CN112852770B

CN112852770B - Alcohol dehydrogenase mutant and application thereof in preparing chiral diaryl alcohol compound by efficient asymmetric reduction

Info

Publication number: CN112852770B
Application number: CN202011216302.4A
Authority: CN
Inventors: 孙周通; 曲戈; 刘贝贝; 毕悦欣
Original assignee: Tianjin Institute of Industrial Biotechnology of CAS
Current assignee: Tianjin Institute of Industrial Biotechnology of CAS
Priority date: 2020-11-04
Filing date: 2020-11-04
Publication date: 2022-01-07
Anticipated expiration: 2040-11-04
Also published as: CN112852770A

Abstract

The invention discloses an alcohol dehydrogenase mutant and application thereof in preparing chiral diaryl alcohol compounds by efficient asymmetric reduction. The alcohol dehydrogenase mutant of the present invention is a protein obtained by mutating the amino acid residue at position 42 and/or 84 and/or 85 and/or 86 of the amino acid sequence of alcohol dehydrogenase TbSADH. The invention applies directed evolution technology and method to carry out enzyme modification on alcohol dehydrogenase TbSADH, obtains a series of mutants which have enzyme activity on diaryl ketones represented by (4-chlorphenyl) pyridine-2-ketone, generates (S) - (4-chlorphenyl) pyridine-2-methanol (the conversion rate is more than 99 percent, the ee percent is more than 99 percent (S)) and (R) - (4-chlorphenyl) pyridine-2-methanol (the conversion rate is 98 percent, the ee percent is 99 percent (R)), and has better industrial application prospect in preparing chiral alcohol compounds by biocatalysis.

Description

Alcohol dehydrogenase mutant and application thereof in preparing chiral diaryl alcohol compound by efficient asymmetric reduction

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a series of alcohol dehydrogenase mutants obtained by modifying alcohol dehydrogenase TbSADH derived from high-temperature anaerobic bacillus (Thermoanaerobacter brockii) by using a directed evolution technology and a method, and an optical pure chiral bisaryl alcohol compound prepared by using the mutants as a biocatalyst through efficient asymmetric reduction.

Background

The chiral diaryl secondary alcohol is a structural unit of a plurality of bioactive molecules, is an important medical and fine chemical intermediate, and the process for synthesizing the chiral diaryl alcohol by one-step asymmetric reduction reaction of prochiral diaryl ketone has the advantage of high atom economy. Wherein, (S) - (4-chlorophenyl) pyridine-2-methanol and (R) - (4-chlorophenyl) pyridine-2-methanol can be used as important raw materials for synthesizing antihistamine drugs carbinoxamine (Barouh et al, J.Med.chem.,1971,14(9):834-836) and bepotastine besilate (Takahashi et al, Clin.exp.Dermatol.,2004,29(5): 526-532).

The (S) - (4-chlorophenyl) pyridine-2-methanol and (R) - (4-chlorophenyl) pyridine-2-methanol are mainly synthesized by chemical and biological enzyme methods. Among the current chemical synthesis methods are: taking 2-cyanopyridine as an initial substrate, adding 4-chlorobenzene magnesium bromide, concentrated sulfuric acid and a metal catalyst Pd (Phe)₃P)₄Etc. the ee value of (S) - (4-chlorphenyl) pyridine-2-methanol can reach 98 percent through three-step synthesis under the action of the same, but the N-group on the pyridine ring needs to be protected and deprotected (Corey)&Helal, Tetrahedron Letters,1996,37(32): 5675-. Also used in recent years are Noyori organometallic catalysts such as: the chiral ketone substrate (4-chlorphenyl) pyridine-2-methanone can be directly reduced into (S) - (4-chlorphenyl) pyridine-2-methanol and (R) - (4-chlorphenyl) pyridine-2-methanol by using a heavy metal catalyst containing Ru, Rh, Ir and the like through hydrogenation, and the ee value reaches 92-99% (Tao et al, J.org.chem.,2012,77(1): 612. sub.616; Yang et al, Org.Lett. sub.2015, 17(17): 4144. sub.4147; ACS Catal.2019,9, 5562. sub.5566; Org.Lett.2019,21, 5392. sub.5396). Although these chemical syntheses can produce products with high ee values, the entire process requires the use of, for example, concentrated sulfuric acid, H₂(8-10bar), high pressure and organic metal reagent, etc., which not only can cause harm to the environment, but also has higher labor protection requirement.

The current methods for synthesizing (S) - (4-chlorophenyl) pyridine-2-methanol and (R) - (4-chlorophenyl) pyridine-2-methanol by a biological enzyme method are as follows: the corresponding products were synthesized by asymmetric reduction of (4-chlorophenyl) pyridine-2-methanone using calcium alginate-immobilized baker' S yeast or the chiral product was obtained by selective hydrolysis of (4-chlorophenyl) pyridine-2-methanol acetate (Takemoto & Achiwa, chem.Pharm. Bull.,1994,42(4): 802. sub.805; Takemoto et al, Phytochemistry,1996,42(2): 423. sub.426), but the ee value for the (S) - (4-chlorophenyl) pyridine-2-methanol product was only 28%. The authors also reported studies on the synthesis of (S) - (4-chlorophenyl) pyridine-2-methanol from prochiral ketone (4-chlorophenyl) pyridine-2-methanone using immobilized plant cells with ee values of up to 48% (Takemoto et al, Phytochemistry,1996,42(2): 423-. However, these whole cell transformation methods are inefficient, and there is no report on the amino acid sequence of the relevant enzyme. In 2007, Truppo et al reported a study of asymmetric reduction of prochiral ketone to synthesize (S) - (4-chlorophenyl) pyridine-2-methanol using a commercial carbonyl reductase (KRED), but the ee value was only 60%, the enzyme protein sequence was unknown, and the species source was not reported (Truppo et al, org.Lett.,2007,9(2): 335-338). Ni et al screened a Kluyveromyces sp.CCTCCMM2011385 strain by traditional enrichment culture in 2012, which can catalyze (4-chlorophenyl) pyridine-2-methanone to produce (S) - (4-chlorophenyl) pyridine-2-methanol (86.7% ee) (CN 102559520A). However, the wild strain has low content of active enzyme, can only catalyze 2g/L of substrate at most, has low product concentration and high separation cost, and thus cannot meet the practical application. Li Zheng is equal to 2013, a carbonyl reductase PasCR derived from Pichia pastoris GS115 is researched, the carbonyl reductase PasCR can asymmetrically reduce and catalyze diaryl ketone compounds, and the highest conversion rate is only 50% (Li Zheng et al, BioEngineers, 2013, 29: 68-77). In 2016, Zhou et al isolated and purified Kluyveromyces alcohol dehydrogenase (named KPADH), and screened by protein engineering to obtain KPADH mutants M131F, S196Y and S237A, the wild-type alcohol dehydrogenase KPADH and three mutants can catalyze (4-chlorophenyl) pyridine-2-ketone to generate (R) - (4-chlorophenyl) pyridine-2-methanol with the highest conversion rate of 99%, and the ee value of (R) -product is 74.7% -96.1% (Zhou et al, Catal, Sci. Technol.,2016,6(16): 6320-. In 2018, Xu et al can catalyze (4-chlorophenyl) pyridine-2-ketone to generate (R) - (4-chlorophenyl) pyridine-2-methanol by designing and modifying KPADAH, and the ee value of the (4-chlorophenyl) pyridine-2-ketone reaches 99.4 percent, but the yield is 88.6 percent (Xu et al, ACS catalysis, 2018,8, 8330-membered ring-opening benzene 8345).

Disclosure of Invention

The technical problem to be solved by the invention is how to prepare the chiral diaryl alcohol compound.

In order to solve the technical problems, the invention firstly provides an alcohol dehydrogenase TbSADH mutant.

The alcohol dehydrogenase TbSADH mutant provided by the invention is a protein obtained by mutating the amino acid residue shown at the 42 th position and/or the 84 th position and/or the 85 th position and/or the 86 th position of the amino acid sequence of the alcohol dehydrogenase TbSADH.

The alcohol dehydrogenase TbSADH mutant comprises at least one mutation from M1) to M4) as follows:

m1) knocking out the 84 th amino acid or mutating the 84 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH into serine by proline;

m2) mutating the 42 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from histidine to threonine;

m3) mutating the 85 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from alanine to glycine or serine;

m4) the amino acid at the 86 th position of the amino acid sequence of the alcohol dehydrogenase TbSADH is mutated from isoleucine to glycine or alanine or cysteine or serine or methionine or leucine.

The alcohol dehydrogenase is derived from a thermophilic anaerobic bacterium (Thermoanaerobacter brockii), the name of the alcohol dehydrogenase is TbSADH, and the amino acid sequence of the alcohol dehydrogenase is SEQ ID No.2 in a sequence table; the alcohol dehydrogenase TbSADH mutant is a protein obtained by mutating the amino acid residue shown in the 42 th position and/or the 84 th position and/or the 85 th position and/or the 86 th position of the alcohol dehydrogenase TbSADH.

Wherein the amino acid residue at position 42 is located in the large pocket of the catalytic center of alcohol dehydrogenase TbSADH; the 85 th and 86 th amino acid residues are both positioned in a small pocket of the catalytic center of the alcohol dehydrogenase TbSADH; while amino acid residue 84 is not located in the TbSADH catalytic center. The amino acid residues are key sites influencing the enantioselectivity and the enzyme catalytic activity of the alcohol dehydrogenase TbSADH, and the conversion rate of a substrate can be improved and alcohol products with different chiralities can be selectively obtained by modifying the amino acid residues.

Further, the alcohol dehydrogenase TbSADH mutant is any one of the following N1) -N6):

n1) is protein obtained by mutating 84 th position of amino acid sequence of alcohol dehydrogenase TbSADH;

n2) is protein obtained by mutating 84 th and 85 th positions of the amino acid sequence of alcohol dehydrogenase TbSADH;

n3) is protein obtained by mutating 84 th and 86 th positions of the amino acid sequence of alcohol dehydrogenase TbSADH;

n4) is protein obtained by mutating 84 th and 42 th positions of the amino acid sequence of alcohol dehydrogenase TbSADH;

n5) is a protein obtained by mutating 84 th, 85 th and 86 th positions of an amino acid sequence of alcohol dehydrogenase TbSADH;

n6) is a protein obtained by mutating the 84 th, 85 th and 42 th positions of the amino acid sequence of alcohol dehydrogenase TbSADH.

Further, the alcohol dehydrogenase TbSADH mutant is any one of the following (1) to (24):

(1) protein obtained by knocking out 84 th amino acid proline in the amino acid sequence of alcohol dehydrogenase TbSADH and keeping other amino acid sequences unchanged;

(2) the protein is obtained by mutating the 84 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from proline to serine, mutating the 85 th amino acid from alanine to glycine, and keeping other amino acid sequences unchanged;

(3) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to serine, and mutating 86 th amino acid from isoleucine to glycine, and keeping other amino acid sequences unchanged;

(4) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to serine, and mutating 86 th amino acid from isoleucine to alanine, and keeping other amino acid sequences unchanged;

(5) mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to serine, and mutating 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged to obtain protein;

(6) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to serine, and mutating 86 th amino acid from isoleucine to serine, and keeping other amino acid sequences unchanged;

(7) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to serine, and mutating 86 th amino acid from isoleucine to methionine, and keeping other amino acid sequences unchanged;

(8) protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline to serine, and mutating 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged;

(9) knocking out 84 th amino acid proline of an amino acid sequence of the alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to glycine, and keeping other amino acid sequences unchanged to obtain protein;

(10) knocking out 84 th amino acid proline of an amino acid sequence of the alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to serine, and keeping other amino acid sequences unchanged to obtain protein;

(11) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to glycine, and keeping other amino acid sequences unchanged to obtain protein;

(12) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to alanine, and keeping other amino acid sequences unchanged to obtain protein;

(13) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to serine, and keeping other amino acid sequences unchanged to obtain protein;

(14) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to methionine, and keeping other amino acid sequences unchanged to obtain protein;

(15) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged to obtain protein;

(16) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged to obtain protein;

(17) protein obtained by knocking out proline at amino acid 84 of alcohol dehydrogenase TbSADH and mutating histidine to threonine at amino acid 42 while keeping other amino acid sequences unchanged;

(18) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to glycine, mutating 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged to obtain protein;

(19) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to serine, mutating 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged to obtain protein;

(20) knocking out 84 th amino acid proline of an amino acid sequence of the alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to serine, mutating 42 th amino acid from histidine to threonine, and keeping other amino acid sequences unchanged to obtain protein;

(21) knocking out proline at amino acid 84 of the alcohol dehydrogenase TbSADH, mutating alanine at amino acid 85 to glycine, mutating isoleucine at amino acid 86 to valine, and keeping other amino acid sequences unchanged to obtain protein;

(22) knocking out 84 th amino acid proline of an amino acid sequence of the alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to glycine, mutating 86 th amino acid from isoleucine to threonine, and keeping other amino acid sequences unchanged to obtain protein;

(23) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to glycine, mutating 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged to obtain protein;

(24) a fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of the protein shown in any one of (1) to (23).

In the alcohol dehydrogenase TbSADH mutant, the amino acid sequence of the alcohol dehydrogenase TbSADH is shown as SEQ ID No. 2.

The label may be the label shown in the following table.

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG
	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

In order to solve the technical problems, the invention also provides a biological material related to the alcohol dehydrogenase TbSADH mutant.

The biomaterial related to the alcohol dehydrogenase TbSADH mutant provided by the invention is any one of the following a1) -a 4):

a1) nucleic acid molecules encoding the above-described alcohol dehydrogenase TbSADH mutants;

a2) an expression cassette comprising the nucleic acid molecule of a 1);

a3) a recombinant vector comprising the nucleic acid molecule of a1) or the expression cassette of a 2);

a4) a recombinant microorganism comprising a1) said nucleic acid molecule or a2) said expression cassette or a3) said recombinant vector.

The nucleic acid molecule encoding the alcohol dehydrogenase TbSADH mutant according to a1) above, which is the gene according to 1) to 25) above:

1) DNA molecules obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene;

2) the DNA molecule is obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T and mutating the 254 th position from a base C to a base G;

3) a DNA molecule obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T, mutating the 256 th position from a base A to a base G, and mutating the 257 th position from a base T to a base G;

4) a DNA molecule obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T, mutating the 256 th position from a base A to a base G, mutating the 257 th position from a base T to a base C, and mutating the 258 th position from a base T to a base A;

5) a DNA molecule obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T, mutating the 256 th position from a base A to a base T, and mutating the 257 th position from a base T to a base G;

6) a DNA molecule obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T and mutating the 257 th position from the base T to a base G;

7) the DNA molecule is obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T and mutating the 258 th position from the base T to a base G;

8) a DNA molecule obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild type gene from a base C to a base T, mutating the 256 th position from a base A to a base C, and mutating the 258 th position from a base T to a base G;

9) knocking out 250 th, 251 th and 252 th positions of alcohol dehydrogenase TbSADH wild type gene, and mutating 254 th position from base C to base G to obtain DNA molecule;

10) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 253 rd position from a base G to a base A and mutating the 254 th position from a base C to a base G;

11) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 256 th position from a base A to a base G and mutating the 257 th position from a base T to a base G;

12) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 256 th position from a base A to a base G, mutating the 257 th position from a base T to a base C and mutating the 258 th position from a base T to a base A;

13) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene and mutating the 257 th position from a base T to a base G;

14) knocking out 250 th, 251 th and 252 th positions of alcohol dehydrogenase TbSADH wild type gene, and mutating 258 th position from base T to base G to obtain DNA molecule;

15) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 256 th position from a base A to a base T and mutating the 257 th position from the base T to a base G;

16) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 256 th position from a base A to a base C and mutating the 258 th position from a base T to a base G;

17) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 124 th position from a base C to a base A, mutating the 125 th position from a base A to a base C and mutating the 126 th position from a base T to a base C;

18) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base C and mutating the 258 th position from a base T to a base G;

19) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of a wild-type gene of alcohol dehydrogenase TbSADH, mutating the 253 rd position from a base G to a base A, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base C, and mutating the 258 th position from a base T to a base G;

20) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of a wild-type gene of alcohol dehydrogenase TbSADH, mutating the 253 rd position from a base G to a base A, mutating the 254 th position from a base C to a base G, mutating the 124 th position from a base C to a base A, mutating the 125 th position from a base A to a base C, and mutating the 126 th position from a base T to a base C;

21) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 254 th position from a base C to a base G and mutating the 256 th position from a base A to a base G;

22) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 254 th position from a base C to a base G, mutating the 257 th position from a base T to a base C, and mutating the 258 th position from a base T to a base C;

23) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base T, and mutating the 257 th position from a base T to a base G;

24) a fusion sequence obtained after connecting the 5 'end and/or the 3' end of the DNA molecule defined in 1) -23) with a tag coding sequence;

25) a DNA molecule having 90% or more identity to the DNA molecule defined in 1) -24) and encoding the above-mentioned alcohol dehydrogenase TbSADH mutant.

The wild-type gene of the alcohol dehydrogenase TbSADH is shown as SEQ ID No. 1.

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

The nucleotide sequence encoding the above-described alcohol dehydrogenase TbSADH mutant of the present invention can be easily mutated by a person of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified to have 90% or more identity with the nucleic acid molecule of the present invention are derived from the nucleic acid molecule of the present invention and are identical to the sequence of the present invention, as long as they encode the above-mentioned alcohol dehydrogenase TbSADH mutant and have the same function, and thus fall within the scope of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 90% or more, or 95% or more, or 98% or more, or 99% or more identity to the nucleotide sequence of the present invention encoding the protein consisting of the amino acid sequence shown in SEQ ID No. 2. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above a2), the expression cassette containing the nucleic acid molecule encoding the alcohol dehydrogenase TbSADH mutant refers to a DNA capable of expressing the alcohol dehydrogenase TbSADH mutant in a host cell, and the DNA may include not only a promoter which initiates transcription of a gene encoding the alcohol dehydrogenase TbSADH mutant, but also a terminator which terminates transcription of a gene encoding the alcohol dehydrogenase TbSADH mutant. Still further, the expression cassette may further comprise an enhancer sequence.

In a3), the recombinant vector can be bacterial plasmid (such as expression vector based on T7 promoter expressed in bacteria, specifically pET-28a, etc.), phage, yeast plasmid (such as YEp series vector, etc.) or retrovirus packaging plasmid carrying the gene encoding the alcohol dehydrogenase TbSADH mutant or the expression cassette.

In the above a4), the recombinant microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium, carrying the gene encoding the alcohol dehydrogenase TbSADH mutant, or the expression cassette or the recombinant vector.

In order to solve the above technical problems, the present invention also provides a kit for preparing a diaryl alcohol or a chiral diaryl alcohol.

The kit for preparing a diaryl alcohol or a chiral diaryl alcohol provided by the invention comprises a diaryl ketone and the above alcohol dehydrogenase TbSADH mutant or the above biomaterial.

In order to solve the technical problems, the invention also provides a new application of the alcohol dehydrogenase TbSADH mutant or the biological material or the complete set of products.

The invention provides the application of the alcohol dehydrogenase TbSADH mutant or the biological material or the complete product in any one of the following b1) -b 4):

b1) synthesizing a diaryl alcohol or a chiral diaryl alcohol;

b2) preparing a product for synthesizing the diaryl alcohol or the chiral diaryl alcohol;

b3) catalyzing the diaryl ketone to generate diaryl alcohol or chiral diaryl alcohol;

b4) preparing a product for catalyzing diaryl ketone to generate diaryl alcohol or chiral diaryl alcohol.

In order to solve the technical problems, the invention finally provides a method for synthesizing the diaryl alcohol or the chiral diaryl alcohol.

The synthesis method of the diaryl alcohol or the chiral diaryl alcohol provided by the invention comprises the following steps: and (3) carrying out catalytic reaction by using the alcohol dehydrogenase TbSADH mutant by using diaryl ketone as a substrate to obtain diaryl alcohol or chiral diaryl alcohol.

In the method, the alcohol dehydrogenase TbSADH mutant can perform catalysis in the form of crude enzyme liquid, crude enzyme liquid freeze-dried powder (crude enzyme powder) or whole cells.

Further, the whole cell of the alcohol dehydrogenase TbSADH mutant can be prepared by a method comprising the following steps: expressing the alcohol dehydrogenase TbSADH mutant in a host cell to obtain a recombinant cell, namely the whole cell;

lysing the recombinant cells to obtain the crude enzyme solution;

and (3) freeze-drying the crude enzyme solution to obtain the crude enzyme solution freeze-dried powder (crude enzyme powder).

Still further, the recombinant cell can be prepared according to a method comprising the following steps: introducing the coding gene of the alcohol dehydrogenase TbSADH mutant into a host cell, and obtaining the recombinant cell expressing the alcohol dehydrogenase TbSADH mutant after induction culture.

Further, the gene encoding the alcohol dehydrogenase TbSADH mutant is introduced into the host cell in the form of a recombinant vector. Wherein, the recombinant vector can be a bacterial plasmid (such as an expression vector based on a T7 promoter expressed in bacteria, specifically pET-28a and the like), a bacteriophage, a yeast plasmid (such as YEp series vectors and the like) or a retrovirus packaging plasmid, wherein the bacterial plasmid carries the coding gene of the alcohol dehydrogenase TbSADH mutant.

In one embodiment of the invention, the recombinant vector is specifically a recombinant plasmid obtained by replacing a small fragment between the NcoI and AvrII cleavage sites of pRSFDuet-1 with the gene encoding the alcohol dehydrogenase TbSADH mutant. The coding gene of the alcohol dehydrogenase TbSADH mutant is the gene of 1) to 25) above.

In the method, the host cell may be a prokaryotic cell or a lower eukaryotic cell.

Further, the prokaryotic cell may specifically be a bacterium; the lower eukaryotic cell may specifically be a yeast cell.

Further, the host cell is specifically Escherichia coli.

In one embodiment of the invention, the host cell is e.coli BL21(DE 3). Accordingly, the induction culture is carried out by adding IPTG to the culture system to a final concentration of 0.05-1.0mmol/L (e.g., 0.1mmol/L) and inducing the culture at 20-30 deg.C (e.g., 20 deg.C) for 10-20 hours (e.g., 18 hours).

In the method, when the alcohol dehydrogenase TbSADH mutant is used for catalytic reaction, the reaction system can also contain isopropanol and a coenzyme of the alcohol dehydrogenase TbSADH mutant besides the substrate and the alcohol dehydrogenase TbSADH mutant.

Further, the coenzyme may be NADP⁺Or NAD⁺。

Further, the catalytic reaction using the alcohol dehydrogenase TbSADH mutant is carried out in a phosphate buffer solution having a concentration of 0.001-0.1mol/L (e.g., 0.05mol/L), pH 6-8 (e.g., pH 7.4).

The concentration of the substrate in the reaction system is 1-100mmol/L (such as 10mmol/L or 20mmol/L or 100 mmol/L); the concentration of the crude enzyme powder of the alcohol dehydrogenase TbSADH mutant in the reaction system is 1-100g/L (such as 1g/L or 10 g/L); the concentration of the whole alcohol dehydrogenase mutant cells in the reaction system is 50-500g/L (such as 100 g/L); the isopropyl alcoholThe volume percentage content in the reaction system is 10-30 percent (such as 10 percent); the NADP⁺Or the NAD⁺The concentration in the reaction system is 0.1-1.0mmol/L (e.g., 1.0 mmol/L).

In the method, when the alcohol dehydrogenase TbSADH mutant is used for catalytic reaction, the temperature of the catalytic reaction can be 20-35 ℃, and specifically can be 30 ℃; the reaction time is based on the completion of the reaction, and may be generally 1 to 24 hours, specifically 24 hours.

In the method, the method also comprises a step of extracting the diaryl alcohol from the reaction liquid according to a conventional method in the field after the reaction is finished.

In any of the above products or uses or methods, the bisaryl ketone can be any of: (4-chlorophenyl) pyridine-2-methanone, acetophenone, (4-fluorophenyl) pyridine-2-methanone, (4-tolyl) pyridine-2-methanone, (4-methoxyphenyl) pyridine-2-methanone, (2-tolyl) pyridine-2-methanone, (3-chlorophenyl) pyridine-2-methanone, 4-chlorobenzophenone, and 4-nitrobenzophenone. In a specific embodiment of the present invention, the bisaryl ketone is (4-chlorophenyl) pyridin-2-one, acetophenone, (2-tolyl) pyridin-2-one or (4-tolyl) pyridin-2-one.

The diaryl alcohol or chiral diaryl alcohol may be any one of the following: (4-chlorophenyl) pyridine-2-methanol, phenethyl alcohol, (4-fluorophenyl) pyridine-2-methanol, (4-tolyl) pyridine-2-methanol, (4-methoxyphenyl) pyridine-2-methanol, (2-tolyl) pyridine-2-methanol, (3-chlorophenyl) pyridine-2-methanol, 4-chlorodiphenylmethanol, and 4-nitrodiphenylmethanol. In a specific embodiment of the invention, the diarylalcohol is (4-chlorophenyl) pyridine-2-methanol, phenethyl alcohol, (2-tolyl) pyridine-2-methanol or (4-tolyl) pyridine-2-methanol. The (4-chlorphenyl) pyridine-2-methanol is (S) - (4-chlorphenyl) pyridine-2-methanol and/or (R) - (4-chlorphenyl) pyridine-2-methanol; the phenethyl alcohol is (S) -phenethyl alcohol and/or (R) -phenethyl alcohol; the (2-tolyl) pyridine-2-methanol is (S) - (2-tolyl) pyridine-2-methanol and/or (R) - (2-tolyl) pyridine-2-methanol; the (4-tolyl) pyridine-2-methanol is (S) - (4-tolyl) pyridine-2-methanol and/or (R) - (4-tolyl) pyridine-2-methanol.

The invention adopts rapid semi-rational design to directionally evolve and reform alcohol dehydrogenase TbSADH, obtains a series of mutants which have enzyme activity on diaryl ketones represented by (4-chlorphenyl) pyridine-2-ketone, and the mutants can catalyze asymmetric reduction of a substrate (4-chlorphenyl) pyridine-2-ketone to generate (S) - (4-chlorphenyl) pyridine-2-methanol and/or (R) - (4-chlorphenyl) pyridine-2-methanol. The method has the advantages of high yield, high ee value, less coenzyme usage, no need of additionally adding enzymes for cofactor circulation such as glucose dehydrogenase and the like, mild reaction conditions, simple operation and the like. Experiments prove that: the wild-type alcohol dehydrogenase TbSADH before modification has no activity on a substrate (4-chlorphenyl) pyridine-2-methanone, and the modified optimal (S) specificity mutant can catalyze the (4-chlorphenyl) pyridine-2-methanone to generate (S) - (4-chlorphenyl) pyridine-2-methanol, wherein the conversion rate is more than 99%, and the ee% is more than 99% (S); the most preferred (R) -specific mutant catalyzes the formation of (R) - (4-chlorophenyl) pyridine-2-methanone to (R) - (4-chlorophenyl) pyridine-2-methanol with a conversion of 98% and an ee% of 99% (R).

Drawings

FIG. 1 is a diagram showing the catalytic reduction of (4-chlorophenyl) pyridine-2-methanone to (S) - (4-chlorophenyl) pyridine-2-methanol and the production of (R) - (4-chlorophenyl) pyridine-2-methanol by an alcohol dehydrogenase TbSADH mutant.

FIG. 2 is a chart showing the HPLC detection results of reactions of catalyzing reduction of (4-chlorophenyl) pyridine-2-methanone to (S) - (4-chlorophenyl) pyridine-2-methanol and reaction to produce (R) - (4-chlorophenyl) pyridine-2-methanol by the alcohol dehydrogenase TbSADH mutant. A: liquid chromatography results of (4-chlorphenyl) pyridine-2-ketone and racemic (4-chlorphenyl) pyridine-2-methanol standard substances; b: the liquid chromatography result of the reaction liquid of the negative control group; c: experimental group 1 (mutant catalysis) reaction liquid chromatography results; d: experimental group 2 (mutant catalysis) reaction solution liquid chromatography results. The negative control in the figure is the reaction catalyzed by the empty vector plasmid pRSFDuet-1 (Novagen); mutant 1 is TbSADH mutant P84S/I86C; mutant 2 is TbSADH mutant H42T/. DELTA.P 84/A85S.

FIG. 3 is a graph showing the whole-cell reaction process of TbSADH mutant catalyzing (4-chlorophenyl) pyridine-2-methanone to produce (S) - (4-chlorophenyl) pyridine-2-methanol (substrate concentration 100 mM). The abscissa is the reaction time and the ordinate is the substrate conversion. The mutants tested included P84S/I86A, P84S/I86C, P84S/I86L and A85G/I86L.

FIG. 4 is a graph showing the reaction process of crude enzyme powder of TbSADH mutant catalyzing (4-chlorophenyl) pyridine-2-methanone to produce (R) - (4-chlorophenyl) pyridine-2-methanol (substrate concentration 20 mM). The abscissa is the reaction time and the ordinate is the substrate conversion. The mutants tested included Δ P84/A85G, Δ P84/A85S/H42T and Δ P84/A85G/I86V.

FIG. 5 shows the conversion rate and enantioselectivity results of crude TbSADH mutant enzyme powder catalyzing other diaryl ketone substrates. The mutants tested included P84S/I86A (T84), P84S/I86C (T85), P84S/I86L (T89), Δ P84/A85G (T110), Δ P84/A85S/H42T (T124), and Δ P84/A85G/I86V (T129). Note: mutants that are inactive on the substrate do not appear.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 alcohol dehydrogenase TbSADH mutant and sequence thereof

The alcohol dehydrogenase described in this example is derived from Thermoanaerobacterium (Thermoanaerobacter brockii) named TbSADH, and the amino acid sequence thereof is shown in SEQ ID No. 2. The alcohol dehydrogenase TbSADH mutant in this example is specifically any one of the following (1) to (23):

(1) alcohol dehydrogenase TbSADH mutant Δ P84: a protein obtained by knocking out (delta) the 84 th amino acid proline of the amino acid sequence of the alcohol dehydrogenase TbSADH and keeping other amino acid sequences unchanged;

(2) alcohol dehydrogenase TbSADH mutant P84S/A85G: a protein obtained by mutating the 84 th amino acid of the amino acid sequence of the alcohol dehydrogenase TbSADH from proline (P) to serine (S), and mutating the 85 th amino acid from alanine (A) to glycine (G), while keeping other amino acid sequences unchanged;

(3) alcohol dehydrogenase TbSADH mutant P84S/I86G: protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to serine (S), and mutating 86 th amino acid from isoleucine (I) to glycine (G), and keeping other amino acid sequences unchanged;

(4) alcohol dehydrogenase TbSADH mutant P84S/I86A: protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to serine (S), and mutating 86 th amino acid from isoleucine (I) to alanine (A), and keeping other amino acid sequences unchanged;

(5) alcohol dehydrogenase TbSADH mutant P84S/I86C: protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to serine (S), and mutating 86 th amino acid from isoleucine (I) to cysteine (C), and keeping other amino acid sequences unchanged;

(6) alcohol dehydrogenase TbSADH mutant P84S/I86S: protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to serine (S), and mutating 86 th amino acid from isoleucine (I) to serine (S), and keeping other amino acid sequences unchanged;

(7) alcohol dehydrogenase TbSADH mutant P84S/I86M: protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to serine (S), and mutating 86 th amino acid from isoleucine (I) to methionine (M), and keeping other amino acid sequences unchanged;

(8) alcohol dehydrogenase TbSADH mutant P84S/I86L: protein obtained by mutating 84 th amino acid of amino acid sequence of alcohol dehydrogenase TbSADH from proline (P) to serine (S), and mutating 86 th amino acid from isoleucine (I) to leucine (L), and keeping other amino acid sequences unchanged;

(9) alcohol dehydrogenase TbSADH mutant delta P84/A85G: a protein obtained by knocking out (delta) the 84 th amino acid proline (P) of the amino acid sequence of the alcohol dehydrogenase TbSADH, mutating the 85 th amino acid from alanine (A) to glycine (G), and keeping other amino acid sequences unchanged;

(10) alcohol dehydrogenase TbSADH mutant delta P84/A85S: a protein obtained by knocking out (delta) the 84 th amino acid proline (P) of the amino acid sequence of the alcohol dehydrogenase TbSADH, mutating the 85 th amino acid from alanine (A) to serine (S), and keeping other amino acid sequences unchanged;

(11) alcohol dehydrogenase TbSADH mutant delta P84/I86G: the protein is obtained by knocking out (delta) of 84 th amino acid proline (P) of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine (I) to glycine (G), and keeping other amino acid sequences unchanged;

(12) alcohol dehydrogenase TbSADH mutant delta P84/I86A: a protein obtained by knocking out (delta) the 84 th amino acid proline (P) of the amino acid sequence of the alcohol dehydrogenase TbSADH, mutating the 86 th amino acid from isoleucine (I) to alanine (A), and keeping other amino acid sequences unchanged;

(13) alcohol dehydrogenase TbSADH mutant delta P84/I86S: the protein is obtained by knocking out (delta) of 84 th amino acid proline (P) of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine (I) to serine (S), and keeping other amino acid sequences unchanged;

(14) alcohol dehydrogenase TbSADH mutant delta P84/I86M: the protein is obtained by knocking out (delta) of 84 th amino acid proline (P) of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine (I) to methionine (M), and keeping other amino acid sequences unchanged;

(15) alcohol dehydrogenase TbSADH mutant delta P84/I86C: the protein is obtained by knocking out (delta) the 84 th amino acid proline (P) of the amino acid sequence of the alcohol dehydrogenase TbSADH, mutating the 86 th amino acid from isoleucine (I) to cysteine (C), and keeping other amino acid sequences unchanged;

(16) alcohol dehydrogenase TbSADH mutant delta P84/I86L: the protein is obtained by knocking out (delta) of 84 th amino acid proline (P) of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine (I) to leucine (L), and keeping other amino acid sequences unchanged;

(17) alcohol dehydrogenase TbSADH mutant delta P84/H42T: a protein obtained by knocking out (delta) the 84 th amino acid proline (P) of the amino acid sequence of the alcohol dehydrogenase TbSADH, mutating the 42 th amino acid from histidine (H) to threonine (T), and keeping other amino acid sequences unchanged;

(18) the alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86L: the protein is obtained by knocking out (delta) of 84 th amino acid proline (P) of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine (A) to glycine (G), mutating 86 th amino acid from isoleucine (I) to leucine (L), and keeping other amino acid sequences unchanged;

(19) the alcohol dehydrogenase TbSADH mutant delta P84/A85S/I86L: the protein is obtained by knocking out (delta) of 84 th amino acid proline (P) of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine (A) to serine (S), mutating 86 th amino acid from isoleucine (I) to leucine (L), and keeping other amino acid sequences unchanged;

(20) the alcohol dehydrogenase TbSADH mutant delta P84/A85S/H42T: a protein obtained by knocking out (delta) the 84 th amino acid proline (P) of the amino acid sequence of the alcohol dehydrogenase TbSADH, mutating the 85 th amino acid from alanine (A) to serine (S), mutating the 42 th amino acid from histidine (H) to threonine (T), and keeping other amino acid sequences unchanged;

(21) the alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86V: the protein is obtained by knocking out (delta) of 84 th amino acid proline (P) of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine (A) to glycine (G), mutating 86 th amino acid from isoleucine (I) to valine (V) and keeping other amino acid sequences unchanged;

(22) the alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86T: the protein is obtained by knocking out (delta) of 84 th amino acid proline (P) of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine (A) to glycine (G), mutating 86 th amino acid from isoleucine (I) to threonine (T), and keeping other amino acid sequences unchanged;

(23) the alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86C: the protein is obtained by knocking out (delta) the 84 th amino acid proline (P) of the amino acid sequence of the alcohol dehydrogenase TbSADH, mutating the 85 th amino acid from alanine (A) to glycine (G), mutating the 86 th amino acid from isoleucine (I) to cysteine (C), and keeping other amino acid sequences unchanged.

Example 2 preparation of engineering bacteria of alcohol dehydrogenase TbSADH Gene or its mutant

Preparation of alcohol dehydrogenase TbSADH gene wild type gene engineering strain

1. Optimization of alcohol dehydrogenase TbSADH gene sequence

The method comprises the steps of carrying out codon optimization on an alcohol dehydrogenase TbSADH gene derived from high-temperature anaerobic bacillus (Thermoanaerobacter brockii) by taking escherichia coli as a host cell, carrying out whole-gene synthesis after optimization, wherein the sequence of the optimized alcohol dehydrogenase TbSADH gene is shown as SEQ ID No. 1.

2. Construction of recombinant vector pRSFDuet-1-TbSADH

The DNA fragment shown in SEQ ID No.1 was homologously recombined onto the pRSFDuet-1 plasmid (Novagen) using the primers pRSF-NcoI (ggt ata tct tat taa agt taa aca aaa ttaa tta cagg) and pRSF-AvrII (taa cct agg ctg ctg cca ccg ctg agc aac). A recombinant plasmid, which was sequenced to show that a small fragment between the cleavage sites NcoI and AvrII of the pRSFDuet-1 plasmid (Novagen) was the DNA fragment shown in SEQ ID No.1, was designated pRSFDuet-1-TbSADH.

3. Construction of recombinant bacterium

And (2) electrically transferring the recombinant vector pRSFDuet-1-TbSADH into E.coli BL21(DE3) competent cells, carrying out inverted culture in an LB solid plate containing kanamycin (Kan) resistance for 12-16h, selecting positive transformants, carrying out colony PCR and DNA sequencing verification, and verifying the correct transformants to be alcohol dehydrogenase TbSADH gene wild-type genetic engineering strains.

Preparation of di-alcohol dehydrogenase TbSADH gene mutant engineering strain

Engineering strains of alcohol dehydrogenase TbSADH gene mutants expressing the respective alcohol dehydrogenase TbSADH mutants in example 1 were constructed, respectively. Part of the alcohol dehydrogenase TbSADH mutant is obtained by designing a degenerate primer to construct a mutant library and screening, and the other part of the alcohol dehydrogenase TbSADH mutant is obtained by designing a mutant primer. The designed degenerate primer and mutant primer sequences are shown in tables 1 and 2, respectively.

TABLE 1 primer sequences used for mutant library construction

TABLE 2 mutants and primer sequences used

Note: the mutation sites are underlined.

The specific construction method comprises the following steps:

1. PCR reaction

And (3) taking the recombinant vector pRSFDuet-1-TbSADH constructed in the step one as a template, and respectively carrying out two rounds of PCR reactions by adopting primers corresponding to the mutants. The PCR reaction system and procedure were as follows:

the first round of PCR system is 50 μ L, and comprises the following components: 50ng of template (pRSFDuet-1-TbSADH); PrimeStar DNA polymerase (2.5U/. mu.L) 0.5. mu.L; dNTP (2.5mmol/L) 4. mu.L; 2 XPS Buffer 25 uL; ddH₂18.5 mu L of O; 1 μ L of the pre-primer (10 μ M); the rear primer (10. mu.M) was 1. mu.L. Specific sequences of primers used for each mutant are shown in tables 1 and 2.

First round PCR procedure: pre-denaturation at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 30 seconds; annealing at 56 deg.C for 15 seconds; extension at 72 ℃ for 40 seconds; final extension 72 ℃ for 10 min. The number of cycles was 32.

The second round of PCR system was 50. mu.L, comprising the following components: 50ng of template (pRSFDuet-1-TbSADH); PrimeStar DNA polymerase (2.5U/. mu.L) 0.5. mu.L; dNTP (2.5mmol/L) 4. mu.L; 2 XPS Buffer 25 uL; ddH₂18.5 mu L of O; first round PCR product 2. mu.L.

Second round PCR procedure: pre-denaturation at 95 ℃ for 2 minutes; denaturation at 95 ℃ for 30 seconds; annealing at 60 ℃ for 15 seconds; extension at 72 ℃ for 7 minutes; final extension 72 ℃ for 10 min. The number of cycles was 28.

2. Obtaining and screening of alcohol dehydrogenase TbSADH gene mutant engineering strain

After the step 1 is completed, adding 2 mu L of Dpn I enzyme into each reaction system, digesting for 2 hours at 37 ℃, taking 1 mu L of Dpn I enzyme to electrically transfer into E.coli BL21(DE3) competent cells, putting the cells into a 37 ℃ incubator for inverted culture for 12-16 hours, when transformants grow out, picking transformants for sequencing verification, and respectively naming the transformants with correct sequencing verification as alcohol dehydrogenase TbSADH gene mutant engineering strains, namely recombinant strain delta P84, recombinant strain P84S/A85G, recombinant strain P84S/I86G, recombinant strain P84G/I86G, recombinant strain P84/I G, recombinant strain delta P72/I86G, recombinant strain delta P G/G, recombinant strain delta P72/I72, recombinant strain delta P72/G, recombinant strain delta P72/I G and recombinant strain delta P72/I G/G, Recombinant bacteria delta P84/I86L, recombinant bacteria delta P84/H42T, recombinant bacteria delta P84/A85G/I86L, recombinant bacteria delta P84/A85S/I86L, recombinant bacteria delta P84/A85S/H42T, recombinant bacteria delta P84/A85G/I86V, recombinant bacteria delta P84/A85G/I86T and recombinant bacteria delta P84/A85G/I86C.

The recombinant strain delta P84 is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84 into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84 is obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH.

The recombinant strain P84S/A85G is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84S/A85G into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84S/A85G is obtained by mutating the 250 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base C to a base T and mutating the 254 th site from a base C to a base G.

The recombinant strain P84S/I86G is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84S/I86G into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84S/I86G is obtained by mutating the 250 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from base C to base T, mutating the 256 th site from base A to base G, and mutating the 257 th site from base T to base G.

The recombinant strain P84S/I86A is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84S/I86A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84S/I86A is obtained by mutating the 250 th position of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH from a base C to a base T, mutating the 256 th position from a base A to a base G, mutating the 257 th position from a base T to a base C, and mutating the 258 th position from a base T to a base A.

The recombinant strain P84S/I86C is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84S/I86C into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84S/I86C is obtained by mutating the 250 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from base C to base T, mutating the 256 th site from base A to base T, and mutating the 257 th site from base T to base G.

The recombinant strain P84S/I86S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84S/I86S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84S/I86S is obtained by mutating the 250 th base C to the base T and the 257 th base T to the base G of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH.

The recombinant strain P84S/I86M is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84S/I86M into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84S/I86M is obtained by mutating the 250 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from a base C to a base T and mutating the 258 th site from a base T to a base G.

The recombinant strain P84S/I86L is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant P84S/I86L into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant P84S/I86L is obtained by mutating the 250 th site of the alcohol dehydrogenase TbSADH wild-type gene of pRSFDuet-1-TbSADH from base C to base T, mutating the 256 th site from base A to base C, and mutating the 258 th site from base T to base G.

The recombinant strain delta P84/A85G is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/A85G into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/A85G is obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH and mutating the 254 th position from a base C to a base G.

The recombinant strain delta P84/A85S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/A85S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/A85S is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 253 rd position from a base G to a base A and mutating the 254 th position from a base C to a base G.

The recombinant strain delta P84/I86G is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/I86G into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/I86G is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 256 th position from the base A to the base G and mutating the 257 th position from the base T to the base G.

The recombinant strain delta P84/I86A is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/I86A into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/I86A is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 256 th position from the base A to the base G, mutating the 257 th position from the base T to the base C, and mutating the 258 th position from the base T to the base A.

The recombinant strain delta P84/I86S is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/I86S into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/I86S is obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH and mutating the 257 th position from a base T to a base G.

The recombinant strain delta P84/I86M is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/I86M into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/I86M is obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH and mutating the 258 th position from a base T to a base G.

Recombinant bacteria delta P84/I86C are obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/I86C into host bacteria. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/I86C is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 256 th position from the base A to the base T and mutating the 257 th position from the base T to the base G.

The recombinant strain delta P84/I86L is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/I86L into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/I86L is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 256 th position from a base A to a base C and mutating the 258 th position from a base T to a base G.

The recombinant strain delta P84/H42T is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/H42T into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/H42T is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 124 th position from a base C to a base A, mutating the 125 th position from a base A to a base C, and mutating the 126 th position from a base T to a base C.

The recombinant strain delta P84/A85G/I86L is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86L into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86L is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base C, and mutating the 258 th position from a base T to a base G.

The recombinant strain delta P84/A85S/I86L is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/A85S/I86L into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/A85S/I86L is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 253 rd position from a base G to a base A, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base C, and mutating the 258 th position from a base T to a base G.

The recombinant strain delta P84/A85S/H42T is a strain obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/A85S/H42T into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/A85S/H42T is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 253 rd position from a base G to a base A, mutating the 254 th position from a base C to a base G, mutating the 124 th position from a base C to a base A, mutating the 125 th position from a base A to a base C and mutating the 126 th position from a base T to a base C.

The recombinant strain delta P84/A85G/I86V is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86V into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86V is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 254 th position from a base C to a base G and mutating the 256 th position from a base A to a base G.

The recombinant strain delta P84/A85G/I86T is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86T into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86T is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 254 th position from a base C to a base G, mutating the 257 th position from a base T to a base C, and mutating the 258 th position from a base T to a base C.

The recombinant strain delta P84/A85G/I86C is obtained by introducing a vector expressing an alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86C into a host strain. The vector for expressing the alcohol dehydrogenase TbSADH mutant delta P84/A85G/I86C is obtained by removing the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild-type gene in pRSFDuet-1-TbSADH, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base T, and mutating the 257 th position from a base T to a base G.

Example 3 expression of alcohol dehydrogenase TbSADH Gene or its mutant and preparation of Whole cell, crude enzyme solution and crude enzyme powder

The wild-type gene engineering strain of the alcohol dehydrogenase TbSADH gene prepared in the first step of the embodiment 2 and the 23 engineering strains of the alcohol dehydrogenase TbSADH gene mutant prepared in the second step are induced to express to obtain the wild-type alcohol dehydrogenase TbSADH and each alcohol dehydrogenase TbSADH mutant in the embodiment 1. The method comprises the following specific steps:

respectively selecting recombinant bacteria transformant containing recombinant plasmid of alcohol dehydrogenase TbSADH gene or mutant thereof to 5mL LB liquid culture medium containing 50 ug/mL kanamycin, shaking at 37 ℃ and 220rpm overnight for 12-16 hours, respectively inoculating to TB liquid culture medium containing 50 ug/mL kanamycin according to 1% (volume percentage content), and culturing at 37 ℃ to OD₆₀₀When the concentration is 0.7, adding IPTG with the final concentration of 0.1mmol/L, inducing expression at 20 ℃, 220rpm for 18h, centrifuging at 4 ℃, 4000rpm for 10min, collecting thalli, re-suspending the collected thalli with potassium phosphate buffer solution (50mM, pH 7.4) to obtain alcohol dehydrogenase TbSADH whole cells, ultrasonically crushing the thalli cells under the ice bath condition to obtain an ultrasonically crushed sample (namely crude enzyme solution), centrifuging the ultrasonically crushed sample at 4 ℃, 12000rpm for 10min, taking supernatant, and performing centrifugation at-8 DEG CFreezing at 0 deg.C, and freeze-drying with vacuum drying instrument to obtain crude enzyme powder.

Example 4 alcohol dehydrogenase TbSADH and its mutant crude enzyme powder catalyzing production of (S) - (4-chlorophenyl) pyridine-2-methanone and/or (R) - (4-chlorophenyl) pyridine-2-methanol from (4-chlorophenyl) pyridine-2-methanone

The wild-type alcohol dehydrogenase TbSADH and each of the alcohol dehydrogenase TbSADH mutants prepared in example 3 were used to catalyze the formation of (S) - (4-chlorophenyl) pyridine-2-methanol and/or (R) - (4-chlorophenyl) pyridine-2-methanol from (4-chlorophenyl) pyridine-2-methanone, respectively, to determine the catalytic effect on the asymmetric reduction of prochiral bisarylketone (4-chlorophenyl) pyridine-2-methanone. A schematic diagram of the catalytic reduction of (4-chlorophenyl) pyridine-2-methanone to (S) - (4-chlorophenyl) pyridine-2-methanol and/or (R) - (4-chlorophenyl) pyridine-2-methanol is shown in FIG. 1.

The components of the asymmetric reduction reaction system and the concentration thereof in the system are respectively as follows: substrate ((4-chlorophenyl) pyridine-2-one) 10mmol/L, recombinant alcohol dehydrogenase TbSADH (alcohol dehydrogenase TbSADH mutant, crude enzyme powder, prepared in example 3) 10g/L, isopropanol 10% (volume fraction), NADP⁺1mmol/L, phosphate buffer (50mmol/L, pH 7.4). The asymmetric reduction reaction was carried out at 30 ℃ for 24 hours.

After the reaction was completed, the conversion was calculated and stereoselectivity analysis was performed. The specific method comprises the following steps: and (3) adding 500 mu L of reaction liquid into 500 mu L of ethyl acetate, shaking for 1-2 min, centrifuging at 12000rpm for 2-5 min, taking the supernatant into a centrifuge tube, adding anhydrous sodium sulfate, drying at 4 ℃ overnight, taking the supernatant into the centrifuge tube, adding 500 mu L of chromatographic grade isopropanol when the organic phase is completely naturally volatilized, and carrying out liquid phase analysis on the conversion rate and the ee value. The HPLC detection conditions were as follows: chiralpak AD-H (5 μm, 250 mm. times.4.6 mm) liquid chromatography column, mobile phase n-hexane: isopropanol (90:10, V/V), flow rate of 1mL/min, column temperature of 30 ℃, ultraviolet detection wavelength of 220nm, and sample injection amount of 1 muL.

The HPLC detection result spectrum of the reaction for catalyzing and reducing (4-chlorophenyl) pyridine-2-methanone to generate (S) - (4-chlorophenyl) pyridine-2-methanol and/or (R) - (4-chlorophenyl) pyridine-2-methanol by the alcohol dehydrogenase TbSADH mutant is shown in figure 2. The retention time of (S) - (4-chlorophenyl) pyridine-2-methanol was 9.8min, and the retention time of (R) - (4-chlorophenyl) pyridine-2-methanol was 12.2 min. The optical purity of the product was evaluated by the enantiomeric excess (ee): ee ═ (AS-AR)/(AS + AR) × 100%; the conversion calculation formula is: c (conversion) ═ (AS + AR)/(AS + AR + S) × 100%; AS: peak area values of (S) - (4-chlorophenyl) pyridine-2-methanol obtained by liquid chromatography; AR: peak area values of (R) - (4-chlorophenyl) pyridine-2-methanol obtained by liquid chromatography; s: and analyzing the peak area value of the obtained substrate (4-chlorphenyl) pyridine-2-ketone by liquid chromatography.

The results are shown in Table 3. As shown in Table 3, the mutant of alcohol dehydrogenase TbSADH can catalyze asymmetric reduction catalysis (4-chlorophenyl) pyridine-2-ketone to generate (S) - (4-chlorophenyl) pyridine-2-methanol and/or (R) - (4-chlorophenyl) pyridine-2-methanol, the conversion rate is 1-99%, and the stereoselectivity is 14-99%.

TABLE 3 analysis of conversion and stereoselectivity of alcohol dehydrogenase TbSADH and its mutant (crude enzyme powder) for asymmetric reduction catalysis of (4-chlorophenyl) pyridine-2-ketone

Note: WT stands for wild type TbSADH and "-" for too low an ee value for conversion is not shown.

Example 5 Whole cell/crude enzyme powder amplification reaction of alcohol dehydrogenase mutant catalyzing production of (S) - (4-chlorophenyl) pyridine-2-methanone and/or (R) - (4-chlorophenyl) pyridine-2-methanol

The catalytic effect of (4-chlorophenyl) pyridine-2-methanol and/or (R) - (4-chlorophenyl) pyridine-2-methanol on prochiral bisarylketone (4-chlorophenyl) pyridine-2-ketone before asymmetric reduction is determined by respectively using whole cells or crude enzyme powder of the alcohol dehydrogenase TbSADH mutant (P84S/I86A, P84S/I86C, P84S/I86L, delta P84/A85G, delta P84/A85S/H42T, delta P84/A85G/I86V) with high conversion rate in Table 3 to catalyze (4-chlorophenyl) pyridine-2-ketone. A schematic diagram of the catalytic reduction of (4-chlorophenyl) pyridine-2-methanone to (S) - (4-chlorophenyl) pyridine-2-methanol and/or (R) - (4-chlorophenyl) pyridine-2-methanol is shown in FIG. 1. The A85G/I86L alcohol dehydrogenase TbSADH mutant in the patent with application publication No. CN 111100851A is used as a control.

The components of the asymmetric reduction reaction system and the concentration thereof in the system are respectively as follows: substrate ((4-chlorphenyl) pyridine-2-ketone) 20mmol/L (mutants used are delta P84/A85G, delta P84/A85S/H42T, delta P84/A85G/I86V) or 100mmol/L (mutants used are P84S/I86A, P84S/I86C, P84S/I86L and A85G/I86L in the invention patent of CN 111100851A), recombinant alcohol dehydrogenase TbSADH (whole cell 100g/L or crude enzyme powder 1mg/mL), isopropanol 10% (volume fraction), phosphate buffer (50mmol/L, pH 7.4), and asymmetric reduction reaction conditions are 30 ℃.

After the reaction was completed, the conversion was calculated and stereoselectivity analysis was performed. The specific method comprises the following steps: and (3) adding 500 mu L of ethyl acetate into 500 mu L of reaction liquid, shaking for 1-2 min, centrifuging at 12000rpm for 2-5 min, taking supernatant into a centrifuge tube, heating or naturally volatilizing the ethyl acetate, then adding 500 mu L of chromatographic grade isopropanol, and performing liquid phase analysis on the conversion rate and the ee value. The HPLC detection conditions were as follows: chiralpak AD-H (5 μm, 250 mm. times.4.6 mm) liquid chromatography column, mobile phase n-hexane: isopropanol (90:10, V/V), flow rate of 1mL/min, column temperature of 30 ℃, ultraviolet detection wavelength of 220nm, and sample injection amount of 5 muL.

The time-conversion data for the reactions of mutants P84S/I86A, P84S/I86C, P84S/I86L and A85G/I86L are shown in Table 4, with different reaction time periods selected: samples were collected at 1h, 2h, 3h, 4h, 6h, 8h, 10h and the corresponding conversions were examined (only conversion data are shown here since all 4 mutants had enantioselectivities above 99%). The substrate conversion rate in the reaction process of the TbSADH mutant is shown in FIG. 3 (T84, T85, T89 and T28 respectively represent P84S/I86A, P84S/I86C, P84S/I86L and A85G/I86L), the reaction rate of the alcohol dehydrogenase TbSADH mutant P84S/I86L to generate (S) - (4-chlorophenyl) pyridine-2-methanol is fastest, the reaction can basically achieve the complete conversion (2.02g) of the substrate (4-chlorophenyl) pyridine-2-ketone when the reaction is carried out for 2h, and the catalytic activity of 3 mutants (P84S/I86A, P84S/I86C and P84S/I86L) is far higher than that of A85G/I86L.

The time-conversion data for the reactions of mutants Δ P84/A85G, Δ P84/A85S/H42T and Δ P84/A85G/I86V are shown in Table 5, with different reaction time periods selected: samples were collected at 3h, 5h, 8h, 12h, 16h and 28h and tested for the corresponding conversion (since all 3 mutants had enantioselectivities above 95%, only conversion data are shown here). The substrate conversion rate in the reaction process of the alcohol dehydrogenase TbSADH mutant is shown in figure 4 (T110, T124 and T129 respectively represent delta P84/A85G, delta P84/A85S/H42T and delta P84/A85G/I86V), the reaction rate of the TbSADH mutant delta P84/A85S/H42T for generating (R) - (4-chlorophenyl) pyridine-2-methanol is fastest, and the reaction reaches 16H and basically reaches the complete conversion of the substrate (4-chlorophenyl) pyridine-2-ketone.

TABLE 4 analysis of time-conversion of 100mM amplification of (4-chlorophenyl) pyridine-2-methanone by Whole-cell asymmetric reduction of alcohol dehydrogenase

TABLE 5 analysis of time-conversion of 20mM amplification of (4-chlorophenyl) pyridine-2-one by asymmetric reduction of crude alcohol dehydrogenase powder

Example 6 catalysis of crude enzyme powder of alcohol dehydrogenase mutant on other bisaryl ketone substrates

Other diaryl ketone substrates are catalyzed by crude enzyme powder of wild-type alcohol dehydrogenase TbSADH and alcohol dehydrogenase TbSADH mutants (P84S/I86A, P84S/I86C, P84S/I86L, delta P84/A85G, delta P84/A85S/H42T and delta P84/A85G/I86V) prepared in example 3 respectively, so that the substrate spectrum of the mutants is widened. The diaryl ketone substrate is acetophenone (structure formula is shown as number 2 in figure 5), (2-tolyl) pyridine-2-ketone (structure formula is shown as number 3 in figure 5), (4-tolyl) pyridine-2-ketone (structure formula is shown as number 4 in figure 5), and the acetophenone, pyridine-2-ketone, and pyridine-2-ketone, respectively, the generated products are phenethyl alcohol, pyridine-2-methanol, and pyridine-2-methanol.

The components of the asymmetric reduction reaction system and the concentration thereof in the system are respectively as follows: substrate 10mmol/L, recombinant alcohol dehydrogenase TbSADH (alcohol dehydrogenase TbSADH mutant, crude enzyme powder, prepared in example 3) 1mg/mL, isopropanol 10% (volume fraction), NADP⁺1mmol/L, phosphate buffer (50mmol/L, pH 7.4). The asymmetric reduction reaction is carried out for 12 hours at the temperature of 30 ℃.

After the reaction was completed, the conversion was calculated and stereoselectivity analysis was performed. The specific method comprises the following steps: and (3) adding 500 mu L of ethyl acetate into 500 mu L of reaction liquid, shaking for 1-2 min, centrifuging at 12000rpm for 2-5 min, taking supernatant into a centrifuge tube, heating or naturally volatilizing the ethyl acetate, then adding 500 mu L of chromatographic grade isopropanol, and performing liquid phase analysis on the conversion rate and the ee value. The HPLC detection conditions are shown in Table 5 below.

TABLE 5 HPLC detection method for asymmetric reduction catalysis of other bisaryl ketone substrates by alcohol dehydrogenase TbSADH and mutants thereof

^aDetection conditions are as follows: the flow rate is 1 mL/min; the temperature is 30 ℃; the detection wavelength is 220 nm.

The HPLC detection results are shown in FIG. 5 (T84, T85, T89, T110, T124 and T129 respectively represent P84S/I86A, P84S/I86C, P84S/I86L, delta P84/A85G, delta P84/A85S/H42T and delta P84/A85G/I86V). As can be seen from FIG. 5, the mutant of alcohol dehydrogenase TbSADH has good catalytic activity on other 3 diaryl ketone substrates, except that it has good catalytic activity and enantioselectivity on substrate (4-chlorophenyl) pyridine-2-ketone.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences

<120> alcohol dehydrogenase mutant and application thereof in preparing chiral bisaryl alcohol compound by efficient asymmetric reduction

<160>2

<170>PatentIn version 3.5

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atgaaaggtt ttgcaatgct cagtatcggt aaagttggct ggattgagaa ggaaaagcct 60

gctcctggcc catttgatgc tattgtaaga cctctagctg tggccccttg cacttcggac 120

attcataccg tttttgaagg agccattggc gaaagacata acatgatact cggtcacgaa 180

gctgtaggtg aagtagttga agtaggtagt gaggtaaaag attttaaacc tggtgatcgc 240

gttgttgtgc cagctattac ccctgattgg cggacctctg aagtacaaag aggatatcac 300

cagcactccg gtggaatgct ggcaggctgg aaattttcga atgtaaaaga tggtgttttt 360

ggtgaatttt ttcatgtgaa tgatgctgat atgaatttag cacatctgcc taaagaaatt 420

ccattggaag ctgcagttat gattcccgat atgatgacca ctggttttca cggagctgaa 480

ctggcagata tagaattagg tgcgacggta gcagttttgg gtattggccc agtaggtctt 540

atggcagtcg ctggtgccaa attgcgtgga gccggaagaa ttattgccgt aggcagtaga 600

ccagtttgtg tagatgctgc aaaatactat ggagctactg atattgtaaa ctataaagat 660

ggtcctatcg aaagtcagat tatgaatcta actgaaggca aaggtgtcga tgctgccatc 720

atcgctggag gaaatgctga cattatggct acagcagtta agattgttaa acctggtggc 780

accatcgcta atgtaaatta ttttggcgaa ggagaggttt tgcctgttcc tcgtcttgaa 840

tggggttgcg gcatggctca taaaactata aaaggcgggc tatgccccgg tggacgtcta 900

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Met Lys Gly Phe Ala Met Leu Ser Ile Gly Lys Val Gly Trp Ile Glu

1 5 10 15

Lys Glu Lys Pro Ala Pro Gly Pro Phe Asp Ala Ile Val Arg Pro Leu

20 25 30

Ala Val Ala Pro Cys Thr Ser Asp Ile His Thr Val Phe Glu Gly Ala

35 40 45

Ile Gly Glu Arg His Asn Met Ile Leu Gly His Glu Ala Val Gly Glu

50 55 60

Val Val Glu Val Gly Ser Glu Val Lys Asp Phe Lys Pro Gly Asp Arg

65 70 75 80

Val Val Val Pro Ala Ile Thr Pro Asp Trp Arg Thr Ser Glu Val Gln

85 90 95

Arg Gly Tyr His Gln His Ser Gly Gly Met Leu Ala Gly Trp Lys Phe

100 105 110

Ser Asn Val Lys Asp Gly Val Phe Gly Glu Phe Phe His Val Asn Asp

115 120 125

Ala Asp Met Asn Leu Ala His Leu Pro Lys Glu Ile Pro Leu Glu Ala

130 135 140

Ala Val Met Ile Pro Asp Met Met Thr Thr Gly Phe His Gly Ala Glu

145 150 155 160

Leu Ala Asp Ile Glu Leu Gly Ala Thr Val Ala Val Leu Gly Ile Gly

165 170 175

Pro Val Gly Leu Met Ala Val Ala Gly Ala Lys Leu Arg Gly Ala Gly

180 185 190

Arg Ile Ile Ala Val Gly Ser Arg Pro Val Cys Val Asp Ala Ala Lys

195 200 205

Tyr Tyr Gly Ala Thr Asp Ile Val Asn Tyr Lys Asp Gly Pro Ile Glu

210 215 220

Ser Gln Ile Met Asn Leu Thr Glu Gly Lys Gly Val Asp Ala Ala Ile

225 230 235 240

Ile Ala Gly Gly Asn Ala Asp Ile Met Ala Thr Ala Val Lys Ile Val

245 250 255

Lys Pro Gly Gly Thr Ile Ala Asn Val Asn Tyr Phe Gly Glu Gly Glu

260 265 270

Val Leu Pro Val Pro Arg Leu Glu Trp Gly Cys Gly Met Ala His Lys

275 280 285

Thr Ile Lys Gly Gly Leu Cys Pro Gly Gly Arg Leu Arg Met Glu Arg

290 295 300

Leu Ile Asp Leu Val Phe Tyr Lys Arg Val Asp Pro Ser Lys Leu Val

305 310 315 320

Thr His Val Phe Arg Gly Phe Asp Asn Ile Glu Lys Ala Phe Met Leu

325 330 335

Met Lys Asp Lys Pro Lys Asp Leu Ile Lys Pro Val Val Ile Leu Ala

340 345 350

Claims

1. An alcohol dehydrogenase TbSADH mutant, characterized in that: the alcohol dehydrogenase TbSADH mutant is any one of the following (1) to (15):

the amino acid sequence of the alcohol dehydrogenase TbSADH is shown in SEQ ID NO. 2;

(2) knocking out 84 th amino acid proline of an amino acid sequence of the alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to glycine, and keeping other amino acid sequences unchanged to obtain protein;

(3) knocking out 84 th amino acid proline of an amino acid sequence of the alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to serine, and keeping other amino acid sequences unchanged to obtain protein;

(4) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to alanine, and keeping other amino acid sequences unchanged to obtain protein;

(5) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to methionine, and keeping other amino acid sequences unchanged to obtain protein;

(6) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged to obtain protein;

(7) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged to obtain protein;

(8) protein obtained by knocking out proline at amino acid 84 of alcohol dehydrogenase TbSADH and mutating histidine to threonine at amino acid 42 while keeping other amino acid sequences unchanged;

(9) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to glycine, mutating 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged to obtain protein;

(10) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to serine, mutating 86 th amino acid from isoleucine to leucine, and keeping other amino acid sequences unchanged to obtain protein;

(11) knocking out 84 th amino acid proline of an amino acid sequence of the alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to serine, mutating 42 th amino acid from histidine to threonine, and keeping other amino acid sequences unchanged to obtain protein;

(12) knocking out proline at amino acid 84 of the alcohol dehydrogenase TbSADH, mutating alanine at amino acid 85 to glycine, mutating isoleucine at amino acid 86 to valine, and keeping other amino acid sequences unchanged to obtain protein;

(13) knocking out 84 th amino acid proline of an amino acid sequence of the alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to glycine, mutating 86 th amino acid from isoleucine to threonine, and keeping other amino acid sequences unchanged to obtain protein;

(14) knocking out 84 th amino acid proline of an amino acid sequence of alcohol dehydrogenase TbSADH, mutating 85 th amino acid from alanine to glycine, mutating 86 th amino acid from isoleucine to cysteine, and keeping other amino acid sequences unchanged to obtain protein;

(15) a fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of a protein represented by any one of (1) to (14).

2. The biomaterial related to the alcohol dehydrogenase TbSADH mutant of claim 1, which is any one of the following a1) -a 4):

a1) a nucleic acid molecule encoding the alcohol dehydrogenase TbSADH mutant of claim 1;

a2) an expression cassette comprising the nucleic acid molecule of a 1);

3. The biomaterial of claim 2, wherein: the nucleic acid molecule encoding the alcohol dehydrogenase TbSADH mutant of claim 1 is any one of 1) to 15) below:

2) knocking out 250 th, 251 th and 252 th positions of alcohol dehydrogenase TbSADH wild type gene, and mutating 254 th position from base C to base G to obtain DNA molecule;

3) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 253 rd position from a base G to a base A and mutating the 254 th position from a base C to a base G;

4) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 256 th position from a base A to a base G, mutating the 257 th position from a base T to a base C and mutating the 258 th position from a base T to a base A;

5) knocking out 250 th, 251 th and 252 th positions of alcohol dehydrogenase TbSADH wild type gene, and mutating 258 th position from base T to base G to obtain DNA molecule;

6) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 256 th position from a base A to a base T and mutating the 257 th position from the base T to a base G;

7) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 256 th position from a base A to a base C and mutating the 258 th position from a base T to a base G;

8) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 124 th position from a base C to a base A, mutating the 125 th position from a base A to a base C and mutating the 126 th position from a base T to a base C;

9) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base C and mutating the 258 th position from a base T to a base G;

10) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of a wild-type gene of alcohol dehydrogenase TbSADH, mutating the 253 rd position from a base G to a base A, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base C, and mutating the 258 th position from a base T to a base G;

11) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of a wild-type gene of alcohol dehydrogenase TbSADH, mutating the 253 rd position from a base G to a base A, mutating the 254 th position from a base C to a base G, mutating the 124 th position from a base C to a base A, mutating the 125 th position from a base A to a base C, and mutating the 126 th position from a base T to a base C;

12) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 254 th position from a base C to a base G and mutating the 256 th position from a base A to a base G;

13) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 254 th position from a base C to a base G, mutating the 257 th position from a base T to a base C, and mutating the 258 th position from a base T to a base C;

14) a DNA molecule obtained by knocking out the 250 th, 251 th and 252 th positions of the alcohol dehydrogenase TbSADH wild type gene, mutating the 254 th position from a base C to a base G, mutating the 256 th position from a base A to a base T, and mutating the 257 th position from a base T to a base G;

15) a fusion sequence obtained after connecting a tag coding sequence to the 5 'end and/or the 3' end of the DNA molecule defined in 1) to 14);

4. A kit for preparing a bisaryl alcohol comprising a bisaryl ketone and the alcohol dehydrogenase TbSADH mutant of claim 1 or the biomaterial of claim 2 or 3.

5. Use of the alcohol dehydrogenase mutant of claim 1 or the biological material of claim 2 or 3 or the kit of parts of claim 4 in any of the following b1) -b 4):

b1) synthesizing diaryl alcohol;

b2) preparing synthetic diaryl alcohol;

b3) catalyzing the diaryl ketone to generate diaryl alcohol;

b4) preparing a product for catalyzing diaryl ketone to generate diaryl alcohol;

the diaryl ketone is (4-chlorphenyl) pyridine-2-ketone;

the diaryl alcohol is (4-chlorphenyl) pyridine-2-methanol.

6. A method for synthesizing diaryl alcohol comprises the following steps: carrying out catalytic reaction by using the alcohol dehydrogenase TbSADH mutant of claim 1 by using diaryl ketone as a substrate to obtain diaryl alcohol;

the diaryl ketone is (4-chlorphenyl) pyridine-2-ketone;

the diaryl alcohol is (4-chlorphenyl) pyridine-2-methanol.

7. The method of claim 6, wherein: the catalytic reaction condition is that the reaction is carried out for 1 to 24 hours at the temperature of between 20 and 35 ℃; the concentration of the substrate in the reaction system is 1-100 mmol/L.