Protease and Enzyme Engineering

1. Protein engineering: based on the study of the relationship between protein structure and function, the use of genetic engineering technology or chemical modification technology to transform existing proteins into new proteins of modern biotechnology.
2. enzyme engineering: the use of enzymes, organelles or cell-specific catalytic function, through the appropriate reactor industrialized production of human products or to achieve a particular purpose of a technical science.
3. The main contents of enzyme engineering research: 1) chemical enzyme engineering 2) biological enzyme engineering 3) immobilized enzymes and cells 4) enzyme reactors and sensors 5) non-aqueous phase catalysis of enzymes
4. Protein fusion: recombination of part of the gene coding for one protein to another protein gene, or combining fragments of different protein genes together to produce new fusion proteins by gene cloning and expression.
5. The role of protein fusion: 1) for the isolation and purification of the expression product; 2) to improve the solubility of the expression product; 3) to improve protein stability.
6. Protein crystallography: the engineering of the structural study of biological macromolecules using X-ray diffraction technology, an important part of structural biology.
8. targeted mutation: the local nucleotide sequence of a specific gene is altered in a targeted manner by means of molecular cloning, which is usually used to study the functional structure of proteins as well as for the modification of target proteins.
10. Research scope of enzyme engineering:
1) Development and production of various types of natural enzymes;
2) Enzyme isolation and purification and identification techniques;
3) Immobilization techniques;
4) Cross-pollination using other fields of biotechnology;
5) development and application of multi-enzyme reactors.
11. Stability and stabilization of enzymes:
(i) Causes of enzyme inactivation:
(1) Some specific amino acid residues in the active center of the enzyme are chemically modified, so that the enzyme activity is lost (microscopic);
(2) Influence of the external environment, spatial barriers in the active center of the enzyme, so that it can not bind to the substrate;
3) changes in the higher structure of the enzyme (changes in helix and folding);
4) breakage of the polypeptide chain (very strong);
(ii) Stabilization of the enzyme:
(1) low temperature preservation (the enzyme itself is not denatured, not easy for other enzymes to degrade the target protein);
2) Addition of salts (high concentration of (NH4)2SO4);
3) Addition of ligands such as substrate coenzymes;
4) addition of strong denaturants (to protect the primary structure and revive it when used);
5) crystallization.
12. Superiority of microorganisms as enzyme sources:
1) Easy access to enzymes needed for enzymes;
2) easy access to high yielding strains;
3) short production cycle;
4) low cost of production;
5) easy management of production;
6) More ways to improve microbial enzyme production.

13. Immobilized enzyme: It refers to the enzyme that exists in a certain space in a blocked state, which can continuously carry out the reaction, and the enzyme can be recovered and reused after the reaction.
14. Advantages of immobilized enzyme:
1) It is extremely easy to separate the immobilized enzyme from the product and substrate;
2) Can perform repeated batch reactions and loaded columns in a continuous reaction over a long period of time;
3) the ability to increase the stability of the enzyme in most cases;
4) the reaction process of the enzyme can be strictly controlled;
(5) No enzyme residue in the product solution, simplifying the purification process;
6) It is more suitable for multi-enzyme reaction than free enzyme;
7) the yield of the product can be increased and the quality of the product can be improved; 8) the efficiency of enzyme use is improved and the cost is reduced.
15. Disadvantages of immobilized enzyme:
1) There is a loss of enzyme activity when immobilized;
2) Increased cost of production and large initial investment in the plant;
3) can only be used for soluble substrates and is more suitable for small molecule substrates;
4) Not suitable for multi-enzyme reactions compared to intact bacteriophage, especially those requiring cofactors;
5) the separation procedures that intracellular enzymes must undergo.
16. Principles of preparation of immobilized enzymes:
1) Care must be taken to maintain the catalytic activity and specificity of the enzyme;
2) Immobilization should be conducive to automation and continuity of production;
(3) The immobilized enzyme should have the smallest spatial site resistance, as far as possible not to hinder the proximity of coal and substrate, in order to improve the product yield;
4) The enzyme and carrier must have a strong binding force so that the immobilized enzyme can be recovered and the storage facilitates repeated use;
(5) The immobilized enzyme should have maximum stability, and the selected carrier should not react chemically with the waste product or reaction solution;
(6) immobilized enzyme cost should be low, conducive to industrial use.
17. Methods of enzyme immobilization:
(i) Non-covalent binding method:
(1) crystallization method: applicable to enzymes with low enzyme activity, the concentration changes after crystallization, and there is no loss in constant continuous use;
(2) decomposition method: the enzyme is made into a dry powder, which is dispersed in the insoluble phase in water, even if the dry powder is suspended in the solvent, advantage: recovery is more convenient, disadvantage: the dry powder is easy to absorb water, the particles become larger, the activity is reduced, and the enzyme vitality in organic solvents will be affected;
(3) Physical adsorption: a method in which the enzyme is physically adsorbed on an insoluble carrier; Advantage: the enzyme active center is not easily destroyed, fewer changes in the senior structure, less loss of enzyme activity, also suitable for immobilized cells; Disadvantage: weak interaction between the enzyme and carrier, the enzyme is easy to fall off;
(4) Binding to the water-soluble carrier through ionic bonding; Advantages: simple operation, mild conditions, the senior structure and active center can not be destroyed, also applicable to immobilized cells; Disadvantages: the carrier and the enzyme binding force is weaker, anionic or cationic buffer, the ionic concentration of the influence of the larger, the enzyme is more prone to fall off from the carrier;
(ii) Chemical binding method:
(1) covalent binding method: the enzyme and carrier covalent binding; method: the carrier-related gene activation, and then coupling reaction with the enzyme-related genes, in the carrier binding is relatively strong, generally will not be fixed substrate concentration changes and change; disadvantages: reaction conditions are more intense, often lead to changes in the high-level structure, destruction of the active center, is only applicable to the immobilization of the enzyme, is not suitable for the immobilization of the cell;
(2) Cross-linking method: on the use of multifunctional reagents or bifunctional reagents, so that the enzyme and enzyme or microorganisms and microbial cells cross-linking immobilization method, generally by reducing the concentration of cross-linking agent and the reaction time to maintain enzyme activity;
(iii) Embedding method:
(1) grid type: the enzyme or microorganisms embedded in a fine grid of polymer gel, this method is the immobilization of microbial cells with more methods; Advantages: do not need to combine with the enzyme protein reaction, the enzyme activity recovery rate is high;
(2) microcapsule type: the enzyme molecule is encapsulated in a capsule, the polymer membrane is semi-permeable, this capsule is impermeable and can exist in some anhydrous organic phase.
18. Properties of immobilized enzymes:
(i) Change in enzyme activity after immobilization:
Causes: 1) The spatial conformation of the enzyme molecule changes during immobilization, even affecting the amino acids in the active center;
The spatial freedom of the enzyme molecule is restricted after immobilization, which directly affects the vacuolar effect of the active center on the substrate;
The internal diffusion resistance makes the approach of substrate molecules to the active center resisted;
When embedded, the enzyme is surrounded by a polymer semipermeable membrane, and the macromolecular substrate cannot approach the enzyme through the membrane;
Effect of immobilization on enzyme stability:
Thermal stability increases;
2) Increased stability to various organic reagents and enzyme reagents;
(3) Stability to different pH values, the stability of protease, storage stability and operational stability have an impact;
(4) The reasons for the increased stability of immobilized enzyme: immobilized enzyme and carrier can be connected at multiple points, which can prevent the protease molecule from stretching and deformation; the enzyme’s vitality can be relieved after immobilization and released; the enzyme’s self degradation can be inhibited;
(iii) The optimum temperature changes —- increases;
(iv) Optimum pH change: range widening;
(v) Change in Km (change in Mie constant —- becomes smaller, affinity increases).
19.Methods of immobilization:
1) Co-immobilization of coenzyme and enzyme in the same carrier results in a system that is permanently free of additional coenzyme;
2) immobilize the coenzyme directly on the enzyme molecule.
20. cell immobilization: cells that are restricted from free movement, i.e., cells are constrained or limited to certain spatial boundaries by physical, chemical and other factors, but the cells still retain catalytic activity and have the viability to be used repeatedly and continuously.
1. Advantages and disadvantages of cell immobilization:
Advantages: 1) Immobilized cells maintain the original state and natural environment of the intracellular enzyme system, and thus are more stable;
Maintain the original multienzyme system in the cell, for multi-step catalytic advantage is more obvious, do not need coenzyme regeneration;
More obvious advantages for immobilized proliferating cell fermentation; high density of immobilized cells, can proliferate, shorten the fermentation production cycle; good fermentation stability, can be repeated for a longer period of time for continuous use; the fermentation broth contains fewer organisms is conducive to the isolation and purification of the product to improve the quality of the product;
Disadvantages: 1) The presence of a variety of enzymes in the cell will form unwanted by-products;
The presence of cell membranes, cell walls, and also carriers can form a diffusion limitation;
3) the size of the pores formed by the carrier affects the permeability of the polymer substrate.
2. chemical modification: where the covalent structure of a protein is altered by the inclusion or removal of a chemical gene, we refer to this phenomenon as chemical modification.
3. Factors affecting the functional group reactivity of proteins: 1) polarity of microregions: 2) hydrogen bonding effect; 3) electrostatic effect; 4) site-blocking effect.
4. Enzyme protein functional level hyper-reactivity: refers to a protein side chain gene and individual reagents can occur rapid reaction.
5. Factors affecting hyper-reactivity: 1) alteration of the pK value of the protein function; 2) greater reactivity of the functional group of the protein; 3) attraction of the reagent by electrostatic interactions and proper orientation; 4) stereochemical adaptations between the reagent and protein regions close to the modification site.
6. Determinants of modifier reactivity: 1) selective adsorption; 2) electrostatic interactions; 3) site-blocking factors; 4) catalytic factors: 5) microregion polarity (polarity of the local environment).
7. Control of modification reaction specificity:
(i) Selection of reagents:
(1) There are several cases of modification of amino acids:
a. Modification of all amino groups without modifying other genes;
b.Modification of alpha amino group by counter modification;
c. modification of the amino group with catalytic activity; d. change the charged state and solubility of proteins, change the charged state of proteins to choose reagents that can carry the maximum charge under neutral conditions, change the solubility of proteins the reaction is carried out in water, the choice of chemical reagents for the water-soluble; 3) quantitative determination of the reaction product; 4) consideration of the size of the reagent: the choice of reagent size is smaller to facilitate the modification, without cause large changes in the conformation of the protein;
Selection of reaction conditions:
The reaction conditions will not cause irreversible denaturation of the protein;
The choice of reaction conditions is conducive to the specific modification of proteins;
(iii) reaction specificity: 1) can use the specificity of some genes in the protein; 2) choose different reaction pH; 3) use some product instability; 4) affinity labeling; 5) differential labeling: in the system when there are enzyme molecules, substrates, inhibitors; 6) use of the difference in protein state.
8. Affinity reagent: also known as site-specific inhibitors, the reagent acts on a gene at the site being acted upon, and does not interact with other genes outside of the site being acted upon, and this type of modifier is called an affinity reagent.
9. Affinity labeling: affinity reagents generally have a similar structure to the substrate, the active site of the enzyme has a high degree of affinity for the active site of the amino acid residues can be covalently labeled, this type of chemical modification of the specificity of the labeling of the affinity, also known as the specificity of irreversible inhibition.
10. immobilized enzyme: on a certain space, in a closed state, continuous action can occur, and finally recycled.
11. how immobilization affects enzyme stability through the following three effects: 1) creates a spatial barrier; 2) creates a diffusion restriction; 3) multipoint covalent linkage.
12. methods of stabilization:
(i) immobilization (chemical binding, embedding method, etc.): 1) generating spatial barriers: inhibiting chemical inactivation; 2) generating diffusion limitation: embedding the enzyme inside the porous particles, where the substrate contacts the surface of the porous particles before diffusing into the interior to interact with the enzyme, uncontrolled by the concentration of the substrate; 3) multipoint covalent linkage: linking the enzyme multipointly and covalently to the surface of the carrier, or cross-linking the enzyme with a bifunctional reagent or lowering the enzyme to be encapsulated in the carrier The tight pore can make the enzyme conformation more solid, thus preventing the enzyme conformation from folding state to stretching state excessively.
13. Ribonuclease: It is a description of RNA with catalytic activity, whose chemical nature is that ribonucleic acid has the catalytic function of an enzyme, and the substrate can be a different molecule or some parts of the same RNA molecule.
14. natural nucleases: (a) shear-type nucleases (catalyze the cutting of self and heterogeneous RNA, nucleic acid endonuclease): 1) hammerhead-type nuclease; 2) hairpin-type nuclease; 3) protein-RNA complex enzyme; (b) splicing-type nuclease: including group Ⅰ intron and group Ⅱ intron; to achieve self-scissoring of RNA; with the activity of nucleic acid endonuclease and ligase.
15. In vitro selection: starting from a large-capacity random molecular library constructed from randomly ordered RNA or DNA molecules, in which a very small number of molecules with specific functions are screened.
16. Aptamer: RNA or DNA fragments that can bind specifically and efficiently to ligands such as organic substances or proteins are called RNA aptamers or DNA aptamers, respectively.
17. Screening aptamer program: 1) chemically synthesize a library of DNA molecules, in a position on the molecular chain to introduce a completely random or partially mutated order, the ends of the molecule is a fixed order, in order to PCR amplification; 2) after a few rounds of PCR amplification, in vitro transcription to form a library of randomly ordered RNA; 3) these RNA molecules through the combination of target molecules affinity chromatography columns, according to the RNA and target molecule binding ability size will be distinguished, binding strong RNA molecules were finally eluted down; 4) eluted RNA molecules after reverse transcription, PCR amplification, transcription, in the next screening cycle, after 5 to 10 cycles, to obtain the library enriched with target molecules with high affinity of RNA molecules.
18. Nuclease screening process: 1) by transcribing a random sequence of RNA to construct a random library of DNA; 2) random library with catalytic activity molecules can catalyze the substrate and carry out covalent ligation; 3) the reaction product through the immobilized in the substrate RNA 5 ‘end of the sequential complementary pairing of oligonucleotide affinity columns, selective adsorption of the random library of those who can catalyze the RNA molecules in the ligation reaction; 4) After high salt elution: reverse transcription, PCR amplification, transcription, and entry into the next round of screening; 5) In the next screening cycle, mutations are introduced into the active molecules at a certain frequency by error-prone PCR, which increases the molecular polyselectivity of the random library obtained by screening; 6) After several rounds of screening, RNA molecules with catalytic activity are enriched, and the relatively less active molecules are eliminated.
19. deoxyribonuclease: a single-stranded DNA fragment with catalytic function synthesized using in vitro molecular evolution techniques has efficient catalytic activity and structure recognition.
1. In vitro selection method: 1) synthesize DNA molecules artificially without transcription and reverse transcription, and directly carry out PCR amplification; 2) obtain single-stranded DNA molecules: PCR amplification connects a biotin to the primer, and the PCR product is passed through the affinity column of the biotin protein to realize the separation of positive and negative strands; 3) introduce divalent metal ions as cofactors; 4) introduce some additional functional groups into the DNA in order to target these deoxyribonucleases. additional functional groups into the DNA to increase the structural and functional affinity.
2. Classification of deoxyribonucleases: (deoxyribonucleases that cleave RNA) (deoxyribonucleases that cleave DNA) (deoxyribonucleases with kinase activity) (deoxyribonucleases with ligase function) (catalyze porphyrin cyclohexyl metal chelation reaction)
3. antisense nucleic acid: a DNA or RNA molecule that binds to an mRNA molecule, forming a spatial site barrier that prevents it from binding to the ribosome and thus translating into a protein in addition to degrading the mRNA.
4. rational design of enzyme molecules: study of natural enzymes or actually mutability using various methods of biochemistry, crystallography, spectroscopy, etc. to obtain enzyme molecular characteristics. Spatial structure. The relationship between structure and function, as well as amino acid residues and other information, and then use this as the basis for enzyme modification.
5. Non-rational design of the enzyme molecule: without the need for accurate information on the structure of the enzyme molecule, the enzyme molecule is transformed by random mutation, genetic recombination, blank screening and other methods, and the mutant enzyme of the required nature is selected directionally.
6. Directed evolution of enzyme molecule: that is, the development direction of enzyme molecule; it is to start from one or more existing parental enzymes (natural or artificially obtained), to construct an artificial mutant enzyme library through mutation or recombination of genes, and to ultimately obtain the evolved enzyme with certain characteristics of the leading expectations through screening. Directed evolution = random mutation + forward recombination + selection (or screening).
7. Error-prone PCR: When using Taq enzyme for PCR amplification of the target gene, the mutation frequency of Taq enzyme is changed by adjusting the reaction conditions, such as increasing the concentration of Mg2+, adding Mn ions, changing the concentration of dNTP in the system, etc., so as to randomly introduce mutations to the target gene at a certain frequency to construct a mutation library, and then selecting or screening for the required mutants.
8. Continuous error-prone PCR: Useful mutated genes obtained from one PCR amplification as templates for the next PCR amplification, and carry out random mutagenesis continuously and repeatedly, so that the small mutations after each time will accumulate and produce important intentional mutations.
9. DNA reorganization: Combine the positive mutations that have been obtained in different genes to form a new mutation gene pool, also known as sexual PCR.
10. Operation of DNA reorganization: DNA fragments isolated from the positive mutation gene pool are randomly cut by deoxyribonuclease Ⅰ to obtain random fragments, after several PCR cycles without primers, in the process of PCR cycle, the random fragments are used as each other’s templates and primers for amplification, until the full-length genes are obtained, which leads to recombination between fragments from different genes, and recombination of intentional mutations in the parents. Undertake recombination.

11. Staggered extension method: In the PCR reaction, the conventional annealing and extension are combined into one step and its reaction time is greatly shortened so that only a very short nascent chain can be synthesized. The denatured nascent chain is then used as a primer to anneal with different templates that are simultaneously present in the system while the extension continues. This process is repeated until a full-length gene fragment is produced, which results in spaced nascent DNA molecules containing different template sequences. Such nascent DNA molecules contain a large number of combinations of mutations that will facilitate the generation of new enzyme properties.
12. Gene library: an organism’s genomic DNA with restriction endonuclease partially digested, the enzyme section will be inserted into the carrier DNA molecules, all the carrier molecules inserted into the genomic DNA fragments of the carrier molecule collection will contain the entire genome of this organism, which also constitutes the organism’s gene library.
13. Representativeness of the library: whether the DNA molecules contained in the library can completely reflect all the possible changes and alterations of the exogenous genes, which is the most important indicator of the quality of the library. It is the most important indicator of the quality of the library. The indicator of the representativeness of the library is the library capacity of the library.
14. Library capacity: refers to the number of independent recombinant clones contained in the original mutation library constructed.
15. Vectors for mutation library construction: (λ phage vector system) (plasmid vector system) (mammalian cell expression vector system).
16. Enzyme mimicry: Also known as artificial enzyme or enzyme model, it is an applied science that mimics the shape and size of the enzyme’s active site and its microenvironment and other structural features as well as the enzyme’s mechanism of action and stereochemistry at the molecular level.
17. The (catalytic group) and (substrate) of the enzyme model must have stereochemical features that match each other, which is quite important for the formation of good reaction specificity and catalytic potency.
18. “Subject-guest” chemistry: The field of chemistry in which the subject (enzyme) and the guest (substrate) form stable complexes through ligand and other secondary bonds is called “subject-guest” chemistry.
19. Classification of simulated enzymes: (a) according to type: 1) simple enzyme models; 2) mechanistic enzyme models; 3) simple synthetic enzyme-like compounds; (b) according to properties: 1) subject-guest enzyme models; 2) micellar enzyme models; 3) peptidases; 4) semi-synthetic enzymes; 5) molecularly imprinted enzymes; 6) antibody enzymes.
20. Antibody enzyme: the product of high selectivity of antibody and high efficient catalytic ability of enzyme, the essence is a class of immunoglobulin with catalytic ability, also known as catalytic antibody, specificity exceeds the catalytic speed of enzyme reaction specificity, and some of them can reach the catalytic speed of enzyme as well.
21. Molecular imprinting: the process of preparing a polymer that is selective for a compound, the compound is called an imprinted molecule, also called a template molecule.
22. Principle of molecular imprinting (preparation method of molecular imprinting): 1) select the functional monomer of the imprinted molecule, so that the two have a complementary reaction; 2) in the imprinted molecule —- monomer complexes around the polymerization reaction; 3) remove the imprinted molecule from the polymer by extraction; 4) the formation of a polymer retained within the imprinted molecule with the exact same shape, size of the cavity, the polymer can be highly selective re bind the imprinted molecules with high selectivity.
23. Types of surface molecular imprinting: 1) molecular imprinting on the surface of inorganic materials as carriers; 2) surface modification of solid materials; 3) protein surface imprinting.
24. Bio-imprinting: a kind of molecular imprinting, refers to the natural biological materials (such as proteins and saccharides) as the skeleton, on which molecular imprinting, and the process of generating the imprinted molecules with specific recognition of the cavity.
25. Principle of bioimprinting: the flexibility of the conformation of biomolecules is canceled in the anhydrous organic phase, and their conformations are fixed, thus the conformational changes produced by the interaction between the template molecule and the biomolecule in the aqueous solution can only be preserved when they are moved into the anhydrous organic phase.
26. Conversion of proteins into semi-synthetic enzymes by bio-imprinting: 1) Partial denaturation of the protein to disturb the conformation of the starting protein; 2) Addition of the imprinted molecule so that the imprinted molecule is fully bound to the partially deformed protein; 3) After the interaction of the imprinted molecule with the protein, cross-linking of the imprinted protein with cross-linking agent; 4) Removal of the imprinted molecule by dialysis.

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Compound Glucoamylase	9032-08-0
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alpha-Amylase	9000-90-2
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Lipase	9001-62-1
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