BIOTECHNOLOGY - V. H. Gerasymenko - 2006

Part II. Special Biotechnologies

Chapter 20. BIOTECHNOLOGY OF ENZYME PRODUCTION

20.1. SOURCES OF ENZYMES

Today, the industry produces about 20 individual Enzymes and nearly 40 so-called technical enzyme preparations (Table 20.2). These are mixtures containing, alongside the target enzyme, a significant amount of Proteins with similar physicochemical properties. Separating these proteins often requires labor-intensive and costly chromatographic Methods, which dramatically increases the final product's cost. Therefore, pure enzymes find limited application primarily in medicine and scientific research, whereas Industrial processes typically rely on enzyme preparations.

Class="center">Table 20.2.

Assortment of technical enzyme preparations

(according to M. V. Hernet, A. M. Egorov, 1982)

The sources of enzymes include animal, plant, and microbial Tissues. Although certain animal tissues—such as the Pancreas or gastric mucosa—are rich in enzymes, large-scale production of enzymes of animal and plant origin is unfeasible due to raw material limitations. For instance, in the former Soviet Union, cheese production required an average of 250 tonnes of Proteolytic Enzymes (rennet) annually, the isolation of which demanded 10 million calf stomachs.

Furthermore, animal and plant tissues have stringent cultivation requirements, which significantly increases the cost of enzyme production.

Among the enzyme preparations isolated from animal and plant raw Materials are a complex enzyme preparation containing amylase, lipase, proteinases, Ribonuclease, etc., obtained from pancreatic extract; Papain from the juice of the papaya tree Carica papaya; bromelain from pineapple stems; and ficin from the juice or leaves of trees of the genus Ficus.

The most accessible and practically inexhaustible source for obtaining enzymes on an industrial scale is microorganisms: prokaryotes (Bacteria, actinomycetes, rickettsia) and some eukaryotes (Yeasts, filamentous Fungi, Algae, Protozoa). They contain enzymes of all currently known types, and modern microbiological methods allow for The production of large quantities of enzymes in a short time.

Microbiological production of enzymes has significant advantages over tissue-based methods. First of all, microorganisms reproduce extremely rapidly. In some species, Cell Division occurs every hour or even every ten minutes. Consequently, a small initial amount of bacteria can yield a substantial microbial biomass within a few days—rates that are completely unattainable with plants and animals.

Secondly, microorganisms are capable of growing on relatively simple and inexpensive nutrient media, which are often byproducts of other manufacturing processes. Microorganisms can utilize CARBOHYDRATES, alcohols, and carboxylic acids as carbon sources. Nitrogen sources can be of three types: organic (proteins, Peptides, Amino Acids), Mineral Substances (ammonium salts, ammonia), and atmospheric nitrogen. In industrial media, various grades of starch (corn, potato, rice), corn steep liquor, soybean meal, Yeast biomass hydrolysates, etc., are most commonly used as sources of organic carbon and nitrogen. The Nutritional Requirements of microorganisms for macro- and microelements are met using Salts of inorganic acids.

Thirdly, an important feature of microbial enzyme production is that each microorganism contains a wide range of enzymes. This allows the same microbial species to be used as a producer for various enzymes. By applying techniques such as the induction of enzyme Biosynthesis, the production of a specific enzyme by a given microbial species can be substantially increased. The Essence of this method is that introducing specific substances—Inducers (often specific substrates of the target enzymes) into the nutrient medium alters cellular metabolic pathways, prompting the preferential Synthesis of the desired enzyme.

The rate of enzyme biosynthesis by microorganisms is quite high. However, the productivity of producer strains can be increased 2-to-5-fold through Selection, optimization of cultivation conditions, and the isolation of high-yielding mutant microbial strains. Using recombinant DNA technologies, it is possible to obtain microbial strains in which enzymes account for up to 50% of the total synthesized protein mass. All of this has made it possible to dramatically increase the production of enzyme preparations and reduce enzyme costs by approximately 100 to 1000 times.

For instance, combining mutagenesis and selection methods with Introduction/32.html">Genetic Engineering allowed Japanese researchers to achieve a 200-fold increase in the yield of α-amylase from Bacillus subtilis. When an enzyme hydrolyzes starch at higher temperatures, the production of sugars accelerates. Therefore, the goal was set to increase the production of thermostable α-amylase. Japanese scientists introduced the Gene for this enzyme from a thermophilic bacterium into the hay bacillus, resulting in a significant boost in thermostable α-amylase yield.

Most enzymes synthesized by microorganisms are extracellular products released into the surrounding liquid medium—these are extracellular enzymes. The mycelium of a three-day culture typically retains no more than 10-15% of the enzymes. Sometimes, enzymes are not secreted into the external environment but remain bound to cellular Organelles, functioning as intracellular enzymes. Extracellular enzymes are predominantly Hydrolases.

Enzymes can be inducible or constitutive. Inducible enzymes are those synthesized by microorganisms only in the presence of specific substances or inducers in the nutrient medium. For example, the enzyme lactase can be synthesized only when lactose is present in the medium. Most hydrolytic enzymes belong to the inducible category, and The biosynthesis of most of them is inhibited by glucose.