Application of enzyme engineering technology in biopharmaceuticals
Enzyme engineering is a science that uses enzymes, enzyme-containing organelles or cells (microorganisms, animals, plants) in certain reaction devices, utilizes the biocatalytic function of enzymes, and transforms the corresponding raw materials into useful substances with the help of engineering means and uses them in social life. It includes the preparation of enzyme preparation, curing of enzyme, modification and transformation of enzyme and enzyme reactor. Its application is mainly concentrated in pharmaceutical industry, food industry and light industry.
1. Enzyme immobilization technology and its application
By embedding the enzyme in the gel, microcapsules, or through covalent bonds, ionic bonds adsorption connected to the solid-phase carrier, or through the cross-linking agent to make the enzyme molecules cross-linking each other and other methods to make the enzyme insoluble confined in a limited space of the technical process. This technique allows the enzyme to be used repeatedly in batch reactions, continuously in sequential reactions or the enzyme to be easily separated from the product. The methods of immobilization include basic methods such as adsorption, covalent binding, embedding, microencapsulation and cross-linking, as well as novel immobilization techniques such as crosslinked enzyme crystals, crosslinked enzyme aggregates, silica matrix embedding and lipid embedding [1]. The embedding method is more commonly used in the pharmaceutical field, followed by the adsorption method. A variety of immobilized enzymes have been used for large-scale industrial production, such as: aminoacylase, penicillin acylase, aspartoacylase, aspartate-β-decarboxylase.
2.Chemical modification of enzymes
Chemical modification of enzyme refers to the main chain of enzyme protein molecule by “cutting”, “shearing” and its chemical modification.
Chemical modification refers to the process of “cutting” and “shearing” the main chain of the enzyme protein molecule and its chemical modification, which is a technical process of combining certain chemical substances or groups to the enzyme molecule by chemical means to change the catalytic properties and functions of the enzyme. Through the chemical modification of enzyme, it can improve the activity of enzyme, increase the stability of enzyme, eliminate or reduce the antigenicity of enzyme and so on.
3. Non-aqueous phase catalysis of enzymes and directional evolution of enzymes
The technical process of enzyme catalytic reaction in non-aqueous medium (organic solvent medium, gas medium, supercritical fluid medium, ionic liquid medium, etc.) is called non-aqueous phase catalysis of enzyme [5]. Enzyme catalysis in nonaqueous media has the remarkable features of increasing the solubility of nonpolar substrates or products, carrying out synthetic reactions that cannot be carried out in aqueous solution, reducing the feedback inhibition of the products to the enzyme, and improving the substrate selectivity, group selectivity, regioselectivity, and enantioselectivity of asymmetric reactions of chiral compounds. Directed evolution of enzyme technology [5] is a simulation of the natural evolutionary process (natural random mutation and natural selection, etc.), artificial random mutation of genes in vitro, the establishment of mutant gene libraries, through the special environment of artificially controlled conditions, directed selection to obtain the enzyme with excellent catalytic properties of the mutant technological process.
4. Production and application of enzyme preparation
4.1 Production of enzymes
4.1.1 Nuclease and antibody enzyme
Ribonucleic acid enzyme is a class of ribonucleic acid (RNA) composed of enzymes, with a high degree of nucleic acid sequence specificity from the
and has a strong application value. As long as you know the nucleotide sequence of a certain nucleic acid enzyme, you can design and synthesize the nucleic acid composition that catalyzes its self-cleavage and breakage, and based on the entire sequence of these genomes, you can design and synthesize the prevention and treatment of . Nucleotides for human, animal and plant viral diseases caused by these viruses, such as the ability to combat influenza, hepatitis, AIDS and tobacco mosaic disease. Nucleases can also be used as tools to study nucleic acid mapping and gene expression [4]. Antibody enzymes, also known as catalytic antibodies, are a class of antibody molecules with biocatalytic functions that can be obtained by induction and modification methods. Antibody enzymes have been used in the study of enzyme action mechanism, synthesis and disassembly of chiral drugs, preparation of anticancer drugs, etc.
4.1.2 Enzyme-labeled drugs
Recently, it has become possible to design drugs based on their possible targets of action in the organism (e.g., enzymes or receptors), and the resulting drugs are called enzyme-labeled drugs. This design approach is now known as the mainstream of drug design and plays a great role in the design of new drugs. Angiotensin peptide converting enzyme (ACE) inhibitors are a successful example of enzyme-labeled drugs, and ACE inhibitors have become important and commonly used antihypertensive drugs. Recent studies have found that HIV infection and transmission is mainly caused by proteases on the surface of HIV particles. Therefore, the study of HIV protease has become a hotspot, and it is hoped that the study of HIV protease inhibitors will lead to the search for ways to prevent HIV infection and treat AIDS.
4.2 Application of enzyme engineering technology in pharmaceutical process
Enzyme engineering technology in the production of small investment, process simplicity, low energy consumption, high product yield, high efficiency, high efficiency and low pollution and other advantages, become the main force in the application of chemical and pharmaceutical industries. In the past, chemical synthesis, microbial fermentation and extraction of biological materials and other traditional technologies to produce drugs, can be produced by modern enzyme engineering. Even expensive drugs that are impossible to obtain by traditional technology, such as human insulin, 6-APA and 7-ADCA, can be obtained. Immobilized genetically engineered bacteria, engineered cells and the clever combination of immobilization technology and continuous bioreactor will lead to fundamental changes in the entire fermentation industry and chemical synthesis industry.
4.2.1 Application of enzyme engineering to prepare biological metabolites
The application of immobilized cells can produce a variety of primary metabolites or intermediates in large quantities, such as sugar, organic acids and amino acids. The products are D-fructose, glycerol, 1,6-diphosphate fructose, citric acid, malic acid, alanine, aspartic acid, phenylalanine, tryptophan, lysine and so on.
4.2.2 Application of enzyme engineering to produce antibiotics and vitamins
Application of enzyme engineering can prepare cephalosporin Ⅳ (cephalosporin acylase), 7-ADCA (penicillin V acylase), deacetyl cephalosporin (cephalosporin acetate lyase). In recent years also immobilized production of Penicillium flavum (penicillin synthetase system) cell production of penicillin research, the synthesis of penicillin and cephalosporin precursors of the latest process is also used in enzyme engineering.
4.2.3 Application of enzyme engineering production of amino acids and organic acids
Production of DL-amino acids (amino acylase), L-lysine (diaminoheptanoic acid dehydroxylase or α-amino-ε-caprolactam hydrolase and racemization enzyme), uric anhydride acid (L-histidine aminolysis enzyme), L-tyrosine and L-dopa (β-tyrosinase) and other organic acids.
4.2.4 Application of enzyme engineering production of nucleotide drugs
Adenine nucleotides (AMP) by the protein-producing Pseudomonas aeruginosa extracted nucleic acid with hot water, and then hydrolyzed by nuclease. Deoxyribonucleotides are produced by extracting deoxyribonucleic acid (RNA) from fish white, followed by enzymatic hydrolysis by 5′-phosphodiesterase. Existing nucleic acid-rich plants and animals (pollen, etc.) extracted ribonucleic acid (RNA), and then 5′-phosphodiesterase enzyme digestion to phosphoryl glycoside (AMP), phosphoryl cytidine (CMP), phosphoryl uridine (UMP) and phosphoryl uridine (GMP) to produce a mixture of nucleotides. Inosinic acid was produced by acyloside deaminase.ATP and AMP were produced by carbamoylphosphate kinase, kinase plus acetate kinase, respectively.
5.Prospect of Enzyme Engineering Technology for Pharmaceuticals
As an important part of bioengineering, enzyme engineering has been recognized by the world for its important role and significant research results. To give full play to the catalytic function of enzyme, to expand the application range of enzyme and to improve the application efficiency of enzyme is the main goal of enzyme engineering application research. 21st century enzyme engineering development theme is: research and development of new enzyme, optimal production of enzyme and high efficiency application of enzyme. In addition to the commonly used technologies, we should also take advantage of the latest knowledge of genetics and proteomics, DNA rearrangement and cellular, phage surface display technology for the research and development of new enzymes, the most impressive new enzymes are nucleic acid enzymes, antibody enzymes and telomerase and so on. Immobilization, molecular modification and non-aqueous phase catalysis are to be used to realize the efficient application of enzymes, and the curing technology is to be widely applied to biochips, biosensors, bioreactors, clinical diagnostics, drug design, affinity chromatography as well as the study of protein structure and function, so as to enable enzyme technology to play a greater role in the field of pharmaceuticals.
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| Compound Glucoamylase | 9032-08-0 |
| Pullulanase | 9075-68-7 |
| Xylanase | 37278-89-0 |
| Cellulase | 9012-54-8 |
| Naringinase | 9068-31-9 |
| β-Amylase | 9000-91-3 |
| Glucose oxidase | 9001-37-0 |
| alpha-Amylase | 9000-90-2 |
| Pectinase | 9032-75-1 |
| Peroxidase | 9003-99-0 |
| Lipase | 9001-62-1 |
| Catalase | 9001-05-2 |
| TANNASE | 9025-71-2 |
| Elastase | 39445-21-1 |
| Urease | 9002-13-5 |
| DEXTRANASE | 9025-70-1 |
| L-Lactic dehydrogenase | 9001-60-9 |
| Dehydrogenase malate | 9001-64-3 |
| Cholesterol oxidase | 9028-76-6 |