What is the bio-enzyme production process?

1.Enzyme production

Enzymes are produced from microorganisms, animals and plants, but the main source is microorganisms. Since microorganisms have more advantages than plants and animals, generally excellent enzyme-producing strains are selected to produce enzymes through fermentation. In order to increase the enzyme concentration in the fermentation broth, excellent strains are selected, genetically engineered bacteria are developed, and fermentation conditions are optimized. Industrial production needs special performance of new enzymes, such as high temperature resistant α-amylase, alkaline-resistant protease and lipase, etc. Therefore, we need to research and develop strains to produce special performance of new enzymes.

2.Enzyme preparation

Enzyme separation and purification technology is the core of current biotechnology “post-treatment process”. Using a variety of separation and purification techniques, from microbial cells and their fermentation broth, or animal and plant cells and their culture broth in the separation and purification of enzymes, made of highly active enzyme preparations of different purity, in order to make enzyme preparations more widely used in all aspects of the national economy, must improve the activity of enzyme preparations, purity and yield, the need to study the new separation and purification techniques.

3.Enzyme and cell immobilization

Enzyme and cell immobilization research is the central task of enzyme engineering. In order to improve the stability of enzymes, reuse enzyme preparations, expand the application range of enzyme preparations, using a variety of immobilization methods for the immobilization of enzymes, the preparation of immobilized enzymes, such as immobilized glucose isomerase, immobilized carbamoylase, etc., the determination of immobilized enzymes, and immobilized enzymes for the application of various aspects of the development of research. Immobilized enzyme still has strong vitality. It is highly valued by various fields such as biochemistry, chemical engineering, microbiology, polymers and medicine.

Immobilized cells are developed on the basis of immobilized enzymes. Various immobilization methods are used to immobilize microbial cells, animal cells and plant cells to make a variety of immobilized biological cells. The study of the enzymatic properties of immobilized cells, especially the kinetic properties, and the research and development of immobilized cells in various applications is a hot topic in enzyme engineering nowadays.

Immobilization technology is an important milestone in the modernization of enzyme technology, and it is a breakthrough technology to overcome the shortcomings of natural enzymes in industrial applications and to give full play to the characteristics of enzyme reaction. It can be said that there is no modern enzyme technology without the development of immobilization technology.

4. Enzyme molecular modification

Also known as enzyme molecular modification. In order to improve the stability of the enzyme, reduce antigenicity, extend the half-life of medicinal bacteria in the body, using various modification methods to modify the structure of the enzyme molecule, in order to create a natural enzyme does not have some excellent characteristics (such as higher stability, no antigenicity, resistance to protease hydrolysis, etc.), and even create a new enzyme activity, to expand the application of the enzyme, so as to increase the value of the enzyme application, and achieve greater economic and social benefits. and social benefits.

Enzyme molecular modification can be carried out from two aspects:

(1) Use protein engineering technology to modify the enzyme molecule structure gene, expecting to obtain the new enzyme (mutant enzyme) with excellent characteristics and high activity which has a reasonable primary structure and space structure.

(2) Chemical or enzymatic modification of the primary structure of enzyme proteins, or chemical modification of the enzyme molecule with chemical modification of the side chain group. In order to change the enzymatic properties. These enzymes are particularly useful in basic research in enzymology and in medicine.

The microorganisms used in the production of enzymes are filamentous fungi, yeasts, and bacteria in three major groups, mainly with aerobic bacteria. The strains and uses of several major industrial enzymes are listed below:

Amylase

Amylases hydrolyze starch to produce pasty malt oligosaccharides and maltose. Production is dominated by deep fermentation with Bacillus subtilis and Bacillus licheniformis of the genus Bacillus, the latter of which produces heat-resistant enzymes. Deep and semi-solid fermentations with strains of Aspergillus and Rhizopus are also used for food processing [6] . Amylases are mainly used in sugar production, textile desizing, treatment of fermentation raw materials and food processing. Glucoamylase can hydrolyze starch into glucose, which is now almost entirely produced by deep fermentation of Aspergillus niger, and is used in sugar production, alcohol production, and fermentation raw material processing.

Protease

The use of strains and production of the most varieties. With lichen-shaped bacillus, short small bacillus and bacillus subtilis to deep fermentation production of bacterial protease; with streptomyces, Aspergillus deep fermentation production of neutral protease and Aspergillus acidic protease, used for leather dehairing, fur softening, pharmaceuticals, the food industry; with Trichoderma spp. some of the bacteria in semi-solid fermentation production of rennet in the manufacture of cheese instead of rennet extracted from the stomach of the original calf.

Glucose isomerase

A species developed rapidly in the 70s. Streptomyces cells are first obtained by deep fermentation, and after immobilization, the glucose solution is converted into a syrup containing about 50% fructose, which can be used in the food industry instead of sucrose. With amylase, glucoamylase and glucose isomerase, etc. will be made of corn starch into syrup has become one of the emerging sugar industry.

Selection of Expression Systems
1 E. coli expression system
①pET expression system is preferred
② Protein recombinant expression and purification can be performed using solubilized tags, MBP is preferred as a first choice.
③ Preferred pET24 or pET28 (if purification is needed) with BL21(DE3), TB medium 37° growth to 1-1.5 OD, 18 ℃ growth for 1 hour to 3 OD , 0.5 mM IPTG induction for 19 hours to OD up to 10.
④ Rescue measures for high expression and low solubility: cooling down to as low as 15 ℃; changing the medium to 2xYT or ZYP5052 (self-induced), changing the expression host; truncation of the N-terminal and/or C-terminal 2-10 amino acid residues; expression by fusion with highly soluble proteins, such as MBP; chemically inducing molecular chaperones, co-expressing molecular chaperones/interacting proteins, or providing ligands.
⑤ Advantages and disadvantages of E. coli expression system
Advantages: most convenient, most effective
Disadvantages: weak secretion expression ability; difficulty in disulfide bond formation; no post-translational modification

2 Yeast Expression System (Picrosporum)
Advantages: stable integration of exogenous genes; the promoter of alcohol oxidase gene is strong, and the expression can be strictly regulated by methanol; recombinant proteins can be expressed in intracellular or extracellular forms; contains post-translational modification functions common to eukaryotic expression systems; there are commercial hosts/vectors, which is easy to operate; easy to amplify, and the fermentation density is extremely high.
② Disadvantages: unique form of post-translational processing; problem of excessive glycosylation.

3 The first choice is the expression system reported in the literature, the second choice is the E. coli system, and the yeast system is used if the expression in E. coli is inactive. According to the source of the gene to determine the expression system, the gene plus affinity tags to facilitate purification.

Enzyme modification
①Rational design: based on the protein structure and function information on the coding gene change and recombination expression test.
Procedure: firstly, obtain the enzyme structure from BRENDA database, then modify the enzyme structure and predict the enzyme structure with Alphafold2, then do docking analysis between enzyme and ligand molecule with PyMOL software, and finally, according to the new enzyme structure, mutate the gene sequences directionally, and then recombinantly express it to obtain a new enzyme (to improve the enzyme’s thermal stability, catalytic efficiency, and substrate specificity).
② Irrational design: high-frequency mutation or recombination of coding sequences and recombinant expression and high-throughput testing

Ab initio design process: 1. force field development and sampling algorithm 2. high throughput testing 3. structure identification

Directed evolution of proteins
①Select original genes
②Establish a diversity gene mutation library
③Link multiple mutated genes in vectors and express them in corresponding strains respectively
④ Select a high-quality mutant gene by screening, and then express the mutant gene in large quantities.

Methods of generating mutant gene libraries
①In vivo high-frequency random mutation: use E. coli XL1-Red as gene replication host (defective DNA damage repair system)
Cultivated to plateau stage, 1 base mutation occurs in 2Kb on average.
②Random Mutagenesis kit: mutation rate 0.1 -1.6% /PCR, equivalent to 1-16 base mutations/gene
③ Point-by-point saturation mutagenesis

Natural Evolution: Spontaneous Mutation, Recombination, Natural Selection
Agricultural evolution: spontaneous mutation, breeding, screening
Laboratory evolution: accelerated mutation rate, molecular breeding, screening

DNA Shuffling
Protein engineering experimental strategy
①Select the starting gene to establish an inactivation system and/or a screening method.
②If the structure-function relationship is known, adopt rational design method first.
③Random mutation fine-tuning and high-throughput screening
④Design from scratch only if there is no suitable starting gene.

Protein Research Techniques
1 Physical and chemical properties related to protein separation and purification
Molecular size (dialysis, ultrafiltration, gel filtration, centrifugation)
②Molecular shape (gradient centrifugation, electrophoresis)
(iii) Charged properties (electrophoresis, ion exchange chromatography)
(iv) Solubilization properties (salinization, organic solvent precipitation)
⑤ Differences in specific binding to ligands (immunoaffinity chromatography, bioaffinity chromatography, metal chelate affinity chromatography)
(vi) Adsorption properties (hydrophobic chromatography)
(vii) denaturation and denaturation (denaturation and denaturation of urea)

2 Protein expression system
① Bacterial expression system: short cycle, high efficiency, low cost, but no post-translational modification. Mainly used for the production of prokaryotic proteins, simple eukaryotic proteins.
Selection of expression vector: pet series, pGEX series, PQE30……
Selection of expression strains: BL21(DE3), Rosetta, M15……
Induction conditions: IPTG concentration, temperature and time duration
Purification: Ni-NTA (His tag), Strep-beads, GST…..

② Yeast expression system: short cycle, high efficiency, low cost, post-translational modification exists. There will be inappropriate glycosylation, high mannose modification. Mainly used to produce intracellular/secretory proteins, disulfide-binding proteins, glycosylated proteins.

(iii) Bacteriophage expression system: high gene capacity, protein soluble, suitable for production of toxic proteins, post-translational modifications similar to mammalian. Cultivation conditions are harsh and it lacks some glycosylation modifications. Mainly used for the production of membrane proteins, large size proteins, viral vaccines, signaling proteins, cytokines, kinases.
The baculovirus genome has a huge capacity for exogenous gene insertions of up to 38 kb.
Baculoviruses are produced in insect cells and do not replicate in mammalian cells.
Advantages: baculoviruses can be efficiently transduced in mammalian cell lines, including primary and stem cells.
Safety (does not replicate in mammalian cells) and lack of observable cytopathic effects
Frozen stored pre-transduced cells can also be used as test preparation cells
Portability (for analysis of pharmacologically relevant cell types)
Speed of test development (no need to spend time generating stable cell lines)

④ Mammalian expression systems: long cycle time, low efficiency, presence of post-translational modifications, high protein bioactivity. Mainly used to produce complex eukaryotic proteins, proteins that require precise PTM.
Transient expression of proteins: PEI or liposome transfection reagents
Stable cell line development: Flp-In™ Jump-In™ cell engineering platforms
Inducible Expression: Tetracycline-regulated expression
Viral delivery-mediated expression: lentiviral expression system for functional analysis; adenoviral expression system for protein production

3 Protein Purification
Why do we need to purify proteins?
To characterize the structure and function of proteins of interest.
(ii) To study protein regulation and protein interactions
③ generate antibodies
Purification Methods
Based on physical/chemical properties
Protein size: dialysis, ultrafiltration, gel filtration chromatography
Protein charge: ion exchange chromatography
Protein hydrophobicity: hydrophobic interaction chromatography

Based on biological properties: affinity chromatography

①Dialysis
Influencing factors: dialysis tube MWCO; buffer volume; time of dialysis; dialysis buffer replacement frequency

Ultrafiltration
Centrifugal filtration, protein size is smaller than the pore size of the filter tube, it is centrifuged to the bottom of the tube.

③ Gel filtration chromatography
Separate proteins of different sizes

④Ion exchange chromatography
Cation exchange column: gel is negatively charged, protein is positively charged
Anion exchange column: gel is positively charged, protein is negatively charged
Medium
Inert support: agarose, dextran
Charged groups: carboxymethyl: negatively charged, diethylamino: positively charged
Balancing ions: negatively charged groups: H+ or Na+ positively charged groups: OH- or Cl-
Stepwise or gradient elution of proteins by increasing salt concentration or changing pH.

⑤ Affinity chromatography
Affinity chromatography is a method of separating biomolecules from a mixture based on highly specific macromolecular binding interactions between a biomolecule and another substance.
Examples include: antigen binding to antibodies, enzyme binding to ligands, glutathione and GST fusion proteins binding, anti-biotin proteins binding to biotin-binding molecules, and metal ions binding to polyhistidine fusion proteins.

Immobilized metal chelate chromatography (IMAC) using metal ions (Ni 2+; Co 2+; Cu 2+) bound to poly-(His) ₆ tagged proteins.

Purification of Strep-tagged proteins
Beads can specifically adsorb proteins with the strep tag, and then with biotin the protein can be eluted from the beads. (The principle is similar to the GST pull down experiment)

proteinA/protein G affinity chromatography
Genetically engineered protein A and protein G can specifically bind to the Fc region of mammalian IgG. By binding protein A and protein G to the column material, IgG and its subclasses and fragments can be purified by affinity chromatography.
protein A: molecular weight is 42kDa, encoded by spa gene, with 5 isotypic immunoglobulin-binding structural domains, each consisting of 3 alpha helices.
protein G: with a molecular weight of 65 kDa, encoded by the spg gene, binds the Fc and Fab segments of the antibody as well as the albumin in the serum. The genetically engineered protein G removes the binding site for albumin and retains only the Fc binding domain, which is more potent than protein A. The protein A/protein G is a genetically engineered protein that binds albumin.
proteinA/protein G: is a genetically engineered binding protein. It consists of 4 protein A and 2 protein G immunoglobulin binding domains, which has a wider binding range than protein A or protein G alone, and combines their advantages into one, which can be applied to the purification of IgG from almost all species.

How to identify the purified protein?
SDS-PAGE; HPLC; mass spectrometry; Western blot; Binding assays; Functional assays; Structural elucidation.

4 Electron microscopy
Single-particle cryo-electron microscopy (Single-ParticleAnalysis, SPA)
Cryo-electron tomography microscopy (cryo-ET)

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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