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Enzyme Engineering
This is a mind map about enzyme engineering, including enzyme molecular modification, enzyme immobilization, application in the food industry, etc.
Edited at 2023-11-24 01:40:30
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Enzyme Engineering
- bacteria
This is a mind map about bacteria, and its main contents include: overview, morphology, types, structure, reproduction, distribution, application, and expansion. The summary is comprehensive and meticulous, suitable as review materials.
- Plant asexual reproduction
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- Outline
Enzyme Engineering
The chemical nature of enzymes
A type of special protein produced by living cells, with catalytic activity and high specificity,
Also known as: biocatalyst
Enzyme Engineering
concept
Also known as: enzyme technology
The process of converting corresponding raw materials into required products using the catalytic effect of enzymes in a certain bioreactor is a science and technology formed by combining enzymology theory and chemical engineering.
Classification
Chemical Enzyme Engineering (Primary Enzyme Engineering)
natural enzymes
chemical modification enzymes
Immobilized Enzyme
Artificial synthetic enzyme
Biological enzyme engineering (Advanced Enzyme Engineering)
The product of the combination of enzymology & modern molecular biology technology mainly based on gene recombination technology
clonase
mutant enzyme
new enzyme enzyme
Catalysis in non-aqueous media
Enzyme Reactor & Enzyme Sensor
A brief history
Enzyme preparation & fermentation production
main method
Extracted directly from biological cells
chemical synthesis
Microbial fermentation produces enzymes
Commonly used enzyme-producing microorganisms
bacteria
Actinomycetes
Mold
yeast
…
Cells used in food enzyme preparations Conditions that should be met
Safe, reliable, non-pathogenic bacteria
Good stability, not easy to degrade, not easy to be infected by phages
The enzyme yield is high and has good development and application value.
Easy to culture and manage, enzyme-producing cells are easy to grow and reproduce
Can use cheap raw materials and short fermentation cycle
fermentation method
solid fermentation
Liquid fermentation (main)
Intermittent (batch) fermentation method
continuous fermentation
Process conditions & controls
condition
medium
temperature
pH
Dissolved oxygen
Fermentation time
in principle
(First) conducive to bacterial growth → optimal conditions
(No longer) affects the formation of enzyme → the need for enzyme production
Methods to increase enzyme production
Gene recombination technology, breeding of excellent cells
Strengthen the production process
Control reasonable fermentation conditions
pH, temperature, matrix concentration…
Add substrate/substrate analog/precursor substance
For example: isopropyl-β-D mercaptogalactose → β-galactosidase production increases 1000 times.
Control repressor concentration
The product accumulates to a certain concentration and synthesis is hindered.
Add surfactant
Mainly nonionic surfactants.
Add enzyme production promoter
Calcium magnesium phytate, polyethylene, etc.
Enzyme isolation and purification
cell disruption
machinery broken
principle
Mechanical movement: shear force → tissue and cell disruption.
method
mashing method
grinding method
Homogenization method
physics broken
principle
Various physical factors → destruction of the outer structure of tissues and cells → cell fragmentation.
method
Temperature difference
Pressure difference
ultrasound
chemistry broken
principle
Various chemical reagents → cell membrane → cell disruption.
method
Organic solvents
Benzyl, acetone, butanol, chloroform
Surfactant
Triton, Tween
Enzymatic hydrolysis fragmentation
principle
The catalytic action of the cell's own enzyme system or external enzyme preparations → the destruction of the outer structure of the cell → cell fragmentation.
method
Autolysis method
external enzyme preparation method
extract
purification
dialysis
sucrose gradient solution
centrifuge
Basic principle: Different concentrations of sucrose solutions, protein molecules have different relative molecular weights → different sedimentation speeds → high-speed centrifugation → enzymes & proteins: along the concentration gradient → respective zones (each zone contains only one enzyme/protein)
Enzyme molecular modification
concept
Change the structure of the enzyme molecule →change some properties & functions of the enzyme
main method
chemical modification
concept
Chemical means→binding certain chemical substances/groups to enzyme molecules, or deleting/replacing certain parts of the enzyme molecules,→changing the physical and chemical properties of the enzyme→the purpose of changing the catalytic properties of the enzyme.
method
Macromolecule binding modification
Macromolecules: hydrogen bonding (non-covalent interaction)/covalent connection→enzyme molecule surface→protective layer→stability↑
Side chain group modification (Small molecule modification)
Small molecules covalently modify enzymes → some groups on the enzyme surface
Intramolecular or intermolecular cross-linking
Dual/multifunctional reagent→enzyme is more stable
glutaraldehyde
diimine
Limited hydrolysis modification of peptide bonds (enzyme protein backbone modification)
Defined peptide bond site in the peptide chain: hydrolysis → enzyme spatial structure: change → change enzyme properties & functions
Amino acid substitution modification (Reactive group modification)
Selective modification: amino acid side chain components → change amino acid substitution → change active center amino acid → change enzyme characteristics
Metal ion replacement modification
Metal substitution in the enzyme molecule → changes the specificity, stability and inhibitory effect of the enzyme
physical modification
concept
Physical methods → do not change the constituent units & groups of the enzyme, but only change the spatial conformation of the enzyme molecule (change or rearrangement of secondary bonds) → change some characteristics and functions of the enzyme.
method
High pressure treatment
Increased enzyme activity
optimal conditions change
Appropriate denaturation changes spatial conformation
Appropriately improve stability
Enzyme immobilization
The concept of enzyme immobilization
Enzyme & insoluble carrier→binding→a process that completely or basically limits the catalytic activity of free enzymes, cells or organelles to a certain space.
The immobilized enzyme still has the catalytic activity of the enzyme and can react continuously, and the reacted enzyme can be recycled and reused.
method
adsorption method
adsorption method
physical adsorption
Hydrogen bonding, hydrophobic interaction, π electron affinity → enzyme immobilized on water-insoluble carrier
carrier
organic carrier
Starch, gluten, fiber, chitin...
inorganic carrier
Activated carbon, porous glass/ceramics, alumina, silica gel...
Representative enzymes
α-amylase, glucoamylase, glucose oxidase
Features
Adsorption capacity is low
(Generally <1mg protein/g adsorption)
Less loss of enzyme activity
The binding force with the carrier is weak and easy to fall off
Ion adsorption
Suitable pH & ionic strength → side chain dissociation group of enzyme & water-insoluble carrier containing ion exchange group → electrostatic force → binding
carrier
anion exchanger
cation exchanger
Representative enzymes
β-amylase, glucoamylase, glucose isomerase, DEAE-Sephadex immobilized aminoacylase
Features
advantage
The operation is simple, the treatment conditions are mild, and more highly active immobilized enzymes can be obtained;
Can fully choose carriers with different charges and shapes,
The adsorption process can simultaneously purify the enzyme,
The immobilized enzyme can be reactivated after being inactivated during use, and the carrier can be recycled and reused.
shortcoming
The immobilized enzyme prepared by adsorption method is easy to fall off, which affects the purity of the product and the stability of the operation.
embedding method
Certain method → Enzyme embedded in semi-permeable carrier
Embedding method
Gel embedding method
Enzymes or enzyme-containing bacterial cells are embedded in micropores inside various gels to produce immobilized enzymes/immobilized bacterial cells.
Commonly used gels
Agar gel, calcium alginate gel, carrageenan, gelatin, polyacrylamide gel, photo-cross-linked resin, etc.
Features
The conditions are mild and have little impact on enzyme activity, but the intensity is poor.
semipermeable membrane embedding method
Also known as: microcapsule embedding method, which refers to positioning enzyme molecules in a semipermeable polymer membrane to make microencapsulated enzymes.
Commonly used semipermeable membranes
Polyamide film, collodion film, nitrocellulose, polystyrene, chitosan, etc.
Features
The size of the microcapsules can be controlled, the encapsulation time is short, the contact surface area between the enzyme and the substrate is large, and multiple enzymes can be immobilized in the same capsule, which is conducive to the immobilization of multiple enzymes.
Features
advantage
easy to use,
Only embedded, no chemical reaction →immobilized enzyme with higher activity,
It is suitable for most enzymes, crude enzyme preparations, and even intact microbial cells.
shortcoming
Only small molecule substrates & products can pass through the gel network, but macromolecular substrates are not suitable.
The resistance of the gel network to substance diffusion leads to changes in the kinetic behavior of the immobilized enzyme and reduced activity.
Covalent bonding method/covalent coupling method
Enzyme protein side chain groups & carrier surface functional groups → covalent bonding
Enzyme functional group covalently bound to the carrier
Amino group, phenol group, imidazole group, mercapto group, hydroxyl group, indole group, etc.
carrier
Natural organic carrier (cellulose, agarose)
Synthetic polymers (nylon, polyamino acids)
Inorganic carrier (porous glass, metal oxide)
Combining methods
Diazotization method, alkylation method, arylation method, condensation reaction, azide reaction, thiol-dithiol exchange method, metal coupling reaction, etc.
Features
advantage
The combination is firm and not easy to fall off, which is convenient for continuous use.
shortcoming
The carrier activation operation is complex,
Moreover, the enzyme directly participates in chemical reactions during the preparation process, which can easily cause changes in the enzyme structure, reduce or destroy the activity of the immobilized enzyme, and result in a low recovery rate (about 30%).
Cross-linking method
Dual/multifunctional reagent → between enzyme molecules or between enzyme and carrier or between enzyme and inert protein → cross-linking reaction
Commonly used cross-linking agents
Glutaraldehyde, bisazobenzidine-2,2-disulfonic acid...
Cross-linking reaction
High enzyme concentration → occurs between: enzyme molecules
After: insoluble state
Low enzyme concentration → occurs: inside the enzyme molecule
Cross-linking method
Direct cross-linking method
Glutaraldehyde solution → enzyme solution → insoluble immobilized enzyme
enzyme-assisted protein cross-linking
Dual/multifunctional reagent → inert protein & enzyme → cross-linking
Carrier cross-linking method
Part of the functional group & carrier of the dual/multifunctional reagent → cross-linking, Another part of the functional group & enzyme protein → cross-linking
Adsorption cross-linking method
The enzyme is adsorbed on the adsorbent (silica gel, bentonite, alumina, etc.) → cross-linked with the bifunctional reagent
Features
The combination is strong and can be used for a long time;
However, the conditions are severe and the loss of enzyme activity is large.
The immobilized enzyme particles are small and inconvenient to use.
Improve
Used in combination with adsorption method/embedment method
Immobilized Enzyme
Advantages and Disadvantages
advantage
It is easy to separate the enzyme from the substrate and product, so the product is relatively easy to purify;
Can be reused, improves usage efficiency and has low cost;
In most cases, the stability of the enzyme can be improved;
It can increase the yield of the product and improve the quality of the product;
It is conducive to realizing pipelined, continuous and automated operations, and is easy to be used in conjunction with various separation methods.
shortcoming
Especially during the immobilization process, there is inevitably a certain loss of enzyme activity;
Only suitable for water-soluble, small molecule substrates;
Compared with intact bacteria, it is not suitable for multi-enzyme reactions and requires cofactors.
nature
Vitality: Usually reduced ↓
reason
conformational changes
The interaction between the enzyme and the carrier causes the conformation of the active center of the enzyme to change (mainly occurs in adsorption methods and covalent coupling methods), resulting in a decrease in enzyme activity.
three-dimensional shielding effect
Due to the steric hindrance of the carrier to the active center or regulatory center of the enzyme, the contact between the enzyme and the substrate is affected.
Stability: usually improved ↑
Thermal stability↑
pH-enzyme activity relationship
The optimal pH & enzyme activity-pH curve of the reaction: change ←Based on: Enzyme protein & carrier charge
carrier
negatively charged
Optimum pH: moving towards alkaline direction
Positively charged
Optimum pH: moving towards acidic direction
Improved resistance to proteases↑
Protease has a large molecular weight and cannot enter the immobilized enzyme.
Improved resistance to denaturants and inhibitors↑
Operational stability↑
Can be used for a long time
Longer half-life
Storage stability↑
reason
Immobilization increases the firmness of enzyme conformation
Resist the attack of enzymes by adverse factors
Limit the interactions between enzyme molecules
Applications in the food industry
Improve beer production technology and improve beer quality
New beer brewing process using immobilized biocatalysts
Immobilized enzymes for beer clarification
Add protease & glucose oxidase to improve beer stability
Protease: Improve beer stability
Glucose oxidase: improve beer stability & shelf life
Glucanase improves beer foam retention
Barley → β-glucan: too much → difficult to filter, turbid; sedimentation
Reduce diacetyl content in beer
Improve technology and produce dry beer
Improving the production technology of fruit wine and juice drinks
Juice extraction, clarification, filtration
food preservation
Enzymatic preservation
glucose oxidase
Oxygen removal and preservation
Sugar removal and preservation of egg products
Lysozyme
Production of high fructose corn syrup using immobilized enzymes
Enzymatic production of new oligosaccharides
fructooligosaccharides
isomaltooligosaccharide
α-Glucosidase →
Galacto-oligosaccharides
Enzymatic production of cyclodextrin
The role of cyclodextrin
Stabilize volatile, oxidative and photolytic substances
Change the physical and chemical basis of matter
Change the reaction properties of the included complex molecules
Enzyme method applied in the production of cheese products
Rennet