CN118956817A

CN118956817A - Lipase mutant and its application

Info

Publication number: CN118956817A
Application number: CN202411401209.9A
Authority: CN
Inventors: 阮礼涛; 沈江伟; 张小明; 陈茜; 顾虹
Original assignee: Shanghai Aobo Biomedical Co ltd
Current assignee: Shanghai Aobo Biomedical Co ltd
Priority date: 2020-04-29
Filing date: 2020-04-29
Publication date: 2024-11-15
Also published as: CN113564144A; CN113564144B

Abstract

本发明公开了南极假丝酵母脂肪酶(PAL)和玉米丝黑穗病菌脂肪酶(SRL)突变体及其在不对称水解二酯底物制备系列光学纯产物中的应用。本发明利用半理性设计思路，通过对脂肪酶三维结构分析，考察了系列氨基酸位点对对映体选择性的影响；进一步对正向突变位点进行组合构建系列组合突变体，应用于不对称水解工艺，在室温(10‑40℃)和纯水相条件下制备系列S‑单酯产物，e.e.>95％，克服了野生型脂肪酶只能在较低温度和添加助溶剂的条件下才能达到较高选择性的局限，为脂肪酶不对称水解制备医药中间体产业化奠定基础。The present invention discloses mutants of Antarctic Candida albicans lipase (PAL) and corn head smut fungus lipase (SRL) and their application in asymmetric hydrolysis of diester substrates to prepare a series of optically pure products. The present invention uses a semi-rational design approach and investigates the effects of a series of amino acid sites on enantiomeric selectivity by analyzing the three-dimensional structure of lipase; further, a series of combined mutants are constructed by combining the forward mutation sites, which are applied to the asymmetric hydrolysis process to prepare a series of S-monoester products at room temperature (10-40°C) and pure water phase conditions, with e.e.>95%, overcoming the limitation that wild-type lipase can only achieve high selectivity under the conditions of lower temperature and addition of cosolvent, and laying a foundation for the industrialization of preparing pharmaceutical intermediates by asymmetric hydrolysis of lipase.

Description

Lipase mutant and application thereof

The application discloses a divisional application of China patent office, application number 202010354148.0 and Chinese patent application named lipase mutant and application thereof, which are filed 29 days of 04 month in 2020.

Technical Field

The invention relates to lipase mutants derived from candida antarctica and maize head smut and application thereof in preparing an optically pure medical intermediate by asymmetric hydrolysis, belonging to the technical field of bioengineering.

Background

Lipase is a multifunctional biocatalyst capable of catalyzing hydrolysis, esterification, transesterification and other reactions. Because the catalyst has the advantages of high catalytic activity, good stability, good stereoselectivity, broad-spectrum substrate specificity and the like in the water phase and the organic phase, the catalyst is widely applied to the enzyme catalytic synthesis process to prepare various optical pure high-grade medical intermediates. Lipase is used as a biocatalyst in key intermediate preparation processes such as Paroxetine (Paroxyetine), escitalopram (Escitalopram), duloxetine (Duloxetine), rismine (RICASTIGMINE) and ibuprofen (Ibuprophen). Studies show (US 20100087525) that lipase has a broad substrate spectrum, and the selectivity and catalytic activity of lipase can be obviously affected by adjusting the reaction temperature and the cosolvent, so that the active pocket of lipase has strong plasticity. Therefore, the lipase can be modified by utilizing technologies such as directed evolution, semi-rational design and the like, the stereoselectivity and the catalytic activity of the lipase are improved, and the application range of the lipase is further widened.

Directed evolution (Directed evolution) is to simulate natural evolution process in experiments, artificially generate gene diversity through rapid mutation, recombination and other modes, and then apply screening pressure according to specific needs and purposes to screen out enzyme proteins meeting application expectations. This technology has been widely used in biology such as enzyme engineering and other crossing fields, but research on enzyme-catalyzed stereoselective transformation has been slow to develop relative to other enzyme property transformation due to the difficulty in constructing a screening model for enzyme stereoselective transformation, and the efficiency of analysis and detection by HPLC or GC is difficult to match with the screening of a large amount of mutation library, so that research on enzyme directed evolution of stereoselective transformation remains a challenge. With the progress of understanding the structure and function of proteins, the modification of enzymes is gradually transferred to functionally rich small mutant libraries constructed based on semi-rational design techniques. Semi-rational design refers to the use of related information such as protein sequence, structure, function and the like to pre-screen possible beneficial targets and amino acid residues for enzyme modification by computer assistance, so that a high-quality mutation library is constructed, the screening workload is reduced, and the enzyme modification efficiency is improved.

The preparation of chiral intermediates by lipases mainly has two schemes of chiral resolution and asymmetric hydrolysis. The resolution process needs to carry out optical resolution on the racemate, so the theoretical yield of the target configuration product is lower than 50%; the asymmetric hydrolysis process does not need to discard one optical isomer, so that the problems of low productivity and racemization reaction yield caused by enantiomer racemization recovery in the resolution process are fundamentally solved, and the theoretical yield can reach 100%. In recent years, asymmetric hydrolysis of lipases has gained attention from more and more researchers, but the practical application effect is unsatisfactory. Zaida et al prepared (R) -5-ethoxy-5-oxo-3-phenylpentanoic acid (Tetrahedron: asymmetry 22 (2011) 2080-2084) by asymmetric hydrolysis of 3-phenylpentanoic acid diethyl ester with immobilized lipase, and reacted with dioxane as a cosolvent at low temperature to give a product with an e.e. value of 92%. Wei et al prepared (R) -3- (4-fluorophenyl) -5-methoxy-5-oxopentanoic acid (Journal of Molecular Catalysis B: enzymatic 97 (2013) 270-277) by asymmetric hydrolysis of dimethyl 3- (4-fluorophenyl) glutarate with lipase, reacted at 30℃with dioxane as a co-solvent to give a product e.e. of only 71.6%. Liu et al selected 3- (4-fluorophenyl) -glutaric acid dimethyl ester as model substrate, screened commercial lipase for asymmetric hydrolysis process study, the results are shown in Table 1, 10% acetonitrile is added as cosolvent, reaction is carried out at room temperature, and the stereoselectivity of the product can not meet the process requirements (Process Biochemistry 47 (2012) 1037-1041).

Table 1 literature reports lipase screening

Lipase enzyme	e.e.(％)	Configuration of
			PPL	56.3	S
CRL	11.1	S
			RNL	3.6	R
HLE	26.4	R
			Lipase AK	0	-
Lipase PS	3.7	S
			Lipase AYS	0	-
Lipozyme TL	63.0	S
			Novozym 435	91.8	R

Teva (US 20100087525) reports a process route for asymmetric hydrolysis of substrate 3-isobutyl glutarate diester by using commercial lipase, by feeding substrate 3-isobutyl glutarate dimethyl ester and cosolvent tertiary amyl alcohol, and making reaction at-2 deg.C, the final substrate concentration is about 135g/L, the conversion rate is 99%, the product is S configuration, and the e.e. value is 96%. When the reaction is carried out at a low temperature of-2 ℃ by adding 3-isobutyl dimethyl glutarate at one time, the e.e. value of the product is reduced to 89%. If the cosolvent tertiary amyl alcohol is removed from the reaction system, the e.e. value of the product under the same reaction conditions is reduced to 70%. In summary, the lipase asymmetric hydrolysis process still has the following problems: 1) The asymmetric hydrolytic lipase with strict stereoselectivity has insufficient sources and cannot meet the process development requirements; 2) The stereoselectivity is unstable and greatly influenced by the reaction temperature, the cosolvent and the substrate addition mode; 3) The lipase catalytic activity is far lower than the optimal reaction condition due to the low-temperature environment conversion, the enzyme use cost is increased, and the productivity is greatly reduced; 4) The addition of the cosolvent also improves the process cost and the difficulty of separating and extracting the product, and cannot realize industrialized amplification.

According to the research, through a semi-rational design technology, 3-isobutyl diethyl glutarate is used as a model substrate, candida antarctica lipase (Pseudozyma ANTARCTIA LIPASE, PAL) is used as a research object to carry out mutation site analysis, and the three-dimensional selectivity is modified to obtain a forward single mutant; after the forward mutation sites are combined, a series of mutant strains are constructed, and asymmetric hydrolysis with strict stereoselectivity can be carried out on various diesters under the condition of normal temperature and in a full water phase system; the gene mining technology is utilized to clone and express the maize head smut lipase (Sporisorium reilianum lipase, SRL), and after the PAL forward mutation site is integrated, strict stereoselectivity is shown in asymmetric hydrolysis of a diester substrate, so that an efficient and stable biocatalyst is provided for the asymmetric hydrolysis process of the lipase, and the method has great industrialized application potential.

Disclosure of Invention

The invention provides a series of candida antarctica PAL and maize head smut SRL lipase mutants through a semi-rational design technology, can avoid low-temperature reaction conditions and remove cosolvent dependence, remarkably improves the reaction stereoselectivity and process stability, and provides an efficient and stable biocatalyst for a series of asymmetric hydrolysis processes of diester substrates.

The technical scheme adopted by the invention is as follows:

The selection of the key mutation sites of the candida antarctica lipase PAL is determined by constructing and screening a mutant library.

The invention provides a series of candida antarctica lipase PAL mutants, and compared with the wild candida antarctica lipase PAL, the mutant sites and mutant forms corresponding to the mutants are formed by single-point mutant forms and all possible combination modes shown in the table 2.

TABLE 2 beneficial mutation sites and mutant forms thereof

Amino acid position	Parent amino acids	Beneficial mutant forms
			140	L	V,I,M,A,F,W,G,S,C,T,D,N,E,P,H,K
154	V	A,I,L,M,F,W,S,C,T,Q,Y
			281	A	I,L,M,F,V,S,C,T
282	A	V,I,L,M,F,G,S,C,N,K,Y,R

The PAL amino acid sequence of the wild candida antarctica lipase is shown as SEQ ID NO. 1, and the corresponding coding gene is shown as SEQ ID NO. 2. The mutant L140X1,V154X2,A281X3,A282X4,L140X1/V154X2,L140X1/A281X3,L140X1/A282X4,V154X2/A281X3,V154X2/A282X4,A281X3/A282X4,L140X1/V154X2/A281X3,L140X1/V154X2/A282X4,L140X1/A281X3/A282X4,V154X2/A281X3/A282X4,L140X1/V154X2/A281X3/A282X4, wherein X1 represents a beneficial mutant form at the L140 site, X2 represents a beneficial mutant form at the V154 site, X3 represents a beneficial mutant form at the a281 site, and X4 represents a beneficial mutant form at the a282 site.

The amino acid sequence of lipase SRL of wild maize head smut is shown as SEQ ID NO. 3, and the corresponding coding gene is shown as SEQ ID NO. 4. Based on the primary and tertiary structure alignment of SRL and PAL lipases, positions 145, 159, 287 and 288 of SRL lipase correspond to amino acid positions 140, 154, 281 and 282 of PAL lipase, respectively. The SRL lipase mutant L145X1,V159X2,A287X3,G288X4,L145X1/V159X2,L145X1/A287X3,L145X1/G288X4,V159X2/A287X3,V159X2/G288X4,A287X3/G288X4,L145X1/V159X2/A287X3,V159X1/V159X2/G288X4,L145X1/A287X3/G288X4,V159X2/A287X3/G288X4,L145X1/V159X2/A287X3/G288X4, wherein X1 represents a beneficial mutant form at the L145 position, X2 represents a beneficial mutant form at the V159 position, X3 represents a beneficial mutant form at the A287 position, and X4 represents a beneficial mutant form at the G288 position.

The invention also relates to a recombinant vector containing the coding gene and recombinant genetic engineering bacteria obtained by utilizing the recombinant vector to transform. The recombinant vector is constructed by connecting the mutant nucleotide sequence of the invention to various vectors by a conventional method. The vector may be any of a variety of vectors conventional in the art, such as various plasmids, phage or viral vectors, and the like.

The invention also provides a genetically engineered bacterium containing the coding gene or the recombinant vector. The genetically engineered bacterium can be obtained by transforming the recombinant expression vector of the invention into a host microorganism. The host microorganism can be various host microorganisms conventional in the art, so long as the recombinant expression vector can stably self-replicate and the carried mutant genes of the invention can be effectively expressed, such as various yeasts, escherichia coli, bacillus, actinomycetes or molds and the like.

The invention also relates to application of the lipase and the mutant thereof in preparing an S-monoester product by asymmetrically hydrolyzing X-substituted glutarate diester. The application is that a substrate, mutant enzyme and buffer solution are put into a reactor for catalytic reaction; the concentration of the substrate is 1-300g/L; the buffer solution is citric acid-sodium citrate with pH of 4.0-9.0, phosphate, tris-HCl buffer solution and the like; the reaction temperature for the application is 10-40 ℃. The reaction formula is shown below.

The beneficial effects of the invention are mainly as follows: a series of highly selective lipases and mutants thereof which can prepare (S) -3-isobutyl-5-methoxy-5-oxopentanoic acid, (S) -3-isobutyl-5-ethoxy-5-oxopentanoic acid, (S) -3- (4-fluorophenyl) -5-methoxy-5-oxopentanoic acid, and (S) -3- (4-chlorophenyl) -5-methoxy-5-oxopentanoic acid and other intermediates under the conditions of normal temperature (10-40 ℃) and equal industrialization acceptable conditions of pure water are obtained, and the reaction e.e. >95% and the conversion rate >99% are the lipases which report that the normal temperature water phase condition specificity asymmetrically hydrolyzes a series of diester substrates to generate S-monoester products for the first time. After the mutant is applied to various asymmetric hydrolysis processes of medical intermediates, a split-despin recovery process can be avoided in the preparation process, the productivity and substrate utilization rate are improved, the stereoselectivity is strict, the production efficiency can be obviously improved, and the method has great industrialized application potential.

Drawings

FIG. 1 shows active pocket structure analysis and mutation site selection

Detailed Description

Gene source: the Pseudozyma antarctia lipase PAL gene and the Sporisorium reilianum lipase SRL gene related to the patent are obtained through total gene synthesis, and the related mutants are obtained through site-directed mutagenesis.

Analysis of three-dimensional structure of enzyme: three-dimensional structural analysis of the enzyme was performed using the Pymol visualization software.

Enantioselectivity assay: the reaction products (S) -3-isobutyl-5-methoxy-5-oxopentanoic acid, (S) -3-isobutyl-5-ethoxy-5-oxopentanoic acid, (S) -3- (4-fluorophenyl) -5-methoxy-5-oxopentanoic acid and (S) -3- (4-chlorophenyl) -5-methoxy-5-oxopentanoic acid and their corresponding enantiomers were determined by high performance liquid chromatography under the detection conditions referred to U.S. Pat. No. 3, 20100087525.

The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

Example 1: selection of mutation sites

The active pocket of the lipase PAL consists of an acyl pocket and an alcohol pocket, and a catalytic triplet D187-H224-S105 and an oxyanion hole are arranged at the joint of the two pockets. The amino acid residues T138, L140, V154 and I189 constituting the acyl pocket and the amino acid residues L278, A281, A282 constituting the alcohol group binding pocket may influence the binding conformation of the enzyme to the substrate, and the above 7 sites were selected as candidate mutation sites as shown in FIG. 1.

Example 2: construction of recombinant expression vector and engineering bacteria

The lipase sequences of PAL (SEQ ID NO: 1) and SRL (SEQ ID NO: 3) are selected, ecoR I and Hind III are designed according to the characteristics of the expression vector pET22b, and lipase genes PAL (SEQ ID NO: 2) and SRL (SEQ ID NO: 4) are synthesized by a total gene synthesis method through the conventional operation of genetic engineering. The pal and srl gene fragments synthesized in example 1 were subjected to double digestion and recovery treatment using EcoR I and Hind III restriction enzymes, and the fragments were ligated overnight at 16℃with the commercial vector pET22b treated with the same restriction enzymes using T4 DNA ligase, thereby constructing recombinant expression vectors pET22b-pal and pET22b-srl. Transforming the constructed recombinant expression vector into E.coli BL21 (DE 3) competent cells, coating the competent cells on LB plates containing ampicillin with a final concentration of 50 mu g/mL, and culturing the competent cells at 37 ℃ overnight; colony PCR identification is carried out by randomly picking clones from colonies growing on a flat plate, and positive clone sequencing verification shows that recombinant bacteria E.coli BL21 (DE 3)/pET 22b-pal and E.coli BL21 (DE 3)/pET 22b-srl are successfully constructed.

Example 3: preparation of somatic cells containing recombinant Lipase or mutants thereof

Inoculating genetically engineered bacteria E.coli BL21 (DE 3)/pET 22b-PAL and E.coli BL21 (DE 3)/pET 22b-SRL or mutants thereof into a 2 XYT culture medium containing 50 mug/mL ampicillin, culturing at 37 ℃ until the concentration OD ₆₀₀ is 0.4-0.6, adding IPTG with the final concentration of 0.1mmol/L into the 2 XYT liquid culture medium, performing induction culture at 18 ℃ for 18-24h, centrifuging the culture solution at 4 ℃ for 5min at 8000rpm, discarding the supernatant, and collecting wet bacterial bodies containing recombinant PAL and SRL lipase or mutants thereof.

Example 4: preliminary screening of effect of candidate site amino acid on enantioselectivity

For the 7 candidate sites determined in example 1, a preliminary screening mutation library was constructed by site-directed mutagenesis, amplified using high-fidelity enzyme PRIMERSTAR, and the PCR product was directly digested with Dpn I, transformed e.coli BL21 (DE 3), screened by spreading Amp plates, sequencing-verified to obtain mutants, the corresponding mutant enzymes were obtained as described in example 3, and the effect of each candidate site amino acid change on enantioselectivity was probed using diethyl 3-isobutylglutarate as a model substrate. The enantioselectivity of the PAL mutants was examined against wild-type PAL, and as a result, it was found that the modification of the enantioselectivity at positions 140, 154, 281 and 282 was significantly affected, and thus, the above 4 positions were subjected to further site-directed saturation mutation. Candidate sites were also determined by PAL enantioselectivity studies for SRL lipase, affecting significant sites 145, 159, 287 and 288.

Example 5: construction of lipase site-directed saturation mutation library and selective determination

Site-directed mutagenesis was used to construct a series of point mutants at positions 140, 154, 281, 282 and 145, 159, 287, 288 of PAL lipase. The PCR products were directly digested with Dpn I using pET22b-pal and pET22b-srl plasmids as templates, respectively, amplified with high fidelity enzyme PRIMERSTAR, transformed into E.coli BL21 (DE 3), screened with Amp plates, sequenced and verified to obtain mutants, and the corresponding mutant enzymes were obtained as described in example 3. The enantioselectivities of the obtained series of mutants were compared using diethyl 3-isobutylglutaric acid as a model substrate, and the results are shown in Table 3. The enantioselectivity change rule of the SRL lipase 145, 159, 287 and 288 series single-point mutants is the same as that of PAL lipase 140, 154, 281 and 282, and the enantioselectivity of the SRL mutants is slightly poorer than that of the PAL mutants under the condition that the corresponding site amino acids are the same.

TABLE 3 Lipase enantioselectivity assay

Note that: the product configuration is R-type, e.e. value is labeled "-"; the product configuration is S-shaped, e.e. values 0-30% are marked as "+"; e.e. values 30-60% are marked as "++"; e.e. value 60-90% marked as' ++ "; e.e. value >90% sign is "+". ++ + +' and its use "

Example 6: preparation of (S) -3-isobutyl-5-methoxy-5-oxopentanoic acid by transformation of lipase combined mutant

A combined mutant strain E.coli BL21 (DE 3)/pET 22b-pal (L140V/A281V) was constructed by single point mutation according to example 5, and cells were prepared for catalytic reaction in the same manner as in example 3. Into a 50mL conversion flask was added 9.9mL of 100mM buffer (pH 8.0), 100. Mu.L of the substrate dimethyl 3-isobutylglutarate was added, 0.39g of wet cells were allowed to react at 30℃and at 150rpm on a shaking table, after completion of the reaction, the reaction was extracted with n-hexane, and the conversion and the enantiomeric ratio of (S) -3-isobutyl-5-methoxy-5-oxopentanoic acid in the reaction extract were analyzed by HPLC, whereby the conversion was >99%, and the product (S) -3-isobutyl-5-methoxy-5-oxopentanoic acid e.e. >95% was shown.

Example 7: preparation of (S) -3-isobutyl-5-ethoxy-5-oxopentanoic acid by transformation of lipase combination mutants

A combined mutant strain E.coli BL21 (DE 3)/pET 22b-srl (L145M/V159L/A287V) was constructed by single point mutation according to example 5 and cells were prepared for catalytic reaction as in example 3. Into a 50mL conversion flask, 9.9mL of 100mM buffer (pH 8.0) was added, and 100. Mu.L of the substrate diethyl 3-isobutylglutaricate and 0.39g of wet cells were added, and the reaction conditions and sample treatment were the same as in example 6. The detection result shows that the conversion rate is >99%, and the product (S) -3-isobutyl-5-ethoxy-5-oxopentanoic acid e.e. >95%.

Example 8: preparation of (S) -3- (4-fluorophenyl) -5-methoxy-5-oxopentanoic acid by transformation of lipase combined mutant

A combined mutant strain E.coli BL21 (DE 3)/pET 22b-pal (L140I/A281V) was constructed by single point mutation according to example 5, and cells were prepared for catalytic reaction in the same manner as in example 3. Into a 50mL conversion flask, 9.9mL of 100mM buffer (pH 8.0) was added, and 0.1g of substrate dimethyl 3- (4-fluorophenyl) -glutarate and 0.39g of wet cell were added, and the reaction conditions and sample treatment were the same as in example 6. The detection result shows that the conversion rate is >99%, and the product (S) -3- (4-fluorophenyl) -5-methoxy-5-oxopentanoic acid e.e. >95%.

Example 9: preparation of (S) -3- (4-chlorophenyl) -5-methoxy-5-oxopentanoic acid by transformation of lipase combined mutant

A combined mutant strain E.coli BL21 (DE 3)/pET 22b-srl (V159A/A287I/G288W) was constructed by single point mutation according to example 5 and the cells were prepared for catalytic reaction as in example 3. Into a 50mL conversion flask, 9.9mL of 100mM buffer (pH 8.0) was added, and 0.1g of dimethyl 3- (4-chlorophenyl) -glutarate and 0.39g of wet cell were added, and the reaction conditions and sample treatment were the same as in example 6. The detection result shows that the conversion rate is >99%, and the product (S) -3- (4-chlorophenyl) -5-methoxy-5-oxopentanoic acid e.e. >95%.

Claims

1. A lipase mutant of corn head smut fungus, characterized in that the mutant is different from the wild-type corn head smut fungus lipase (whose amino acid sequence is shown in SEQ ID NO:3), the leucine residue at position 145 is mutated to valine, isoleucine, methionine, alanine, phenylalanine, tryptophan, glycine, serine, cysteine, threonine, aspartic acid, asparagine, glutamic acid, proline, histidine or lysine; or the valine residue at position 159 is mutated to alanine, isoleucine, leucine, methionine, phenylalanine, tryptophan, serine, cysteine, threonine, glutamine or tyrosine; or the alanine residue at position 287 is mutated to isoleucine, leucine, methionine, phenylalanine, valine, serine, cysteine or threonine, or the glycine residue at position 288 is mutated to valine, isoleucine, leucine, methionine, phenylalanine, serine, cysteine, asparagine, lysine, tyrosine or arginine, or any combination of beneficial mutation forms of the above mutation sites.

2. A recombinant vector constructed from the mutant encoding gene according to claim 1.

3. A recombinant genetically engineered bacterium transformed by the expression vector of claim 2, wherein the genetically engineered bacterium comprises Escherichia coli, Pichia pastoris or Bacillus subtilis.

4. Use of the mutant according to claim 1 in the asymmetric hydrolysis of a diester compound to prepare an S-type monoester product, characterized in that the diester compound has the following structural formula:

R is CH ₃ , CH ₂ CH ₃ , CH ₂ CH ₂ CH ₃ , CH ₂ CH ₂ CH ₂ CH ₃ , HC=CH ₂ or X is