Halohydrin dehalogenase mutant, its encoding gene, recombinant plasmid containing the encoding gene, genetically engineered bacteria containing the recombinant plasmid and use thereof

Abstract

The present application discloses a halohydrin dehalogenase mutant, its encoding gene, a recombinant plasmid containing the encoding gene, a genetically engineered bacterium containing the recombinant plasmid, and its application, belonging to the field of genetic engineering. The halohydrin dehalogenase mutant of the present application is based on the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1, and its amino acid sequence is subjected to the following combined mutations: asparagine at position 179 is mutated to leucine; threonine at position 197 is mutated to alanine; leucine at position 198 is mutated to tyrosine; serine at position 224 is mutated to alanine; and lysine at position 227 is mutated to arginine. The combined mutations of the present application can produce targeted changes in the amino acid sequence, improve the catalytic activity and stereoselectivity of the halohydrin dehalogenase mutant, and at the same time streamline the number of mutation sites and omit specific transformation operations, saving time, labor, and cost, and facilitating industrial production.

Description

Technical Field

[0001] The present application belongs to the field of genetic engineering technology, and in particular relates to a halohydrin dehalogenase mutant, its encoding gene, a recombinant plasmid containing the encoding gene, a genetically engineered bacterium containing the recombinant plasmid, and applications thereof. Background Art

[0002] (S)-1-chloro-3-phenoxy-2-propanol and (S)-1-azido-3-phenoxy-2-propanol are common pharmaceutical intermediates used in the pharmaceutical industry and in the preparation of high-performance dispersants and sunscreens. However, their synthesis via methods such as asymmetric hydroboration, (transfer) hydrogenation, biocatalytic reduction of R-chloroketones, and dynamic kinetic resolution of halohydrins is significantly limited in production and application due to low optical activity, time-consuming processes, and environmental pollution.

[0003] To overcome the drawbacks of the above-mentioned synthesis method, a method for preparing 1-chloro-3-phenoxy-2-propanol by catalyzing the racemic reaction of 1-chloro-3-phenoxy-2-propanol using a halohydrin dehalogenase in an azide reagent system can be used. Furthermore, to address the low stereoselectivity and low yield of wild-type halohydrin dehalogenases, researchers have developed various halohydrin dehalogenase mutants. For example, patent application number CN2021112534635 discloses a halohydrin dehalogenase mutant that significantly improves stereoselectivity, catalytic activity, and substrate yield by mutating the 21 amino acid sequences of the wild-type halohydrin dehalogenase and adding 6 amino acids to the N-terminus.

[0004] However, the catalytic activity and stereoselectivity of the halohydrin dehalogenase mutants under the existing technology still cannot meet the actual needs, and the mutation modification of the original halohydrin dehalogenase is labor-intensive, complicated and costly, which greatly limits its industrial application. Summary of the Invention

[0005] The purpose of the present application is to provide a halohydrin dehalogenase mutant, its encoding gene, a recombinant plasmid containing the encoding gene, a genetically engineered bacterium containing the recombinant plasmid and its application, aiming to solve the technical problems of low catalytic activity and stereoselectivity of existing halohydrin dehalogenase mutants; large workload, complicated operation and high cost of mutation modification.

[0006] In order to achieve the above objectives, the technical solution of this application is:

[0007] The first aspect of the present application provides a halohydrin dehalogenase mutant. The halohydrin dehalogenase mutant is based on the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1, and the amino acid sequence is subjected to the following combined mutations:

[0008] i: mutate asparagine at position 179 to leucine;

[0009] ii: mutate the threonine at position 197 to alanine;

[0010] iii: mutate leucine at position 198 to tyrosine;

[0011] iv: mutate serine at position 224 to alanine;

[0012] v: Lysine at position 227 was mutated to arginine.

[0013] In an optional implementation of the first aspect, the halohydrin dehalogenase mutant has an amino acid sequence shown in SEQ ID NO.2.

[0014] In an optional implementation of the first aspect, the nucleotide sequence of the halohydrin dehalogenase mutant is shown as SEQ ID NO.3.

[0015] The second aspect of the present application provides a gene encoding the halohydrin dehalogenase mutant described in the first aspect. The encoding gene is shown in SEQ ID NO.4.

[0016] The third aspect of the present application provides a recombinant plasmid comprising a gene encoding the halohydrin dehalogenase mutant described in the second aspect.

[0017] In an optional implementation of the third aspect, the expression vector of the recombinant plasmid is pET28a(+).

[0018] The fourth aspect of the present application provides a genetically engineered bacterium comprising a gene encoding the halohydrin dehalogenase mutant described in the second aspect.

[0019] In an optional implementation of the fourth aspect, the expression host of the genetically engineered bacteria is E. coli BL21 (DE3).

[0020] The fifth aspect of the present application provides the use of the genetically engineered bacteria described in the fourth aspect in catalyzing the synthesis of (S)-1-chloro-3-phenoxy-2-propanol from 1-chloro-3-phenoxy-2-propanol.

[0021] In an optional implementation of the fifth aspect, the application method includes:

[0022] S1: The genetically engineered bacteria are suspended in a Tris-SO4 buffer system containing NaN3 to obtain a splitting reaction system;

[0023] S2: After adding 1-chloro-3-phenoxy-2-propanol to the resolution reaction system, the reaction is shaken at a temperature of 30° C. and a rotation speed of 200 r / min, and the reaction product is purified and dried to complete the catalytic resolution.

[0024] Compared with the prior art, the advantages or beneficial effects of this application include at least:

[0025] The first aspect of the present application provides a halohydrin dehalogenase mutant, which, by subjecting the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1 to the aforementioned combined mutation, produces directional structural and functional changes in the amino acids and their related nucleotide sequences, significantly improving the catalytic activity and stereoselectivity of the halohydrin dehalogenase mutant. Furthermore, the number of mutation sites is simplified and specific transformation operations are omitted, saving time and effort and reducing costs, thereby facilitating industrial production. The test results of the embodiment show that the specific enzyme activity of the halohydrin dehalogenase mutant reaches 7.5 U / g, and the catalytic enantiomeric selectivity reaches 193.09; the ee value of the catalytically generated (S)-1-chloro-3-phenoxy-2-propanol is greater than 99.99%, and the yield reaches 47.49%. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] To more clearly illustrate the technical solutions of the embodiments of this application, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some of the embodiments described in this application. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0027] Figure 1 This is a high performance liquid chromatogram of the substrate 1-chloro-3-phenoxy-2-propanol provided in the examples of the present application;

[0028] Figure 2 This is a high performance liquid chromatogram of the catalytic reaction of the genetically engineered bacteria containing the halohydrin dehalogenase mutant provided in the examples of the present application. DETAILED DESCRIPTION

[0029] The following will be combined with the accompanying drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0030] In the following description of this embodiment, the term "and / or" is used to describe the association relationship between associated objects, indicating that three possible relationships exist. For example, A and / or B can represent: A exists alone, B exists alone, and both A and B exist. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0031] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0032] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0033] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise.

[0034] In a first aspect, the present invention provides a halohydrin dehalogenase mutant. The halohydrin dehalogenase mutant is based on the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO. 1, and the amino acid sequence is subjected to the following combined mutations:

[0035] i: mutate asparagine at position 179 to leucine;

[0036] ii: mutate the threonine at position 197 to alanine;

[0037] iii: mutate leucine at position 198 to tyrosine;

[0038] iv: mutate serine at position 224 to alanine;

[0039] v: Lysine at position 227 was mutated to arginine.

[0040] It should be noted that the amino acid names in the sequence shown in SEQ ID NO. 1 match the single-letter abbreviations of the amino acids in Table 1 below.

[0041] Table 1 - Names and abbreviations of amino acids

[0042]

[0043]

[0044] Therefore, in the embodiment of the present application, the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO. 1 is used as a carrier, and after the above-mentioned combined mutation is performed on the specific sites in the amino acid sequence, the amino acid sequence of the obtained halohydrin dehalogenase mutant is shown in SEQ ID NO. 2. Among them, the amino acid sequence of the mutant in the embodiment of the present application is represented by replacing the corresponding amino acid sites in the amino acid sequence of the original halohydrin dehalogenase with mutant amino acids, and the number of the mutation site corresponds to the amino acid sequence site of the original halohydrin dehalogenase.

[0045] At the same time, the present application embodiment provides a nucleotide sequence based on the amino acid sequence of the original halohydrin dehalogenase described above, and the nucleotide sequence of the original halohydrin dehalogenase is shown in SEQ ID NO. 5. Based on the specific nucleotide sequence of each amino acid, after the above-mentioned combined mutation is performed in the present application embodiment, the nucleotide sequence of the obtained halohydrin dehalogenase mutant is shown in SEQ ID NO. 3. Among them, the nucleotide sequence of the mutant in the embodiment of the present application is represented by replacing the corresponding nucleotide site in the nucleotide sequence of the original halohydrin dehalogenase with the nucleotide of the mutant amino acid, and the number of the mutation site corresponds to the nucleotide sequence site of the original halohydrin dehalogenase.

[0046] Those skilled in the art should understand that the above-mentioned combined mutation modification methods are all implemented in accordance with relevant genetic engineering technologies known in the art. The embodiments of this application do not particularly limit the specific gene mutation technology, and it is subject to the ability of those skilled in the art to obtain the target mutant through the above-mentioned combined mutation.

[0047] In addition, the examples of the present application performed individual mutations on each of the above mutation sites to verify the technical effect of the combined mutation described above. The results showed that individual mutations of each site could not achieve the catalytic synthesis and resolution effect of the combined mutation.

[0048] Among them, the embodiment of the present application performs the above-mentioned combined mutation on the amino acid sequence of the original halohydrin dehalogenase shown in SEQ ID NO.1, so that the amino acid and its related nucleotide sequence produce directional structural and functional changes, thereby greatly improving the catalytic activity and stereoselectivity of the halohydrin dehalogenase mutant, and significantly streamlining the number of mutation sites and omitting specific transformation operations, saving time, labor and cost, and facilitating industrial production.

[0049] In a second aspect, the present invention also provides a gene encoding the halohydrin dehalogenase mutant described in the first aspect. The encoding gene is shown in SEQ ID NO. 4.

[0050] In this embodiment of the present application, the halohydrin dehalogenase mutant described in the first aspect is encoded to form a gene encoding the halohydrin dehalogenase mutant. Specifically, the gene encoding the mutant enzyme is synthesized by a total synthesis method based on genetic engineering technology. The gene encoding the halohydrin dehalogenase mutant is shown in SEQ ID NO. 4; at the same time, for the convenience of recording, the gene encoding the halohydrin dehalogenase mutant is represented as HHDH22 in this embodiment of the present application.

[0051] In a third aspect, the embodiments of the present application further provide a recombinant plasmid comprising a gene encoding the halohydrin dehalogenase mutant described in the second aspect.

[0052] Among them, the embodiment of the present application provides a method for preparing the recombinant plasmid described above, preferably comprising:

[0053] The encoding gene of the halohydrin dehalogenase mutant and the expression vector pET28a of Escherichia coli were double-digested with restriction endonucleases Nco I and Xho I for 3-6 hours, and the digested products were recovered and ligated with T4 DNA ligase at a temperature of 16° C. for 16 hours to obtain a recombinant plasmid containing the encoding gene of the halohydrin dehalogenase mutant; at the same time, for the convenience of recording, the recombinant plasmid is represented as pET28a-HHDH22 in the examples of this application.

[0054] It should be noted that the examples of this application do not impose any particular restrictions on the specific sources of T4 DNA ligase, restriction endonucleases Nco I and Xho I. For example, they can be obtained commercially from Fermetens or synthesized according to genetic engineering methods known in the relevant field.

[0055] In a fourth aspect, the embodiments of the present application further provide a genetically engineered bacterium comprising a gene encoding the halohydrin dehalogenase mutant described in the second aspect.

[0056] Among them, the embodiment of the present application preferably uses Escherichia coli as a host to express the recombinant plasmid to synthesize the genetically engineered bacteria, specifically including:

[0057] S41: The recombinant plasmid pET28a-HHDH22 was transformed into the E. coli BL21 (DE3) recipient bacteria and spread on an LB agar plate containing kanamycin (mass concentration of 50 mg / L), and then cultured at 37°C for 12 hours to allow single colonies to grow on the plate; a single colony was randomly picked and cloned, and then inoculated into LB liquid culture medium and cultured for 12 hours. The plasmid was extracted and sequenced, and the genetically engineered bacteria were screened according to the sequencing results, which were represented as positive clones E. coli BL21 (DE3) / pET28a-HHDH22.

[0058] S42: The genetically engineered bacteria were inoculated into 4 mL of LB medium containing 50 mg / L kanamycin, and initially cultured at 37° C. and 200 rpm for 12 h; the genetically engineered bacteria were inoculated into a new 30 mL of LB medium containing 50 mg / L kanamycin at a concentration of 1 vt.%, and cultured at 37° C. and 200 rpm until the optical density (OD) reached 0. 600 ) is 0.6-0.8; then, isopropyl-β-D-thiogalactopyranoside (IPTG) inducer is added to a final concentration of 0.1 mM, and the cells are induced and cultured at a temperature of 25°C and a rotation speed of 200 r / min for 16 hours; finally, the cells are centrifuged at 5000×g for 5 minutes to collect the cells, and the cells are resuspended and washed with Tris-SO4 buffer at pH=7.0, and centrifuged at 13000×g for 1 minute to collect the wet cells, which are then stored at -20°C for later use.

[0059] It should be noted that the LB agar plate is composed of 10.0 g / L tryptone, 5.0 g / L yeast extract, and 10.0 g / L NaCl; the LB solid medium is composed of 10.0 g / L tryptone, 5.0 g / L yeast extract, 10.0 g / L NaCl, and 15.0 g / L agar. Both the LB agar plate and the LB solid medium are autoclaved at 121°C for 20 min.

[0060] The present invention utilizes a gene encoding the above-mentioned halohydrin dehalogenase mutant and expresses the recombinant strain carrying the encoding gene to obtain the genetically engineered bacteria. Studies have shown that the purity of the synthesis and resolution of (S)-1-chloro-3-phenoxy-2-propanol and (S)-1-azido-3-phenoxy-2-propanol catalyzed by this genetically engineered bacteria is higher than 99.99%, and the yield is above 45%. Compared with the catalytic reaction of the original halohydrin dehalogenase, the enantiomeric selectivity is increased by 11.9 times.

[0061] In a specific embodiment, the expression host of the genetically engineered bacteria is preferably E. coli BL21 (DE3).

[0062] In a fifth aspect, the embodiments of the present application also provide the use of the genetically engineered bacteria described in the fourth aspect in catalyzing the synthesis of (S)-1-chloro-3-phenoxy-2-propanol from 1-chloro-3-phenoxy-2-propanol.

[0063] Among them, the specific method of the embodiment of the present application for catalytically resolving the substrate 1-chloro-3-phenoxy-2-propanol by the genetically engineered bacteria includes:

[0064] S51: Weigh 50 mg of genetically engineered bacteria and suspend them in 1 mL of 30 mM Tris-SO 4 buffer system containing NaN 3 (pH=7.0, 50 mM) to obtain a resolution reaction system.

[0065] S51: After adding 1-chloro-3-phenoxy-2-propanol at a concentration of 20 mM to the split reaction system, the reaction was oscillated for 40-80 min at a temperature of 30 ° C and a speed of 200 r / min. During the oscillation reaction, 0.4 mL of the sample was regularly sampled, 900 μL of ethyl acetate was added to the sample, and the reaction was oscillated for 20 min at a temperature of 30 ° C and a speed of 200 r / min. Then, the sample was centrifuged at 13000 × g for 1 min, 500 μL of ethyl acetate was taken out, air-dried overnight, and redissolved with 1 mL of a mixed reagent (n-hexane: isopropanol = 1:1). The sample was filtered through a 0.22 μm organic membrane to complete the catalytic resolution of the substrate 1-chloro-3-phenoxy-2-propanol.

[0066] Test results in the examples show that the genetically engineered bacteria described above preferentially hydrolyze (R)-1-chloro-3-phenoxy-2-propanol. The halohydrin dehalogenase mutant exhibited a specific enzymatic activity of 7.5 U / g, achieved an enantiomeric selectivity of 193.09, and produced (S)-1-chloro-3-phenoxy-2-propanol with an ee value greater than 99.99% and a yield of 47.49%.

[0067] To further illustrate the technical effects of the genetically engineered bacteria containing the halohydrin dehalogenase mutant, the present invention provides an example of a test using the genetically engineered bacteria containing the original halohydrin dehalogenase in the catalytic resolution of the substrate 1-chloro-3-phenoxy-2-propanol. The test method is the same as the specific operation and parameter settings of the catalytic resolution method described above, except that the genetically engineered bacteria containing the halohydrin dehalogenase mutant are replaced with the genetically engineered bacteria containing the original halohydrin dehalogenase.

[0068] The test results show that the genetically engineered bacteria containing the original halohydrin dehalogenase also preferentially hydrolyze (R)-1-chloro-3-phenoxy-2-propanol. Among them, the specific enzymatic activity of the original halohydrin dehalogenase is 3.4 U / g, and the enantiomeric selectivity catalyzed by it is 16.2. Therefore, the enantiomeric selectivity catalyzed by the genetically engineered bacteria containing the halohydrin dehalogenase mutant is 11.9 times that of the genetically engineered bacteria containing the original halohydrin dehalogenase, and the enzymatic activity of the halohydrin dehalogenase mutant is 2.2 times that of the original halohydrin dehalogenase.

[0069] The present invention performs high performance liquid chromatography analysis on the substrate 1-chloro-3-phenoxy-2-propanol, specifically including:

[0070] Agilent-1220 system was used, chromatographic column type: Chiralcel OD-H column (Daicel Co., Japan; 4.6×250 mm L, 5 μm); chromatographic conditions: column temperature: 35°C;

[0071] The chromatographic parameters are: mobile phase is n-hexane: isopropanol = 88:12 (v / v); flow rate is 0.8 mL / min; UV wavelength is 220 nm, the results are Figure 1 As shown. Among them, Figure 1 The high performance liquid chromatogram of the substrate 1-chloro-3-phenoxy-2-propanol is shown.

[0072] according to Figure 1 It can be seen that the retention time of (S)-1-chloro-3-phenoxy-2-propanol in the standard sample 1-chloro-3-phenoxy-2-propanol is about 11.3 minutes, and the retention time of (R)-1-chloro-3-phenoxy-2-propanol is about 18.6 minutes.

[0073] Substrate ee S =[(SR) / (S+R)]×100%,

[0074] Alternatively, substrate ee S =[(RS) / (R+S)]×100%;

[0075] E=ln[(1-c)×(1-ee S )] / ln[(1-c)×(1+ee S )].

[0076] Where R and S are the peak areas of (R)- and (S)-substrates, and c is the conversion rate of rac-substrate.

[0077] At the same time, the present invention uses a genetically engineered bacterium containing a halohydrin dehalogenase mutant to catalyze the separation of the substrate 1-chloro-3-phenoxy-2-propanol. Figure 2 As shown. Among them, Figure 2A high performance liquid chromatogram of a reaction catalyzed by a genetically engineered bacterium containing a halohydrin dehalogenase mutant is shown.

[0078] according to Figure 2 It can be seen that (S)-1-chloro-3-phenoxy-2-propanol peaked at 11.3 min, (R)-1-chloro-3-phenoxy-2-propanol was completely converted without any peak, and the yield was as high as 47.4%.

[0079] In summary, the halohydrin dehalogenase mutant contained in the genetically engineered bacteria has the excellent performance of high specific enzyme activity and high stereoselectivity. Therefore, after the genetically engineered bacteria is used to catalyze the synthesis of (S)-1-chloro-3-phenoxy-2-propanol from 1-chloro-3-phenoxy-2-propanol, the enantiomeric selectivity reaches 193.09, and the ee value of the (S)-1-chloro-3-phenoxy-2-propanol catalyzed by it is greater than 99.99%, and the yield reaches 47.49%.

[0080] The various embodiments in this specification are described in a progressive manner. The same or similar parts between the various embodiments can be referenced to each other, and each embodiment focuses on the differences from other embodiments.

[0081] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A halohydrin dehalogenase mutant, characterized in that The original halohydrin dehalogenase shown in SEQ ID NO.1 was mutated as follows: i: mutate asparagine at position 179 to leucine; ii: mutate the threonine at position 197 to alanine; iii: mutate leucine at position 198 to tyrosine; iv: mutating serine at position 224 to alanine; and v: Lysine at position 227 was mutated to arginine.

2. The halohydrin dehalogenase mutant according to claim 1, characterized in that The amino acid sequence of the halohydrin dehalogenase mutant is shown in SEQ ID NO.

2.

3. A gene encoding a halohydrin dehalogenase mutant according to claim 1 or 2, characterized in that: The coding gene is shown in SEQ ID NO.

4. A recombinant plasmid comprising a gene encoding the halohydrin dehalogenase mutant according to claim 3 .

5. The recombinant plasmid according to claim 4, characterized in that The expression vector of the recombinant plasmid is pET28a(+). A genetically engineered bacterium comprising a gene encoding the halohydrin dehalogenase mutant according to claim 3 .

7. The genetically engineered bacterium according to claim 6, characterized in that The expression host of the genetically engineered bacteria is E. coli BL21 (DE3).

8. Use of the genetically engineered bacterium according to claim 6 or 7 in catalyzing the synthesis of (S)-1-chloro-3-phenoxy-2-propanol from 1-chloro-3-phenoxy-2-propanol.

9. The use according to claim 8, characterized in that The application method comprises: S1: The genetically engineered bacteria are suspended in a Tris-SO4 buffer system containing NaN3 to obtain a splitting reaction system; S2: After adding 1-chloro-3-phenoxy-2-propanol to the resolution reaction system, the reaction is shaken at a temperature of 30°C and a rotation speed of 200 r / min, and the reaction product is purified and dried to complete the catalytic resolution.

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