Prospect: Enzyme, as a biocatalyst, has been widely used in various production fields of light industry. In recent decades, with the continuous technological breakthrough of enzyme engineering, it has been more and more widely used in industry, agriculture, medicine and health, energy development and environmental engineering. Recombinant DNA technology has facilitated the mass production of various enzymes with medical value. The variety of enzymes used in clinic gradually increased. In addition to being used as a conventional treatment, enzymes can also play a medical role as some components of medical engineering. For example, in the extracorporeal circulation device, enzymes are used to remove blood waste, prevent thrombosis and the enzyme controlled drug release system in the body. In addition, enzymes, as clinical in vitro detection reagents, can quickly, sensitively and accurately determine some metabolites in the body, which will also be an important application of enzymes in medical treatment.
1. Enzyme immobilization technology and its application
A technical process in which enzyme insolubility is confined in a limited space by embedding enzymes in gels or microcapsules, or by covalent or ionic adsorption to solid phase carriers, or by cross-linking enzyme molecules with cross-linking agents. The technique allows the enzyme to be used repeatedly in batch reactions, continuously in continuous reactions or easily separated from the product. The immobilization methods include adsorption method, covalent binding method, embedding method, microencapsulation method and cross-linking method, etc. There are also new immobilization technologies such as cross-linked enzyme crystals, cross-linked enzyme aggregates, silicon matrix embedding method and lipid embedding method. In the pharmaceutical field, the embedding method is more used, followed by adsorption method. At present, many immobilized enzymes have been used for large-scale industrial production, such as: aminoacylase, penicillin acylase, aspartase, aspartate-β-decarboxylase.
2. Chemical modification of enzymes
The chemical modification of enzyme refers to the "cutting", "cutting" and chemical modification of the main chain of enzyme protein molecules, which is a technical process that uses chemical means to bind some chemical substances or groups to the enzyme molecules to finally change some catalytic characteristics and functions of the enzyme. Enzyme chemical modification can improve enzyme activity, increase enzyme stability, eliminate or reduce enzyme antigenicity and so on.
3. Non-aqueous catalysis of enzymes and directed evolution of enzymes
The technical process of enzyme catalytic reaction in non-aqueous media (organic solvent medium, gas medium, supercritical fluid medium, ionic liquid medium, etc.) is called non-aqueous phase catalysis of enzyme. Enzymatic catalysis in non-aqueous media has significant characteristics such as improving the solubility of non-polar substrates or products, conducting synthetic reactions that cannot be carried out in aqueous solutions, reducing the feedback inhibition of products on enzymes, and improving the substrate selectivity, group selectivity, region selectivity, and enantiomeric selectivity of asymmetric reactions of chiral compounds. Enzyme directed evolution technology is a technical process that simulates the natural evolution process (natural random mutation and natural selection, etc.), carries out artificial random mutation of genes in vitro, establishes mutant gene library, and obtains mutant enzymes with excellent catalytic properties through directional selection in a special environment under artificial control conditions.
4. Production and Application of enzyme preparations
4.1 Production of enzyme preparations
4.1.1 Nucleases and abases
Nucleases are a class of enzymes composed of ribonucleic acid (RNA), which have a high specificity of nucleic acid sequences and thus have strong application value. As long as the nucleotide sequence of a nuclease is known, it can be designed to synthesize nucleic acid components that catalyze its self-cutting and fragmentation, and according to the full sequence of these genomes, prevention and control can be designed and synthesized. Nucleotides of human, animal and plant viral diseases caused by these viruses, such as influenza, hepatitis, AIDS and tobacco Mosaic. Nucleases can also be used as tools to study nucleic acid mapping and gene expression. Antibody enzymes, also known as catalytic antibodies, are a class of antibody molecules with biocatalytic function, which can be obtained by induction and modification. Abases have been used in the study of enzyme mechanism, synthesis and resolution of chiral drugs, preparation of anticancer drugs and so on.
4.1.2 Enzyme-labeled drugs
Recently, people can design drugs according to the possible target of action in the organism (such as enzymes or receptors), and the resulting drugs are called enzyme-labeled drugs. At present, this design method has been called the mainstream of drug design, and has played a huge 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 are mainly caused by protease on the surface of HIV particles. Therefore, the study of HIV protease has become a hot spot, people hope that the study of HIV protease inhibitors, to find out the prevention of HIV infection and treatment of AIDS.
4.2 Application of enzyme engineering technology in pharmaceutical process
Enzyme engineering technology has the advantages of small production investment, simple process, low energy consumption, high product yield, high efficiency, large benefit and little pollution, and has become the main force in chemical and pharmaceutical industry applications. Drugs produced by traditional techniques such as chemical synthesis, microbial fermentation and biological material extraction can all be produced by modern enzyme engineering. It is even possible to obtain expensive drugs that are impossible to obtain with traditional technology, such as human insulin, 6-APA and 7-ADCA. The immobilization of genetically engineered bacteria, engineered cells and the ingenious combination of immobilization technology and continuous bioreactor will lead to fundamental changes in the whole fermentation industry and chemical synthesis industry.
4.2.1 Preparation of biological metabolites by enzyme engineering The application of immobilized cells can produce a large number of primary metabolites or intermediate products, such as sugars, organic acids and amino acids. The products are D-fructose, glycerin, 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), deacetylated cephalosporin (cephalosporin acetoesterase). In recent years, the production of penicillin by immobilized penicillium flavus (penicillin synthetase line) cells has also been studied, and the latest technology of synthesis of penicillin and cephalosporin precursors has also adopted the method of enzyme engineering.
4.2.3 Application of enzyme engineering to produce amino acids and organic acids
Production of organic acids such as DL-amino acid (aminoacylase), L-lysine (diaminoheptyl dehydroxylase or α-aminoε-caprolactam hydrolase and racemase), urinary anhydride (L-histidine aminohydrolyase), L-tyrosine and L-dopa (β-tyrosinase).
4.2.4 Application of enzyme engineering to produce nucleotide drugs
Adenine nucleotide (AMP) is prepared by extracting nucleic acid from protein-producing candida bacteria with hot water and then hydrolysis by nuclease. Deoxynucleotides were extracted from fish white by deoxyribonucleic acid (RNA) and enzymolysis by 5 '-phosphodiesterase. Ribonucleic acid (RNA) was extracted from existing nucleic acid-rich plants and animals (pollen, etc.), and then enzymolized into AMP, cytidine phosphate (CMP), uridine phosphate (UMP) and uridine phosphate (GMP) by 5 '-phosphodiesterase to obtain mixed nucleotides. Inosine acid is prepared by acylside deaminase. ATP and AMP are prepared by carbamyl phosphokinase, kinase plus acetate kinase, respectively.
5. The prospect of enzyme engineering technology Pharmaceutical Enzyme engineering as an important part of bioengineering, its important role and remarkable research results have been recognized by the world. Giving full play to the catalytic function of enzymes, expanding the application range of enzymes and improving the application efficiency of enzymes are the main goals of enzyme engineering application research. The development theme of enzyme engineering in the 21st century is: the research and development of new enzymes, the optimization of enzyme production and the efficient application of enzymes. In addition to the use of commonly used technologies, but also with the latest knowledge of genetics and proteomics, with the help of DNA rearrangement and cell, phage surface display technology for the research and development of new enzymes, the most eye-catching new enzymes are nucleic acid enzymes, antienzymes and telomerase. Immobilization, molecular modification and non-aqueous catalysis are used to realize the efficient application of enzymes, and solidification technology is widely used in biochips, biosensors, bioreactors, clinical diagnosis, drug design, affinity chromatography and protein structure and function research, so that enzyme technology can play a greater role in the pharmaceutical field.
