Irrational design of methods for modifying enzymes

Irrational design of modified enzymes is mainly through the method of directed evolution, which does not rely on the detailed knowledge of the enzyme structure and mechanism of action. The main ways are as follows:

Error-prone PCR

Principle: In a normal polymerase chain reaction (PCR) process, the probability of the DNA polymerase introducing errors (mutations) when copying DNA is increased by changing the reaction conditions.

For example, increasing the concentration of magnesium ions, changing the proportion of dNTP (deoxyribonucleoside triphosphate), adding manganese ions, etc., so that the amplified gene has random mutations.

The enzymes encoded by these mutated genes may then produce new properties.

Operation and application example: When performing error-prone PCR, a suitable primer is designed first, and then template DNA, primer, DNA polymerase, dNTP and other components containing the target enzyme gene are mixed to perform PCR amplification under the adjusted reaction conditions.

For example, when modifying a lipase for use in the detergent industry, a large number of mutants are generated by error-prone PCR, and then lipase mutants with higher activity and stability are selected in a screening system containing grease and detergent components, so that they can decompose grease stains more effectively in the complex environment of detergent and at higher temperatures.

DNA shuffling

Principle: A set of DNA sequences with certain homology (which can be homologous genes from different species, or different mutant genes of the same species) are cut into small fragments by nuclease, and these small fragments are randomly spliced through their complementary sequences without templates to combine into a new DNA sequence.

Enzymes encoded by new DNA sequences may incorporate the best properties of enzymes from different parents, or produce entirely new functional properties.

Example of operation and application: First obtain a set of related DNA sequences, cut them into small fragments with nucleases (such as DNase I), and then reassemble these fragments by DNA ligase or self-annealing.

For example, DNA reshuffling of several enzyme genes that can degrade different types of organic pollutants is carried out, and the reshuffled gene library is introduced into the microbial host to screen out new enzymes that can efficiently degrade multiple organic pollutants at the same time for bioremediation in environmental governance.

Alternating extension process

How it works: This is a way to simulate gene recombination in vitro. In the PCR reaction system, multiple template DNA is mixed, and in the primer extension stage, the newly synthesized DNA strand can be converted between different templates to produce a recombinant DNA sequence.

It can quickly produce a large number of DNA with different sequence combinations, increasing the diversity of enzymes.

Operation and application example: By adding gene templates and primers of multiple target enzymes into the reaction system, the PCR cycle parameters can be controlled so that the primer extension process can be alternated between different templates.

For example, when modifying enzymes with a variety of substrate specificity, the cross-stretching process is used to generate recombinant genes, and then the enzymes with higher activity to the new substrate combination are screened for simultaneous conversion of multiple substrates in the biosynthesis process.

The method of irrational design of modified enzymes mainly refers to the directed evolution technology of enzymes, which has the following advantages:

No structural details required:

Different from rational design, which requires prior understanding of the spatial structure and catalytic mechanism of the enzyme, irrational design does not require in-depth and accurate cognition of the amino acid sequence, three-dimensional structure and catalytic mechanism of the enzyme, so that experimental design can be carried out.

This greatly reduces the requirements for basic enzymology research, broadens the scope of transformable enzymes, and enables more enzymes with unknown structures and complex mechanisms to become the object of transformation, providing more possibilities for the development of new enzymes.

Closer to natural evolution:

Simulating natural evolutionary mechanisms, such as random mutation, gene recombination and natural selection, can create a large number of mutant libraries in a short time and effectively increase the diversity of enzymes.

This diversity makes enzymes more likely to develop new properties and functions during evolution, so that they can better adapt to a variety of different application needs, such as functioning under different conditions such as temperature, pH, substrate specificity, and so on.

Rapid acquisition of beneficial mutations:

Through targeted selection, mutant enzymes with desired property optimization can be quickly screened, greatly shortening the development cycle from natural enzymes to enzymes with specific properties.

Compared with natural evolution, which takes a long time to accumulate beneficial mutations, directed evolution can be completed in years, months or even days, significantly improving the efficiency of enzyme transformation, and faster meeting the urgent demand for high-performance enzymes in industrial production, pharmaceutical research and development.

Strong operability, low cost:

The experimental operation is relatively simple, mainly based on common molecular biology techniques, such as PCR, gene cloning, etc., and does not require complex equipment and high reagent costs.

At the same time, because there is no need to carry out a lot of structural analysis and theoretical calculation in advance, it also reduces the input of manpower, material resources and time, has a high cost performance, and is suitable for large-scale enzyme transformation research and application.

Multiple features can be optimized simultaneously:

Many characteristics of enzyme can be optimized and improved simultaneously.

For example, it can not only improve the catalytic activity of the enzyme, but also enhance the stability of the enzyme, change the substrate specificity or enantiomer selectivity of the enzyme, so as to obtain an enzyme with better overall performance and better meet the complex requirements of the enzyme in practical applications.

Here are some successful cases of irrational engineered enzymes:

Modification of beta-galactosidase:

Random mutation of β-galactosidase gene of Lactobacillus bulgaricus L3 was performed by error-prone PCR and continuous error-prone PCR.

For example, mutant enzyme W980F significantly improved the yield of hydroquinone transglycosylation products, and significantly enhanced the transglycosylation ability of phenol hydroxyl receptors compared with the original enzyme. Mutant enzyme 252 was also obtained, which could glycosylate resveratrol, and 6 amino acid sites of it were mutated. They were Q40R, K85R, F117S, D433G, V511A and E707G, and further study found that the single point mutation of 117, 511 and 707 could also enable the enzyme to obtain the ability to transside resveratrol.

Transformation of Subtilis protease E:

By using error-prone PCR technique, the PCR reaction conditions were changed to introduce random mutations into Taq DNA polymerase and accumulate mutations after multiple rounds of amplification.

The catalytic efficiency kcat/Km of PC3 obtained after the first directed evolution at 60% and 85% dimethylformamide (DMF) was 256 times and 131 times higher than that of the natural enzyme, and the activity was 157 times higher than that of PC3.

After two further directed evolution, the catalytic efficiency of the mutant was 3 times higher than that of PC3 (60% DMF) and 471 times higher than that of the natural enzyme.

Transformation of cephalosporinase:

Using DNA shuffling technology, cephalosporin genes from 4 different strains were treated as a gene pool, which was cut and recombined.

The resistance of each enzyme gene of the four genes evolved separately to the antibiotic La xefin was increased by about 8 times, and the resistance was increased by 270-540 times and the evolution rate was increased by about 50 times after the four genes were simultaneously involved in the reorganization in a system. The optimal mutant contains 8 gene segments of 3 out of 4 genes and 33 mutation sites.

Modification of D-panthenolactone hydrolase:

Since the lactone hydrolase derived from Fusarium pseudoverticulata was used as the object, single or combined mutants containing 1-14 amino acid sites were obtained through an irrational evolutionary method combining error-susceptible PCR and saturation mutation, and the hydrolase activity was increased by 2.3-128 times compared with the parent enzyme.

Transformation of Candida rufiformis lipase:

DNA family rearrangement technique was used to target the evolution of 6 isozyme genes of Candida rufifolia lipase to construct chimeric gene library, and combined with the multi-strategy of whole gene codon modification, gene dose effect adjustment, molecular chaperone co-expression and process control, the heterologous and efficient expression of the lipase was realized. High density fermentation of recombinants in a 10L fermenter has a maximum enzymatic activity of over 20,000U/mL.

The thermal stability of Candida puccinatus lipase was improved by site-specific mutation, and the mutant recombinants with the maximum Tm value increased by 9.40℃ were obtained.

Modification of nitrile hydrolase:

A semi-rational design directed evolutionary strategy for enzyme activity pockets, ALF-scanning, was developed and applied to the substrate preference modification of nitrile hydrolase.

A nitrile hydrolase mutant V198L/W170G with aromatic nitrile substrate preference and improved catalytic efficiency was obtained by scanning mutation of active pocket residues by alanine, leucine and phenylalanine, combined with site-specific saturation mutation and combined mutation technology. The specific enzyme activity of four aromatic nitrile substrates was increased 11.10, 12.10, 26.25 and 2.55 times, respectively.

Modification of cytochrome P450:

The mutant derived from the directed evolution of Pseudomonas cytochrome P450 can hydroxylate naphthalene through the "peroxide pathway" in the absence of cofactors, and its activity is 20 times higher than that of natural enzymes.