultivation; maintenance of uniformity in the environment of SSF system, and the gaseous atmos-phere, i.e. oxygen consumption rate and carbon dioxide evolution rate.
Submerged fermentation is the cultivation of microorganisms in liquid nutrient broth. Industrial enzymes can be produced using this process. This involves growing carefully selected micro organisms (bacteria and fungi) in closed vessels containing a rich broth of nutrients (the fermentation medium) and a high concentration of oxygen. As the microorganisms break down the nutrients, they release the desired enzymes into solution. Due to the development of large-scale fermentation technologies, the production of microbial enzymes accounts for a significant proportion of the biotechnology industry total output. Fermentation takes place in large vessels (fermenter) with volumes of up to 1,000 cubic metres.
The fermentation media sterilises nutrients based on renewable raw materials like maize, sugars and soya. Most industrial enzymes are secreted by microorganisms into the fermentation medium in order to break down the carbon and nitrogen sources. Batch-fed and continuous fermentation processes are common. In the batch-fed process, sterilised nutrients are added to the fermenter during the growth of the biomass. In the continuous process, sterilised liquid nutrients are fed into the fermenter at the same flow rate as the fermentation broth leaving the system. This will achieve a steady-state production. Parameters like temperature, pH, oxygen consumption and carbon dioxide formation are measured and controlled to optimize the fermentation process.
Firstly, in harvesting enzymes from the fermentation medium one must remove insoluble products, e.g. microbial cells. This is normally done by centrifugation. As most industrial enzymes are extracellular (secreted by cells into the external environment), they remain in the fermented broth after the biomass has been removed. The biomass can be recycled as a fertiliser, but first it must be treated with lime to inactivate the microorganisms and stabilise it during storage.
The enzymes in the remaining broth are then concentrated by evaporation, membrane filtration or crystallization depending on their intended application. If pure enzyme preparations are required, they are usually isolated by gel or ion exchange chromatography. Certain applications require solid enzyme products, so the crude powder enzymes are made into granules to make them more convenient to use. Sometimes liquid formulations are preferred because they are easier to handle and dose along with other liquid ingredients. Enzymes used in starch conversion to convert glucose into fructose are immobilised, typically on the surfaces of inert granules held
in reaction columns or towers. This is carried out to prolong their working life as these enzymes normally go on working for over a year.
Advantages of Submerged Technique
Measure of process parameters is easier than with solid-state fermentation.
Bacterial and yeast cells are evenly distributed throughout the medium.
There is a high water content which is ideal for bacteria.
High costs due to the expensive media
Large reactors are needed and the behaviour of the organism cannot be predicted
There is also a risk of contamination.
A TYPICAL LARGE SCALE MICROBIAL ENZYME PRODUCTION PROCESS
Recovery of the enzyme
It generally depends upon precipitation from an aqueous solution, although some enzymes may be marketed as stabilized solutions. In the bran process, the enzyme is extracted from the koj i (the name given to the mass of material permeated with the mold mycelium) into an aqueous solution by percolation. In the liquid processes, the microbial cells are filtered from the beer. The enzyme may be precipitated by addition of solvents, such as acetone or aliphatic alcohols, to the aqueous enzyme solution, either directly or after concentration by vacuum evaporation at low temperature. The precipitated enzyme may be filtered and dried at low temperature, for example in a vacuum shelf dryer. The dry enzyme powders may be sold as undiluted concentrates on a potency basis or, for most applications, may be diluted to an established standard potency with an acceptable diluent. Some common diluents are salt, sugar, starch, and wheat flour. Most commercial enzymes are quite stable in the dry form, but some require the presence of stabilizers and activators for maximum stability and efficiency in use. In theory, the fermentative production of microbial enzymes is a simple matter, requiring an appropriate organism grown on a medium of optimum composition under optimum conditions.
The stocks in trade of microbial enzyme manufacturers are thus the selected cultures, the composition of media, and the cultural conditions, all of which are usually held confidential. In practice, enzyme manufacturers suffer the samedifficulties in fermentation, frequently in even greater degree, as antibiotics producers. Total loss of fermentation batches may result from contamination, culture variation, failure of cultural control, and other like causes. Furthermore, knowledge and careful application of the best methods for recovery and stabilization
APPLICATIONS OF MICROBIAL ENZYMES IN INDUSTRIES
Detergents were the first large scale application for microbial enzymes. Bacterial proteinases are still the most important detergent enzymes. Some products have been genetically engineered to be more stable in the hostile environment of washing machines with several different chemicals present. These hostile agents include anionic detergents, oxidising agents and high pH.
Amylases are used in detergents to remove starch based stains. Amylases hydrolyse gelatinised starch, which tends to stick on textile fibres and bind other stain components. Cellulases have been part of detergents since early 90s. Cellulase is actually an enzyme complex capable of degrading crystalline cellulose to glucose. In textile washing cellulases remove cellulose microfibrils, which are formed during washing and the use of cotton based cloths. This can be seen as colour brightening and softening of the material. Alkaline cellulases are produced by Bacillus strains and neutral and acidic cellulases by Trichoderma and Humicola fungi.
Starch hydrolysis and fructose production
The use of starch degrading enzymes was the first large-scale application of microbial enzymes in food industry. Mainly two enzymes carry out conversion of starch to glucose: alpha-amylase cuts rapidly the large alpha-1,4-linked glucose polymers into shorter oligomers in high temperature. This phase is called liquefaction and is carried out by bacterial enzymes. In the next phase called saccharification, glucoamylase hydrolyses the oligomers into glucose. This is done by fungal enzymes, which operate in lower pH and temperature than alpha-amylase. Sometimes additional debranching enzymes like pullulanase are added to improve the glucose yield. Beta-amylase is commercially produced from barley grains and used for the production of the disaccharide maltose.
An alternative method to produce fructose is shown in Figure 4. This method is used in Europe and uses sucrose as a starting material. Sucrose is split by invertase into glucose and fructose, fructose separated and crystallized and then the glucose circulated back to the process.
Drinks And Brewing Industries
Enzymes have many applications in drink industry. The use of chymosin in cheese making to coagulate milk protein was already discussed. Another enzyme used in milk industry is beta-galactosidase or lactase, which splits milk-sugar lactose into glucose and galactose. This process is used for milk products that are consumed by lactose intolerant consumers. Enzymes are used also in fruit juice manufacturing. Fruit cell wall needs to be broken down to improve juice liberation. Pectins are polymeric substances in fruit lamella and cell walls. They are closely related to polysaccharides. The cell wall contains also hemicelluloses and cellulose. Addition of pectinase, xylanase and cellulase improve the liberation of the juice from the pulp. Pectinases and amylases are used in juice clarification.
Brewing is an enzymatic process. Malting is a process, which increases the enzyme levels in the grain. In the mashing process the enzymes are liberated and they hydrolyse the starch into soluble fermentable sugars like maltose, which is a glucose disaccharide. Additional enzymes can be used to help the starch hydrolysis (typically alpha-amylases), solve filtration problems caused by beta-glucans present in malt (beta-glucanases), hydrolyse proteins (neutral proteinase), and control haze during maturation, filtration and storage (papain, alpha-amylase and beta-glucanase).
The use of enzymes in textile industry is one of the most rapidly growing fields in industrial enzymology. Starch has for a long time been used as a protective glue of fibres in weaving of fabrics. This is called sizing. Enzymes are used to remove the starch in a process called desizing. Amylases are used in this process since they do not harm the textile fibres .The same effect can be obtained with cellulase enzymes. The effect is a result of alternating cycles of desizing and bleaching enzymes and chemicals in washing machines.
Laccases are produced by white-rot fungi, which use them to degrade lignin - the aromatic polymer found in all plant materials. Laccase is a copper-containing enzyme, which is oxidised by oxygen, and which in an oxidised state can oxidatively degrade many different types of molecules like dye pigments.
Pulp And Paper Industry
Intensive studies have been carried out during the last twenty years to apply many different enzymes in pulp and paper industry. The major application is the use of xylanases in pulp bleaching. Xylanases liberate lignin fragments by hydrolysing residual xylan. This reduces considerably the need for chlorine based bleaching chemicals. Other minor enzyme applications in pulp production include the use of enzymes to remove fine particles from pulp. This facilitates water removal. In the use of secondary (recycled) cellulose fibre the removal of ink is important. The fibre is diluted to 1% concentration with water, flocculating surfactants and ink solvents added and the mixture is aerated.
The ink particles float to the surface. There are reports that this process is facilitated by addition of cellulase enzymes. In paper making enzymes are used especially in modification of starch, which is used as an important additive. Starch improves the strength, stiffness and erasability of paper. The starch suspension must have a certain viscosity, which is achieved by adding amylase enzymes in a controlled process. Pitch is a sticky substance present mainly in softwoods. It is composed of lipids. It is a special problem when mechanical pulps of red pine are used as a raw material. Pitch causes problems in paper machines and can be removed by lipases. This facilitates water removal. In the use of secondary (recycled) cellulose fibre the removal of ink is important in the process
Baking Industry :-
Similar fibre materials are used in baking than in animal feed. It is therefore conceivable that enzymes also affect the baking process. Alpha-amylases have been most widely studied in connection with improved bread quality and increased shelf life. Both fungal and bacterial amylases are used. Overdosage may lead to sticky dough so the added amount needs to be carefully controlled. One of the motivations to study the effect of enzymes on dough and bread qualities comes from the pressure to reduce other additives. In addition to starch, flour typically contains minor amounts of cellulose, glucans and hemicelluloses like arabinoxylan and arabinogalactan. There is evidence that the use of xylanases decreases the water absorption and thus reduces the amount of added water needed in baking. This leads to more stable dough. Especially xylanases are used in whole meal rye baking and dry crisps common in Scandinavia.
Proteinases can be added to improve dough-handling properties; glucose oxidase has been used to replace chemical oxidants and lipases to strengthen gluten, which leads to more stable dough and better bread quality.
Various Important Microbial Enzymes
Carbohydrases are enzymes which hydrolyze polysaccharides or oligosaccharides. Several carbohydrases have industrial importance, but the amylases have the greatest commercial application. The various starch-splitting enzymes are known as amylases, the actions of which
may be expressed in greatly simplifiedform as follows:
The terms "liquefying" and "saccharifying" amylases are general classifications denoting the principal types of amylase action. f-Amylase, which is not of microbial origin, is a true saccharifying enzyme, forming maltose directly from starch by cleaving disaccharide units
from the open ends of chains. The a-amylases from different sources usually have good liquefying ability, but may vary widely in saccharifying ability and thermal
Bacterial amylase preparations generally remain operative at considerably higher temperature than do fungal amylases, and at elevated temperatures give rapid liquefaction of starch. A significant application of the bacterial enzyme is in the continuous process for desizing of textile fabrics Another is in preparing modified starch sizing for textiles and starch coatings for paper
High temperature stability is also important in the brewing industry where microbial amylases have found use in supplementing low diastatic malt, and especially for initial liquefaction of adjuncts such as rice and corn grits Additional specific uses for bacterial amylase is in preparing cold water dispersible laundry starches and in removing wall paper.
Fungal amylases possess relatively low thermal stability but act rapidly at lower temperatures and produce good saccharification. An enormous potential use for fungal amylase is as a saccharifying agent for grain alcohol fermentation mashes. At least two alcohol plants in this country regularly use fungal amylase for this purpose
An extremely important use for fungal amylases isin conversion of partially acid hydrolyzed starch tosweet syrups Amylases find extensive use in baking. Use of fungal amylase by the baker to supplement the diastatic activity of flour is common practice. The fungal amylase has the advantage of low inactivation temperature. This permits use of high levels of the amylase to improve sugar production, which increases gas formation and improves crust color, without danger of excessive dextrinization of the starch during baking
Other applications of microbial amylases where both fungal and bacterial enzymes are utilized are in processing cereal products for food dextrin and sugar mixtures and for breakfast foods, for preparation of chocolate and licorice syrups to keep them from congealing, and for recovering sugars from scrap candy of high starch content. Fungal amylases are also used for starch removal for flavoring extracts and for fruit extracts and juices, and in preparing clear, starch-free
pectin. Microbial amylases are used for modifying starch in vegetable purees, and in treating vegetables for canning
Industrially available proteolytic enzymes produced by microorganisms are usually mixtures of endopeptidases (proteinases) and exopeptidases. In addition to microbial proteases, the plant proteases bromelin, papain, and ficin, and the animal proteases, pepsin and trypsin, have extensive industrial application. Because of the complex structures and high molecular weights of proteins made up of some 20 different amino acids, enzymic proteolysis is extremely
complicated. Most proteases are quite specific with regard to which peptide linkages they can split
Hence, it is necessary to select the appropriate protease complex or combination of enzymes for specific applications. Usually this can only be determined by trial and error methods. By means of such experimentation, however, many and diverse uses have been found for the various proteases. With proper selection of enzymes, with appropriate conditions of time, temperature, and pH, either limited proteolysis or complete hydrolysis of most proteins to amino acids can be brought about.
Microbial proteolytic enzymes from different fungi and bacteria are available. Most fungal proteases will tolerate and act effectively over a wide pH range (about 4 to 8), while with a few exceptions, bacterialproteases generally work best over a narrow range of about pH 7 to 8.
Fungal protease has been used for centuries in the orient for the production of soy sauce, tamari sauce, and miso, a breakfast food After maximum enzyme production has taken place, the koji is covered with brine and enzymatic digestion allowed to take place. Limited use is made of this process for making soy sauce in this country also. In these uses, no attempt is made to separate the enzymes from the producing organisms. For most industrial applications, the microbial proteases are extracted from the growth medium as described in an earlier section of this paper.
One of the largest uses for fungal protease is in baking bread The proper amount of protease action reduces mixing time and increases extensibility of doughs, and improves grain, texture, and loaf volume. However, excess of protease must be avoided, and the time for enzyme action and quantity of enzyme used must be carefully controlled by the baker or sticky, unmanageable doughs will result.
Cereal foods are also treated with proteolytic enzymes to modify their proteins, resulting in better processing