阅读说明：本技术 热稳定性提高的胺脱氢酶突变体及其基因工程菌的构建和应用 (Amine dehydrogenase mutant with improved thermal stability and construction and application of genetically engineered bacterium thereof ) 是由 马富强 郭天杰 张艺凡 杨广宇 于 2020-01-14 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种热稳定性提高的胺脱氢酶突变体及其基因工程菌的构建和应用。本发明提供的胺脱氢酶突变体包括4种单点突变体和11种组合突变体,与野生型胺脱氢酶相比,其单点突变体和组合突变体在42℃下半衰期均更长；尤其是组合突变体,表现出单点突变体热稳定性的叠加效果,其半衰期大约是野生型脱氨氢酶的5倍。通过本发明所提供的构建方法得到的胺脱氢酶突变体的热稳定性更好,在较高的温度下催化合成手性胺时,表现出优良的立体选择性、区域选择性和催化活性,具有较好的应用前景。(The invention belongs to the technical field of biology, and particularly relates to an amine dehydrogenase mutant with improved thermal stability and construction and application of a genetic engineering bacterium thereof. The amine dehydrogenase mutant provided by the invention comprises 4 single-point mutants and 11 combined mutants, and compared with wild amine dehydrogenase, the single-point mutants and the combined mutants have longer half lives at 42 ℃; in particular, the combination mutant showed a superimposed effect of thermal stability of the single-point mutant, with a half-life of approximately 5 times that of the wild-type deaminase. The amine dehydrogenase mutant obtained by the construction method provided by the invention has better thermal stability, shows excellent stereoselectivity, regioselectivity and catalytic activity when catalyzing and synthesizing chiral amine at higher temperature, and has better application prospect.)

1. An amine dehydrogenase mutant having improved thermostability,

the amine dehydrogenase mutant is mutated on an amino acid sequence shown in SEQ ID NO.2, and the mutation site is selected from one or more of A35D, L53R, S210A and T321P.

2. The amine dehydrogenase mutant with improved thermostability according to claim 1, wherein the mutation sites are a35D, L53R, S210A, T321P, a35D/L53R, a35D/S210A, a35D/T321P, L53R/S210A, L53R/T321P, S210A/T321P, a35D/L53R/S210A, a35D/L53R/T321P, a35D/S210A/T321P, L53R/S210A/T321P or a 35D/L53R/S210A/T321P.

3. A gene encoding the thermostable amine dehydrogenase mutant as claimed in claim 1 or 2.

4.A recombinant plasmid comprising the gene of claim 3.

5. A soluble protein, immobilized enzyme or engineered bacterium comprising the improved thermostability amine dehydrogenase mutant according to claim 1 or 2.

6. The method for constructing an amine dehydrogenase mutant having improved thermostability according to claim 1 or 2, comprising the steps of:

searching an amino acid sequence shown by SEQ ID NO.2 in a Pfam database and an NCBI database, removing a repeated identical sequence, selecting an amino acid sequence with the consistency of more than 30% with the amino acid sequence shown by SEQ ID NO.2, then performing multi-sequence comparison through Clusalx1.83 software, arranging the residual amino acid sequence into a fasta file, uploading the fasta file to a Consensus Maker v2.0.0 server, and modifying setting parameters according to needs, wherein the online software generates a Consensus sequence which can be edited at a later stage;

predicting the three-dimensional structure of the obtained protein shown by SEQ ID NO.2 by a swisscodel online tool, observing the crystal structure of the protein shown by SEQ ID NO.2 by adopting PyMOL, and screening out mutation sites related to thermal stability as follows: a35D, L53R, S210A, T321P.

7. The method of constructing an amine dehydrogenase mutant having improved thermostability according to claim 6,

the sequences of the amplification primers of the mutation site A35D are SEQ ID NO.20 and SEQ ID NO. 21;

the sequences of the amplification primers of the mutation site L53R are SEQ ID NO.22 and SEQ ID NO. 23;

the sequences of the amplification primers of the mutation site S210A are SEQ ID NO.24 and SEQ ID NO. 25;

the sequences of the amplification primers of the mutation site T321P are SEQ ID NO.26 and SEQ ID NO. 27.

8. The method of constructing an amine dehydrogenase mutant having improved thermostability according to claim 7,

the amino acid sequence of the single-point mutant corresponding to A35D is SEQ ID NO. 3;

the amino acid sequence of the single-point mutant corresponding to the L53R is SEQ ID NO. 4;

the amino acid sequence of the single-point mutant corresponding to S210A is SEQ ID NO. 5;

the amino acid sequence of the single-point mutant corresponding to the T321P is SEQ ID NO. 6;

the amino acid sequence of the combined mutant corresponding to A35D/L53R is SEQ ID NO. 7;

the amino acid sequence of the combined mutant corresponding to A35D/S210A is SEQ ID NO. 8;

the amino acid sequence of the combined mutant corresponding to A35D/T321P is SEQ ID NO. 9;

the amino acid sequence of the combined mutant corresponding to L53R/S210A is SEQ ID NO. 10;

the amino acid sequence of the combined mutant corresponding to L53R/T321P is SEQ ID NO. 11;

the amino acid sequence of the combined mutant corresponding to S210A/T321P is SEQ ID NO. 12;

the amino acid sequence of the combined mutant corresponding to A35D/L53R/S210A is SEQ ID NO. 13;

the amino acid sequence of the combined mutant corresponding to A35D/L53R/T321P is SEQ ID NO. 14;

the amino acid sequence of the combined mutant corresponding to A35D/S210A/T321P is SEQ ID NO. 15;

the amino acid sequence of the combined mutant corresponding to L53R/S210A/T321P is SEQ ID NO. 16;

the amino acid sequence of the combined mutant corresponding to A35D/L53R/S210A/T321P is SEQ ID NO. 17.

9. Use of the improved thermostability amine dehydrogenase mutant according to claim 1 or 2 in the catalytic synthesis of a chiral amine.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an amine dehydrogenase mutant with improved thermal stability and construction and application of a genetic engineering bacterium thereof.

Background

Chiral amine is a very important amine compound, widely exists in medicines, pesticides, synthetic intermediates, natural products and compounds with biological activity, especially plays an important role in the field of chiral drugs, and about 40 percent of optically active drugs contain chiral amine structures at present. Different enantiomers of chiral amines have very similar physical properties, but the difference of the stereo structures can lead different chiral enantiomers to have different biological activities, so that the metabolic, transformation or activation routes in organisms are different, and the physiological activities and the toxic effects expressed when the chiral amines act on organisms are different. It is therefore of great importance to obtain single isomers with high enantioselectivity or high diastereoselectivity. The traditional chemical synthesis method is to split a racemate by adding a chiral reagent, and the method needs the chiral reagent with equivalent weight, has the highest yield of only 50 percent and low atom economy; compared with a chemical synthesis method, the biological catalysis method has better catalysis effect, and particularly has obvious technical advantages in the aspect of identifying two enantiomers of chiral amine.

Amine dehydrogenase (AmDH) can catalyze prochiral ketone and free amine to asymmetrically synthesize chiral amine under the action of coenzyme, and is an effective biocatalyst. However, amine dehydrogenases are natural biological enzymes, and it is known that natural enzymes all function in a relatively mild environment in the body, but if amine dehydrogenases are applied in an industrially harsh environment (e.g., high temperature, extreme ph, organic solvents, non-natural substrates, product inhibition, etc.), they have poor thermal stability and thus catalytic activity is not high.

The protein engineering is based on the relationship between the structural rule and the biological function of protein molecules, and carries out gene modification or gene synthesis by means of chemistry, physics and molecular biology to modify the existing protein or manufacture a new protein to meet the requirements of human on production and life. The commonly used protein engineering methods include rational design (rational design) and irrational design (irrational design), wherein the rational design requires understanding of the structure, function and function of the protein, but the structure-function relationship of the protein is too complicated, and people still lack sufficient knowledge of the protein, so the accuracy is poor. How to improve the thermal stability of the amine dehydrogenase through protein engineering becomes a problem to be solved urgently.

Disclosure of Invention

Therefore, the technical problem to be solved by the present invention is to overcome the defect of poor thermal stability of the existing amine dehydrogenase, thereby providing an amine dehydrogenase mutant with improved thermal stability, a construction method of a genetically engineered bacterium of the amine dehydrogenase mutant, and an application of the amine dehydrogenase mutant in preparation of chiral amine.

In order to solve the technical problems, the invention adopts the technical scheme that:

the invention provides an amine dehydrogenase mutant with improved thermostability, which is (a 1) or (a 2):

(a1) a derived protein obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 and having the same function with the amino acid sequence shown in SEQ ID NO. 2;

(a2) a derived protein obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO.2 and having at least 90% homology with the amino acid sequence shown in SEQ ID NO. 2;

the amine dehydrogenase mutant is mutated on an amino acid sequence shown in SEQ ID NO.2, and the mutation site is selected from one or more of A35D, L53R, S210A and T321P.

Preferably, the mutant of the amine dehydrogenase with improved thermostability has mutation sites of A35D, L53R, S210A, T321P, A35P/L53P, A35P/S210P, A35P/T321P, L53P/S210P, L53P/T321P, S210P/T321P, A35P/L53P/S210P, A35P/L53P/T321P, A35P/S210P/T321P, L53P/S210P/T321P, or A35P/L53P/S210P/T321P.

The present invention also provides a gene encoding an amine dehydrogenase mutant having improved thermostability as described above.

The invention also provides a recombinant plasmid containing the gene.

The invention also provides a soluble protein, immobilized enzyme or engineering bacterium containing the amine dehydrogenase mutant with improved thermal stability.

The invention also provides a construction method of the amine dehydrogenase mutant with improved thermal stability, which comprises the following steps:

searching an amino acid sequence shown by SEQ ID NO.2 in a Pfam database and an NCBI database, removing a repeated identical sequence, selecting an amino acid sequence with the consistency of more than 30% with the amino acid sequence shown by SEQ ID NO.2, then performing multi-sequence comparison through Clusalx1.83 software, arranging the residual amino acid sequence into a fasta file, uploading the fasta file to a Consensus Maker v2.0.0 server, and modifying setting parameters according to needs, wherein the online software generates a Consensus sequence which can be edited at a later stage;

predicting the three-dimensional structure of the obtained protein shown by SEQ ID NO.2 by a swisscodel online tool, observing the crystal structure of the protein shown by SEQ ID NO.2 by adopting PyMOL, and screening out mutation sites related to thermal stability as follows: a35D, L53R, S210A, T321P.

Preferably, the method for constructing the amine dehydrogenase mutant with improved thermostability,

the sequences of the amplification primers of the mutation site A35D are SEQ ID NO.20 and SEQ ID NO. 21;

the sequences of the amplification primers of the mutation site L53R are SEQ ID NO.22 and SEQ ID NO. 23;

the sequences of the amplification primers of the mutation site S210A are SEQ ID NO.24 and SEQ ID NO. 25;

the sequences of the amplification primers of the mutation site T321P are SEQ ID NO.26 and SEQ ID NO. 27.

Further preferably, a method for constructing the amine dehydrogenase mutant having improved thermostability,

the amino acid sequence of the single-point mutant corresponding to A35D is SEQ ID NO. 3;

the amino acid sequence of the single-point mutant corresponding to the L53R is SEQ ID NO. 4;

the amino acid sequence of the single-point mutant corresponding to S210A is SEQ ID NO. 5;

the amino acid sequence of the single-point mutant corresponding to the T321P is SEQ ID NO. 6;

the amino acid sequence of the combined mutant corresponding to A35D/L53R is SEQ ID NO. 7;

the amino acid sequence of the combined mutant corresponding to A35D/S210A is SEQ ID NO. 8;

the amino acid sequence of the combined mutant corresponding to A35D/T321P is SEQ ID NO. 9;

the amino acid sequence of the combined mutant corresponding to L53R/S210A is SEQ ID NO. 10;

the amino acid sequence of the combined mutant corresponding to L53R/T321P is SEQ ID NO. 11;

the amino acid sequence of the combined mutant corresponding to S210A/T321P is SEQ ID NO. 12;

the amino acid sequence of the combined mutant corresponding to A35D/L53R/S210A is SEQ ID NO. 13;

the amino acid sequence of the combined mutant corresponding to A35D/L53R/T321P is SEQ ID NO. 14;

the amino acid sequence of the combined mutant corresponding to A35D/S210A/T321P is SEQ ID NO. 15;

the amino acid sequence of the combined mutant corresponding to L53R/S210A/T321P is SEQ ID NO. 16;

the amino acid sequence of the combined mutant corresponding to A35D/L53R/S210A/T321P is SEQ ID NO. 17.

The invention also provides application of the amine dehydrogenase mutant with improved thermal stability in catalytic synthesis of chiral amine.

The technical scheme of the invention has the following advantages:

1. the amine dehydrogenase mutant with improved thermal stability provided by the invention comprises a single-point mutant and a combined mutant, and compared with wild amine dehydrogenase, the single-point mutant and the combined mutant have longer half-life at 42 ℃; in particular, the combination mutant showed a superimposed effect of thermal stability of the single-point mutant, with a half-life of approximately 5 times that of the wild-type deaminase. Based on the above, the amine dehydrogenase mutant provided by the invention has better thermal stability and is suitable for catalytic synthesis of chiral amine at higher temperature.

2. The construction method of the amine dehydrogenase mutant with improved thermal stability provided by the invention is different from the rational design based on the precise structure-function relationship of protein, the construction method takes the Consensus Concept as a guiding idea, analyzes information capable of improving the thermal stability of enzyme from the aspect of evolution, performs integrated analysis on an amine dehydrogenase family sequence, and combines the assistance of bioinformatics and crystallography methods to obtain the novel amine dehydrogenase mutant with high stability.

3. The amine dehydrogenase mutant with improved thermal stability provided by the invention has excellent stereoselectivity, regioselectivity and catalytic activity when being applied to catalytic synthesis of chiral amine, and has a good application prospect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of a simulated crystal structure of the ANDD-TDO protein and a schematic diagram of distribution of mutation sites on the crystal structure, provided in example 2 of the present invention.

Detailed Description

In order to facilitate understanding of the objects, technical solutions and gist of the present invention, embodiments of the present invention will be described in further detail below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, this embodiment is provided so that this disclosure will be thorough and complete and will fully convey the concept of the invention to those skilled in the art, and the present invention will only be defined by the appended claims.