Nitrilase mutant with high activity and stereoselectivity and application of nitrilase mutant in synthesis of chiral carboxylic acid

Abstract

The invention discloses a nitrilase mutant with high activity and stereoselectivity and application of the nitrilase mutant in synthesis of chiral carboxylic acid. The mutant is obtained by performing single mutation or multiple mutation on 79th, 166th, 167th and 246th amino acids of an amino acid sequence shown as SEQ ID NO.1. The invention further discloses a preparation method of the nitrilase mutant. According to the invention, the nitrilase OsNIT-mut is subjected to molecular modification through (semi) rational design, and a mutant with synergistically improved hydrolysis reaction specificity and stereoselectivity is obtained. The content of mandelic acid in a product of the mutant OsNIT-mut / V246N / T166G / R79Y / A167N for catalyzing mandelonitrile is 90.5%, the e.e. Value of R-mandelic acid is increased to 95.2%, and the relative hydrolytic activity is 399.1%. The nitrilase mutant can be applied to green industrial catalytic synthesis of chiral mandelic acid through regulation and control of hydrolysis reaction specificity and stereoselectivity, and has important significance.

Description

(I) Technical Field

[0001] This invention relates to a nitrile hydrolase mutant with both high activity and high stereoselectivity and its application in the synthesis of chiral carboxylic acids. (II) Background Technology

[0002] Nitrile compounds are a class of cyano-containing organic chemical raw materials widely used in the chemical, pharmaceutical, pesticide, and materials industries. Nitrile hydrolases catalyze the hydrolysis of nitrile compounds to carboxylic acids, exhibiting unique advantages such as chemoselectivity, stereoselectivity, and regioselectivity, playing a crucial role in nitrile compound conversion. Some nitrile hydrolases, in addition to catalyzing the hydrolysis of nitrile compounds to carboxylic acids, also possess nitrile hydration activity. For example, a nitrile hydrolase derived from the plant rhizosphere microorganism *Pseudomonas* sp. UW4 catalyzes the hydrolysis of indole-3-acetonitrile, with the indole-3-acetamide content in the product being 4.3 times that of indole-3-acetic acid (Appl. Environ. Microbiol. 2014, 80:4640-4649). Furthermore, a nitrile hydrolase derived from *Bradyrhizobium japonicum* catalyzes the hydrolysis of β-aminopropionitrile, with the β-aminopropionamide content reaching as high as 33% (J. Mol. Catal. B: Enzym. 2015, 115:113-118).

[0003] Nitrile hydrolases, possessing both nitrile hydrolytic and hydration activities, hold immense potential in bioorganic synthesis. Improving the reaction selectivity and stereoselectivity of nitrile hydrolases, and specifically generating carboxylic acids, facilitates increased yields and the separation and purification of later products, thus promoting the industrial production of nitrile hydrolases. Therefore, the synergistic regulation of the reaction selectivity and stereoselectivity of nitrile hydrolases is of great significance for their industrial application.

[0004] With the rapid development of enzyme engineering technology, molecular modification can significantly improve the catalytic performance of nitrile hydrolases. In recent years, the regulation of the stereochemistry and reaction selectivity of nitrile hydrolases has also become a research hotspot. For example, the wild-type Rhodococcus zopfii nitrile hydrolase RZNIT exhibits an amide content as high as 88% when catalyzing 2-chloronicotinamide. Based on a modification strategy using characteristic distance regulation, a single site mutation (W167G) increased the hydrolytic activity of the mutant to 20 times that of the wild type, producing only 2-chloronicotinic acid when catalyzing 2-chloronicotinamide (Appl. Environ. Microb. 2022, aem0239721.). Wang et al. used error-prone PCR to mutate nitrile hydrolases, obtaining a double mutant I133M / Y199G that significantly improved the stereoselectivity of o-chloromandelinnis, increasing the optical purity of the product R-o-chloromandelic acid from 89.2% to 99.1% (Appl Environ Microbiol, 2015, 81(24):8469-8477). Our research group previously achieved bidirectional regulation of the reaction selectivity of nitrile hydrolysis (OsNIT) from Oryza sativa through rational design (ACS Catal. 2023, 13:10282-10294). The mutant K200R / R224W / N246V increased the content of mandelic acid synthesized from mandelinnis from 50.9% to 98.5%, but the stereoselectivity of this mutant remained unsatisfactory. Therefore, further design and modification to simultaneously improve its reaction and stereoselectivity will broaden its application in the biosynthesis of chiral carboxylic acids and other chemicals. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a nitrile hydrolase mutant with high activity and high stereoselectivity, its encoding gene, engineered bacteria, and its application in the synthesis of chiral carboxylic acids. This invention uses nitrile hydrolase as the research object and performs site-directed mutagenesis on it. The activity, stereoselectivity, and reaction selectivity of the obtained nitrile hydrolase mutant are synergistically improved. While maintaining hydrolysis activity, it increases the content of carboxylic acids synthesized and improves the optical purity of the product.

[0006] The technical solution adopted in the present invention is:

[0007] This invention provides a nitrile hydrolase mutant with both high activity and high stereoselectivity, which is obtained by single or multiple mutations of amino acids 79, 166, 167, and 246 of the amino acid sequence shown in SEQ ID NO.1.

[0008] Further, preferably, the nitrile hydrolase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO.1 to one of the following: (1) valine at position 246 is reversed to asparagine, V246N; (2) arginine at position 79 is reversed to tyrosine or asparagine, R79Y, R79N; (3) threonine at position 166 is reversed to glycine, T166G; (4) alanine at position 167 is reversed to asparagine, A167N; (5) glycine at position 80 is reversed to lysine, G80K; (6) valine at position 246 is reversed to asparagine, and threonine at position 166 is reversed to glycine, V246N / T166G, with the amino acid sequence as shown in SEQ ID NO. As shown in NO.3; (7) Valine at position 246 is reversed to asparagine, threonine at position 166 is reversed to glycine, and arginine at position 79 is reversed to tyrosine, V246N / T166G / R79Y, and the amino acid sequence is shown in SEQ ID NO.4; (8) Valine at position 246 is reversed to asparagine, arginine at position 79 is reversed to tyrosine, threonine at position 166 is reversed to glycine, and alanine at position 167 is reversed to asparagine, V246N / R79Y / T166G / A167N, and the amino acid sequence is shown in SEQ ID NO.5.

[0009] This invention relates to a coding gene for a nitrile hydrolase mutant, a recombinant vector constructed from the coding gene, and a recombinant engineered bacterium. The recombinant vector is constructed by inserting the nitrile hydrolase mutant gene between the BamHI and HindIII sites of the pET28b vector to construct a recombinant plasmid containing the nitrile hydrolase mutant gene. The engineered bacterium is prepared by transferring the constructed recombinant plasmid into a host bacterium to obtain the recombinant engineered bacterium. The host bacterium can be *Escherichia coli* BL21.

[0010] This invention also provides the application of the nitrile hydrolase mutant in the catalytic synthesis of chiral mandelic acid from mandelic nitrile. The application is as follows: using wet cells obtained by fermentation culture of engineered bacteria containing the nitrile hydrolase mutant encoding gene, or enzyme extracted from the broken wet cells, as a biocatalyst; mandelic nitrile as a substrate; methanol as an adjuvant; and a buffer solution with a pH of 7.5–8.5 as a reaction medium to form a conversion system; the conversion reaction is carried out at 30–60°C and 150–500 rpm; after the reaction, the reaction solution is separated and purified to obtain R-mandelic acid.

[0011] Furthermore, the final concentration of the substrate added is 20-100 mM (preferably 50 mM) based on the volume of the buffer solution, and the amount of catalyst used is based on the weight of the wet bacterial cells, wherein the amount of wet bacterial cells added is 5-20 g / L (preferably 10 g / L) based on the volume of the buffer solution, wherein the water content of the wet bacterial cells is 88-92% by mass; the volume concentration of methanol added is 0.5-5% based on the volume of the buffer solution, preferably 0.5%.

[0012] Furthermore, the buffer solution was a 50 mM Tris-HC1 buffer solution at pH 8.0.

[0013] Furthermore, the preferred reaction conditions are 30°C and 180 rpm for 30 min.

[0014] Furthermore, the method for preparing the wet bacterial cells is as follows: Engineered bacteria containing the nitrile hydrolase mutant encoding gene are inoculated into LB liquid medium containing a final concentration of 50 mg / L kanamycin and cultured with shaking at 37°C and 200 rpm for 8-10 hours to obtain a seed culture; the seed culture is then inoculated into fresh LB liquid medium containing a final concentration of 50 mg / L kanamycin at a volume concentration of 2% and cultured with shaking at 37°C and 180 rpm until the bacterial cell OD... 600 The concentration of the solution was 0.6-0.8. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM. The cells were induced and cultured at 28℃ and 180 rpm for 10-12 h. The cells were then collected by centrifugation at 4℃ and 8000 rpm for 10 min, which yielded wet cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0016] This invention utilizes (semi-)rational design to molecularly modify the nitrile hydrolase OsNIT-mut, obtaining a mutant with synergistically enhanced hydrolysis specificity and stereoselectivity. Before mutation, OsNIT-mut catalyzed mandelic acid at 98.5%, but the ee value of R-mandelic acid was only 46.7%, while the nitrile hydrolysis activity of OsNIT-mut was recorded as 100%. Specifically, the mutant OsNIT-mut / V246N / T166G / R79Y / A167N catalyzed mandelic acid at 90.5%, while the ee value of R-mandelic acid increased to 95.2%, resulting in a relative hydrolysis activity of 399.1%. The regulation of hydrolysis specificity and stereoselectivity of the nitrile hydrolase mutant enables its application in the green industrial catalytic synthesis of chiral mandelic acid, which is of significant importance. (iv) Description of the attached drawings

[0017] Figure 1Comparison of catalytic performance of nitrile hydrolase OsNIT-mut and mutant OsNIT-mut / V246N / T166G / R79Y / A167N in Example 4. (V) Detailed Implementation

[0018] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0019] LB liquid culture medium composition: 10 g / L peptone, 10 g / L NaCl, 5 g / L yeast extract powder, solvent: deionized water, pH: natural. Peptone and yeast extract powder were purchased from Thermo Fisher Scientific, and NaCl was purchased from Sinopharm Reagent Co., Ltd. Sterilization conditions: Autoclaved at 121°C for 20 min using an autoclave. After sterilization, kanamycin was added to a final concentration of 50 mg / L before use for cell culture.

[0020] LB plate medium is prepared by adding 20 g / L agar powder to the above-mentioned components of LB liquid medium, and the sterilization conditions are the same as above.

[0021] Example 1: Construction of the starting strain for the nitrile hydrolase OsNIT

[0022] Using the OsNIT mutant K200R / R224W / N246V (labeled OsNIT-mut) obtained by selectively regulating the nitrile hydrolase OsNIT sequence (GenBank: AB027054) in a previous reaction (ACS Catal. 2023, 13: 10282-10294) as a template, the amino acid sequence is shown in SEQ ID NO.1 and the nucleotide sequence is shown in SEQ ID NO.2. The mutant is cloned between the BamH I and Hind III sites of the pET28b(+) plasmid and introduced into Escherichia coli BL21 host cells to construct the starting strain.

[0023] SEQ ID NO.1

[0024] MAMVPSGSGGGPPVIAEVEMNGGATSGAATVRATVVQASTVFYDTPATLDKAERLIEEAAGYGSQLVVFPEAFVGGYPRGSTFGFGANISIGNPKDKGKEEFRKYHAAAIEVPGPEVTRLAAMAGKYKVFLVMGVIEREGYTLYCSVLFFDPLGRYLGKHRKLMPTALERIIWGFGDGSTIPVYD TPLGKIGALICWENRMPLLRTALYGKGIEIYCAPTADSWQVWQASMTHIALEGGCFVLSAVQFCRRKDYPPPPEYVFTGLGEEPSPDTVVCPGGSVIISPSGEVLAGPNYEGEALITADLDLGEIVRAKFDFDVVGHYARPEVLSLVVNDQPHLPVSFTSAAEKTTAAKSDSTAKPYLEHHHHHH.

[0025] Example 2: Construction of a nitrile hydrolase mutant library

[0026] 1. Screening for mutation sites

[0027] A semi-rational analysis was performed on the loop region structure near the active catalytic pocket of OsNIT-mut to predict amino acid residues that affect enzyme activity and selectivity, including Y77, R79, G80, T166, A167, and V246. A site-directed saturation mutagenesis library was constructed using a site-directed saturation mutagenesis strategy.

[0028] 2. Construction of mutant libraries

[0029] Using the recombinant plasmid pET28b-OsNIT-mut containing the target gene fragment extracted in Example 1 as a template, the template was amplified by overlapping extension PCR using the primers in Table 1.

[0030] The PCR amplification system was as follows (50 μL): template DNA 0.1 ng-1 ng, 2×Phanta Max Buffer 25 μL, dNTPs (10 mM each) 1 μL, upstream and downstream primers 1 μL each, Phanta Max Super-Fidelity DNA Polymerase 1 U, and the remainder ddH2O to be added to the total volume.

[0031] PCR reaction parameters: (1) 95℃ pre-denaturation for 30s; (2) 95℃ denaturation for 30s; (3) 62℃ annealing for 30s; (4) 72℃ extension for 6min, repeat steps (2)-(4) 30 times; (5) 72℃ complete extension for 7min, store at 16℃.

[0032] After the PCR product was positive by 0.9% agarose gel electrophoresis, 1 μL of restriction endonuclease Dpn I was added and digested at 37℃ for 0.5-1 h to remove template plasmid DNA, and then inactivated at 65℃ for 10 min.

[0033] Add 20 μL of template-eliminated PCR product to 100 μL of E. coli BL21(DE3) competent cells, place on ice for 30 min, heat shock at 42 °C for 90 s, add 600 μL of LB liquid medium, thaw at 37 °C for 1 h, then plate on LB plates containing a final concentration of 50 mg / L kanamycin, and incubate at 37 °C overnight to obtain the mutant library.

[0034] Table 1 Primer Design Table

[0035]

[0036] In Table 1, N represents A / C / G / T, and K represents G / T.

[0037] Example 3: Screening of unit point mutants of nitrile hydrolase

[0038] 1. Hydrolytic activity

[0039] (1) The mutant library monoclonal strains obtained in Example 2 were picked up with a sterile toothpick and transferred to a 96-well plate containing 600 μL of LB liquid medium containing kanamycin (kana, 50 mg / L). The plates were incubated at 37°C and 180 rpm for 20 h to obtain seed culture. 30 μL of seed culture was added to each well of a new 96-well plate containing 800 μL of LB liquid medium containing 50 mg / L kana. The plates were incubated at 37°C and 180 rpm for 2-3 h to allow OD to develop. 600 When the concentration reaches 0.6-0.8, add IPTG to each well to a final concentration of 0.1 mM and incubate at 28°C and 180 rpm for 12 h to induce expression of nitrile hydrolase protein. Then centrifuge at 4°C and 4000 rpm for 15 min, discard the supernatant and retain the bacterial cells for later use.

[0040] (2) Step (1) After centrifugation, resuspend the centrifuged bacterial cells in each well of the 96-well plate with 400 μL Tris-HCl (50 mM, pH 8.0), divide it into two equal parts. Add (S)-mandelinnitriles (dissolved in 2 μL methanol, final concentration 50 mM) to each well of the first part, and add (R)-mandelinnitriles (dissolved in 2 μL methanol, final concentration 50 mM) to each well of the second part. React at 30℃ and 180 rpm for 30 min. Terminate the reaction with 20 μL HCl (2 M). After the reaction is completed, centrifuge at 4℃ and 4000 rpm for 15 min and collect the supernatant.

[0041] (3) Dissolve 0.1 g of o-phthalaldehyde and 50 μL of mercaptoethanol in 20 mL of anhydrous ethanol, and dilute by 10 times with anhydrous ethanol to prepare a fluorescent detection solution. Take 20 μL of the first supernatant from step (2) into a black fluorescent microplate, add 150 μL of fluorescent detection solution to each well, react at 37°C for 30 min, and then measure the fluorescence intensity (412 nm excitation wavelength, 467 nm emission wavelength) using a microplate reader to obtain fluorescence intensity 1, and determine the hydrolytic activity of nitrile hydrolase on (S)-mandelian nitrile. Take 20 μL of the second supernatant from step (2) into a black fluorescent microplate, add 150 μL of fluorescent detection solution to each well, react at 37°C for 30 min, and then measure the fluorescence intensity (412 nm excitation wavelength, 467 nm emission wavelength) using a microplate reader to obtain fluorescence intensity 2, and determine the hydrolytic activity of nitrile hydrolase on (R)-mandelian nitrile. The stereoselectivity of nitrile hydrolases was determined by comparing the hydrolytic activities of (S)-mandelic acid and (R)-mandelic acid, and a positive mutant with higher hydrolytic activity to (R)-mandelic acid was obtained for initial screening.

[0042] 2. Reaction selectivity

[0043] (1) Wet bacteria

[0044] The initial screening positive mutants obtained in step 1 were sequenced. Recombinant genetically engineered bacteria with correct sequencing results were inoculated into LB liquid medium containing a final concentration of 50 mg / L kana and cultured at 37°C for 8-10 hours. Then, they were transferred at a 2% (v / v) inoculation rate to fresh LB liquid medium containing a final concentration of 50 mg / L kana and cultured at 37°C and 180 rpm until the bacterial OD reached a certain level. 600 When the concentration is 0.6-0.8, add IPTG to a final concentration of 0.1 mM, and induce culture at 28℃ and 180 rpm for 10-12 h. Centrifuge at 4℃ and 8000 rpm for 10 min to collect the wet bacterial cells (i.e., resting cells, with a water content of 88-92%, used for the hydrolysis of mandelic acid nitrile). Store at -20℃ for later use.

[0045] (2) Detection of catalytic performance of mutants

[0046] The standard reaction system composition and reaction conditions were as follows: 20 mL Tris-HCl buffer solution (50 mM, pH 8.0), 0.2 g wet bacterial cells, methanol added at a volume concentration of 0.5% (v / v), and mandelic acid nitrile added to a final concentration of 50 mM (mandelic acid nitrile was first dissolved in methanol). The reaction solution was reacted at 30℃ and 180 rpm for 30 min. A 1 mL sample was taken, and 50 μL of 2 M HCl was added to terminate the reaction. The mixture was centrifuged at 12000 rpm for 1 min, and the supernatant was collected. The content of each component in the reaction solution was analyzed by high performance liquid chromatography (HPLC), and the relative enzyme activity, the proportion of mandelic acid in the product, and the ee value of the product were calculated. The results are shown in Table 2.

[0047] The hydrolytic activity of nitrile hydrolase is defined as the amount of enzyme required to generate 1 μmol of mandelic acid per minute under standard reaction conditions, i.e., 30℃ and 180 rpm, which is one enzyme activity unit (1U).

[0048] Liquid chromatography analysis was performed using an Agilent 1690 chromatograph with a CHIRALCEL OJRHCD-AW055 column at a column temperature of 25℃ and a detection wavelength of 205nm. The mobile phase was acetonitrile:water (containing 0.1% HAc) = 10:90 (volume ratio), and the flow rate was 0.4mL / min.

[0049] Table 2 Catalytic performance of OsNIT-mut and mutants obtained through screening.

[0050]

[0051]

[0052] Example 4: Construction and Performance Characterization of Combined Mutants

[0053] Using OsNIT-mut / V246N obtained in Example 3 as a template, the combined mutant OsNIT-mut / V246N / T166G (amino acid sequence as shown in SEQ ID NO.3) was obtained as in Example 2. OsNIT-mut / V246N / T166G / R79Y (amino acid sequence as shown in SEQ ID NO.4) and OsNIT-mut / V246N / T166G / R79Y / A167N (amino acid sequence as shown in SEQ ID NO.5) were further obtained.

[0054] The recombinant mutant bacteria were cultured and characterized under the conditions of Example 3, and the results are shown in Table 3. Figure 1 As shown.

[0055] Table 3. Catalytic performance characterization of the OsNIT-mut combinatorial mutant.

[0056]

[0057] Table 3 Figure 1 The results showed that the nitrile hydrolase mutant OsNIT-mut / V246N / T166G / R79Y / A167N catalyzed the amygdalinide reaction with a hydrolysis activity of 399.1% of that of OsNIT-mut, and the optical purity of the R-mandelic acid produced increased from 46.7% to 95.2%, while the mandelic acid content in the product remained at 90.5%.

[0058] The above results indicate that, although the mandelic acid content in the product slightly decreases during the catalytic conversion of mandelic acid from mandelic nitrile by the nitrile hydrolase mutant described in this invention, the stereoselectivity of the R-configuration product is significantly improved. This invention effectively synergistically regulates the specificity and stereoselectivity of the nitrile hydrolase hydrolysis reaction, while preserving the nitrile hydrolysis activity. Therefore, this invention is of great significance for the synthesis of mandelic acid with high optical purity and the development of nitrile hydrolases for the synthesis of carboxylic acids.

Claims

1. A nitrile hydrolase mutant possessing both high activity and high stereoselectivity, characterized in that, The mutant was obtained by single or multiple mutations of amino acids 79, 166, 167, and 246 of the amino acid sequence shown in SEQ ID NO.

1.

2. The nitrile hydrolase mutant as described in claim 1, characterized in that, The nitrile hydrolase mutant is formed by mutating the amino acid sequence shown in SEQ ID NO. 1 to one of the following: (1) valine at position 246 is reversed to asparagine, V246N; (2) arginine at position 79 is reversed to tyrosine or asparagine, R79Y, R79N; (3) threonine at position 166 is reversed to glycine, T166G; (4) alanine at position 167 is reversed to asparagine, A167N; (5) glycine at position 80 is reversed to lysine, G80K; (6) valine at position 246 is reversed to asparagine, V246N ... (7) The valine at position 246 is reversed to asparagine, the threonine at position 166 is reversed to glycine, and the arginine at position 79 is reversed to tyrosine, V246N / T166G / R79Y; (8) The valine at position 246 is reversed to asparagine, the arginine at position 79 is reversed to tyrosine, the threonine at position 166 is reversed to glycine, and the alanine at position 167 is reversed to asparagine, V246N / R79Y / T166G / A167N.

3. A recombinant genetically engineered bacterium containing the coding gene of the nitrile hydrolase mutant of claim 1.

4. The application of the nitrile hydrolase mutant of claim 1 in the catalytic synthesis of chiral mandelic acid from mandelic nitrile.

5. The application as described in claim 4, characterized in that, The application is as follows: using wet cells obtained by fermentation culture of engineered bacteria containing nitrile hydrolase mutant encoding gene or enzyme extracted from wet cell lysis as a biocatalyst, mandelic acid nitrile as substrate, methanol as adjuvant, and buffer solution with pH 7.5-8.5 as reaction medium to form a transformation system, the transformation reaction is carried out at 30-60℃ and 150-500rpm, after the reaction is completed, the reaction solution is separated and purified to obtain R-mandelic acid.

6. The application as described in claim 5, characterized in that, The final concentration of the substrate added is 20-100 mM based on the volume of the buffer solution, and the amount of catalyst added is based on the weight of the wet cell, with the wet cell added amount being 5-20 g / L based on the volume of the buffer solution; the volume concentration of the methanol added is 0.5-5% based on the volume of the buffer solution.

7. The application as described in claim 5, characterized in that, The buffer solution was a 50 mM Tris-HCl buffer solution with a pH of 8.

0.

8. The application as described in claim 5, characterized in that, The reaction conditions were 30℃ and 180rpm for 30min.

9. The application as described in claim 5, characterized in that, The method for preparing the wet bacterial cells is as follows: engineered bacteria containing the nitrile hydrolase mutant encoding gene are inoculated into LB liquid medium containing a final concentration of 50 mg / L kanamycin and cultured with shaking at 37°C and 200 rpm for 8-10 h to obtain seed culture; The seed culture was inoculated at a volume concentration of 2% into fresh LB liquid medium containing a final concentration of 50 mg / L kanamycin, and cultured at 37°C with shaking at 180 rpm until the bacterial cell OD reached its maximum. 600 The concentration of the solution was 0.6-0.

8. Isopropyl-β-D-thiopyranogalactopyranoside was added to a final concentration of 0.1 mM. The cells were induced and cultured at 28℃ and 180 rpm for 10-12 h. The cells were then collected by centrifugation at 4℃ and 8000 rpm for 10 min, which yielded wet cells.

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