Carbonyl reductase mutants and their application in the synthesis of S-bosone

Abstract

The present invention provides a carbonyl reductase mutant and its synthesis S The mutant protein is a non-natural protein, and the mutant protein has the ability to significantly improve the catalytic reduction of the carbonyl group in 1-C-(β-D-pyranosyl)-acetone to generate S The carbonyl reductase mutant strain with enhanced activity in the present invention is applied to S In the production process of ‑configuration bosogen, its carbonyl reduction ability can be improved, and the complete conversion of 100‑240 g / L substrate can be achieved to generate S ‑Configurational Bose.

Description

Technical Field

[0001] The invention belongs to the fields of molecular biology and enzyme engineering, and relates to a carbonyl reductase mutant and an application thereof in synthesizing S-bosone. Background Art

[0002] Carbonyl reductase Carbonyl reductase, also known as alcohol dehydrogenase, is a type of biocatalyst that reversibly catalyzes the reduction of carbonyl groups to alcohols. In order to achieve catalytic activity, carbonyl reductase requires coenzymes to participate in the transfer of hydrogen anions to carbonyl substrates. In the catalytic process of carbonyl reductase, the use of coenzymes is the same as the molar amount of substrates. From the perspective of the economy of the reaction, the corresponding coenzyme regeneration system will be matched in the catalytic reaction of carbonyl reductase. With the development of directed evolution technology, carbonyl reductase is more and more widely used in the field of chiral alcohol preparation.

[0003] It can reversibly catalyze the conversion of ketones and alcohols. Pro-Xylane TM is an important cosmetic raw material with a wide range of biological activities. It uses 1-C-(β-D-pyranosyl)-acetone as a substrate to reduce the carbonyl group. S Configuration and R The key steps of configuring Bose ( Figure 1 ). Currently on the market S Configuration and R Configuration: There are two different configurations of boson. According to a study published by L'Oreal Laboratories in Bioorganic & Medicinal Chemistry Letters 19 (2009) 845–849: S type / R The activity of type = 70 / 30 is significantly higher than the 50 / 50 ratio, which means S The configuration is likely to be the dominant configuration, which is more conducive to the production of glycosaminoglycan (GAG). Subsequent studies have shown that S The cytotoxicity of the configuration Bose is weaker than R Configuration Bose, and the biological activity is higher than R Configuration Bose, therefore S Configurational Bose is superior to R Currently, many companies have developed enzyme-based S- Biocatalytic technology of configuration boson (CN202111296847.5, CN202210266303.2, CN202311146214.5, CN202410146028.X, CN202410288008.6). The present invention obtains a mutant with significantly improved activity in converting 1-C-(β-D-xylopyranosyl)-acetone through directed evolution. S -Lays the foundation for green biomanufacturing of configurational boson. Summary of the invention

[0004] The present invention provides a carbonyl reductase mutant, a coding gene and an application thereof, which improves enzyme activity and substrate structure diversity, etc., so as to meet industrial use requirements.

[0005] The present invention is derived from Kazachstania africana CBS 2517 The enzyme (KaCR, GenBank: XP_003956146.1) was functionally determined and directed evolution was performed to obtain a mutant with significantly improved enzyme activity, catalyzing the production of 1-C-(β-D-pyranosyl)-acetone. S -Configuration Bose, thereby completing the present invention.

[0006] The present invention provides a mutant protein of carbonyl reductase, which has one or more substitution mutations at positions 85, 87, 128, 134, 162, 197, 219, 234, 235 based on the amino acid sequence shown in SEQ ID NO: 1.

[0007] Specifically, based on the amino acid sequence shown in SEQ ID NO: 1, there is a mutation at any of the following sites: the phenylalanine (F) at position 85 mutates to cysteine ​​(C) or threonine (T); the phenylalanine (F) at position 87 mutates to tryptophan (W); the tyrosine (Y) at position 128 mutates to alanine (A) or glycine (G); the phenylalanine (F) at position 134 mutates to valine (V), isoleucine (I) or leucine (L); the histidine (H) at position 162 mutates to alanine (A); the valine (V) at position 197 mutates to threonine (T) or serine (S); the tyrosine (Y) at position 219 mutates to isoleucine (I) or phenylalanine (F); the proline (P) at position 234 mutates to arginine (R); and the valine (V) at position 235 mutates to isoleucine (I).

[0008] Preferably, based on the amino acid sequence shown in SEQ ID NO: 1, there is a mutation at any of the following positions: a combined mutation at positions 85 and 87; a combined mutation at positions 85, 87 and 128; a combined mutation at positions 85, 87 and 134; a combined mutation at positions 85, 87, 134 and 197; a combined mutation at positions 85, 87, 134, 197 and 219.

[0009] The present invention provides a nucleic acid encoding the mutant protein.

[0010] The invention also provides an expression vector containing the nucleic acid.

[0011] The present invention also provides a recombinant host bacteria containing the nucleic acid or the expression vector.

[0012] The present invention also provides the use of the mutant protein in synthesizing S-bosone.

[0013] The present invention provides a method for reducing 1-C-(β-D-xylopyranosyl)-acetone, which comprises the steps of:

[0014] The mutant protein is contacted with the reaction substrate 1-C-(β-D-xylopyranosyl)-acetone, and a coenzyme circulation system is added to perform a catalytic reaction, thereby obtaining the S - configuration Bose; after the reaction is completed, the reduced pressure is concentrated, and the S - Boseraphim.

[0015] Specifically, the pH of the catalytic reaction system is 6.0-8.0; the substrate concentration is 80-240 g / L; and the reaction time is 10-48 hours.

[0016] Preferably, the pH of the catalytic reaction system is pH 6.0-7.0; the substrate concentration is 120-200 g / L; and the reaction time is 12-24 hours.

[0017] Furthermore, the mutant protein is used as a pure protein, or the recombinant host bacteria are cultured to obtain a mutant crude protein solution, or the recombinant host bacteria are used as a biocatalyst to perform a catalytic reaction.

[0018] The enzyme activity of the mutant protein obtained by the present invention is significantly improved compared with the wild type, and can be used to catalyze the production of 1-C-(β-D-xylopyranosyl)-acetone. S -Configuration Bose, has application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Shows R -Configurational Boson and S -Configurational Bose liquid phase diagram.

[0020] Figure 2 The liquid phase spectrum of the KaCR-catalyzed conversion reaction is shown. DETAILED DESCRIPTION

[0021] The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.

[0022] Example 1: Construction of a carbonyl reductase mutant library and screening of mutants

[0023] 1. The first round of vitality transformation

[0024] The relationship between KaCR and coenzyme NADP was obtained by RoseTTAFold All-Atom simulation. + Docking results with substrate 1-C-(β-D-xylopyranosyl)-acetone.

[0025] The amino acid sites within 8 Å were analyzed with the substrate as the center, among which S127 / Y165 / K169 was the catalytic triad. The structure was analyzed and the following sites were selected for the establishment of the mutation library: F85, F87, T125, S126, Y128, F134, H162, V197, V199, F200, N214, T215, E218, Y219, A226, P234 and V235 were subjected to saturation mutation respectively, and the mutation primers were designed using the degenerate codon NNK, and pET21a-KaCR was used as the template. Two-step PCR was used for PCR with the high-fidelity polymerase Pfx.

[0026] 1) Construction of pET21a-KaCR plasmid

[0027] The wild-type amino acid sequence of carbonyl reductase is shown in SEQ ID NO: 1. The corresponding nucleotide sequence was fully synthesized and cloned into the restriction endonuclease sites NdeI and XhoI of the pET-21a vector to obtain the recombinant plasmid pET21a-KaCR, which was further transformed into the expression host E. coli BL21(DE3), pick the positive clones, and obtain the recombinant expression transformants E. coliBL21(DE3) / pET21a-KaCR.

[0028] 2) Construction of carbonyl reductase mutant library

[0029] Using pET21a-NgADH as a template, amplification was performed using the corresponding primers in Table 1. The forward primer 25F was combined with the reverse primer 85-162-R for PCR, and the forward primer 100-F was combined with the reverse primer 197-235-R for PCR. The PCR reaction system and reaction conditions were as follows:

[0030] 3) First step PCR reaction system and reaction conditions

[0031] Round 1: In a PCR reaction system with a total volume of 25 μL, add 15 ng of template, 12.5 μL of 2× PfxMIX, and 0.5 μL of each pair of mutation primers (10 μM), and add sterilized distilled water to 25 μL.

[0032] The first step of PCR reaction procedure: ① pre-denaturation at 98℃ for 2 min, ② denaturation at 98℃ for 30 sec, ③ annealing at Tm-5℃ for 30 sec, ④ extension at 72℃ for 30 sec, ⑤ final extension at 72℃ for 5 min, steps ② to ④ were performed for a total of 30 cycles.

[0033] 4) Second step PCR reaction system and reaction conditions

[0034] Round 2: In a PCR reaction system with a total volume of 50 μL, add 30 ng of template, 25 μL of 2× Pfx MIX, 1 μL of primers as the first-round PCR product fragment, and add sterilized distilled water to 50 μL.

[0035] The second step PCR reaction procedure: ① pre-denaturation at 98℃ for 2min, ② denaturation at 98℃ for 30 sec, ③ annealing at 55℃ for 30sec, ④ extension at 72℃ for 3min, ⑤ final extension at 72℃ for 10min, steps ② to ④ were performed for a total of 25 cycles.

[0036] The PCR product obtained in the above steps was verified by agarose gel electrophoresis analysis, and then digested with restriction endonuclease DpnI at 37°C for 2h. E. coli BL21(DE3) competent cells were spread on plates containing 10 μg / mL ampicillin antibiotics and placed in a 37°C incubator for about 12 hours to grow single colonies and obtain a carbonyl reductase mutant library.

[0037] At the same time, the pET21a-NgADH mutant library plasmid was transferred into E. coliBL21(DE3) competent cells were spread on a plate containing 100 mg / mL ampicillin antibiotics and placed in a 37°C incubator for about 12 hours to grow a single colony and obtain a strain expressing a carbonyl reductase mutant.

[0038] Table 1. Primer sequences for the first round of modification

[0039]

[0040] 5) Inducible expression of carbonyl reductase mutants

[0041] The monoclonal colony obtained after the above step 2 was picked into 4 mL of LB liquid medium (peptone 10 g / L, yeast powder 5 g / L, NaCl 10 g / L) containing ampicillin (100 mg / L), and cultured overnight at 37°C and 200 rpm to obtain a culture solution. The culture solution was inoculated into the fermentation medium (LB liquid medium) at an inoculum volume of 1% (v / v), and cultured in a shaking incubator at 37°C and 200 rpm until the OD 600 The concentration of IPTG was 0.6-0.8, and IPTG was added to a final concentration of 0.1 mM. The cells were induced at 25°C and 200 rpm on a shaker for 8-12 hours. The culture medium was centrifuged at 6000 g to collect the cells, which were crushed under high pressure to obtain the crude enzyme solution of carbonyl reductase for subsequent enzyme activity detection.

[0042] 6) Screening of carbonyl reductase mutants

[0043] The screening method is to detect the decrease of NADPH at 340 nm. The specific reaction method is: substrate 10 mM, NADPH 0.25 mg / mL, crude enzyme solution 10 μL, potassium phosphate buffer to 200 μL. NADPH has a characteristic absorbance at 340 nm. The decrease in absorbance at 340 nm is detected by a microplate reader. If the enzyme activity is relatively high, the consumption of NADPH is fast and the slope of the decline curve is large. The beneficial mutation sites that improved the enzyme activity obtained by screening are 85, 87, 128, 134, 162, 197, 219, 234 and 235, which are mutants 1-13, respectively. The results are shown in Table 2.

[0044] 2. Iterative saturation mutation vitality transformation

[0045] According to the saturation mutation results, a combination mutant of a single mutation site was constructed at the improved site, and the obtained mutant was picked into a test tube containing 4 mL LB medium for culture. The activity of the expressed protein was tested, and the results of obvious improvement in activity are shown in Table 2.

[0046] Mutant 1 with the highest activity improvement was selected as a template, and a mutation library was constructed at 87, 128, 134, 162, 197, 219, 234 and 235 for screening to obtain the optimal mutant 16; mutant 16 with the highest activity was selected as a template, and a mutation library was constructed at 128, 134, 162, 197, 219, 234 and 235 for screening to obtain the optimal mutant 18; mutant 18 with the highest activity was selected as a template, and a mutation library was constructed at 128, 162, 197, 219, 234 and 235 for screening to obtain the optimal mutant 19; mutant 19 with the highest activity was selected as a template, and a mutation library was constructed at 128, 162, 219, 234 and 235 for screening, and no mutants with significantly improved enzyme activity were found.

[0047] Table 2. Relative activities of KaCR and its mutants on substrates

[0048]

[0049] Example 2: Whole-cell catalytic synthesis using mutants S -Configurational Bose

[0050] The mutant 16 was obtained by inducing expression according to the method of Example 1, and the bacterial cells were collected by centrifugation (6000 rpm) and used as biocatalysts. During the reaction, 1 N NaOH was used to control the pH between 6.5 and 7.0.

[0051] 0.9 g of mutant 16 wet cells were resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 80 g / L 1-C-(β-D-xylopyranosyl)-acetone, 450 mM glucose, 0.3 g / L NADP + After 30 U / mL GDH was reacted at 30 °C for 6 hours, HPLC analysis showed a conversion rate of 98.8%.

[0052] 0.9 g of mutant 17 wet cells were resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 80 g / L 1-C-(β-D-xylopyranosyl)-acetone, 450 mM glucose, 0.3 g / L NADP + After the reaction of 30 U / mL GDH at 30 °C for 5 hours, HPLC analysis showed a conversion rate of 98.9%.

[0053] 0.9 g of mutant 18 wet cells were resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 80 g / L 1-C-(β-D-xylopyranosyl)-acetone, 450 mM glucose, 0.3 g / L NADP +After the reaction of 30 U / mL GDH at 30 °C for 3 hours, HPLC analysis showed a conversion rate of 98.9%.

[0054] 0.9 g of mutant 19 wet cells were resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 120 g / L 1-C-(β-D-xylopyranosyl)-acetone, 650 mM glucose, 0.3 g / L NADP + After the reaction of 30 U / mL GDH at 30 °C for 8 hours, HPLC analysis showed a conversion rate of 99.2%.

[0055] 0.9 g of mutant 19 wet cells were resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 240 g / L 1-C-(β-D-xylopyranosyl)-acetone, 1.3 M glucose, 0.5 g / L NADP + After the reaction of 30 U / mL GDH at 30 °C for 16 hours, HPLC analysis showed a conversion rate of 99.1%.

[0056] 0.9 g of mutant 19 wet cells were resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 300 g / L 1-C-(β-D-xylopyranosyl)-acetone, 1.7 M glucose, 0.5 g / L NADP + After the reaction of 30 U / mL GDH at 30 °C for 26 hours, HPLC analysis showed a conversion rate of 98.9%.

[0057] Example 3: Catalytic synthesis using crude enzyme solution of mutants S -Configurational Bose

[0058] The mutants were induced to express according to the method of Example 1, and the cells were collected by centrifugation (6000 rpm), and the crude enzyme solution after the cells were broken was used as a biocatalyst. Other conditions were the same as those of Example 2.

[0059] 0.9 g of mutant 18 wet bacterial cell lysis solution was resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 80 g / L 1-C-(β-D-xylopyranosyl)-acetone, 450 mM glucose, 0.3 g / L NADP + After 30 U / mL GDH was reacted at 30°C for 2 hours, HPLC analysis showed a conversion rate of 99.2%.

[0060] 0.9 g of mutant 19 wet bacterial cell lysis solution was resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 120 g / L 1-C-(β-D-xylopyranosyl)-acetone, 650 mM glucose, 0.3 g / L NADP + After 30 U / mL GDH was reacted at 30°C for 6 hours, HPLC analysis showed a conversion rate of 99.2%.

[0061] 0.9 g of mutant 19 wet bacterial cell lysis solution was resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 240 g / L 1-C-(β-D-pyranosyl)-acetone, 1.3 M glucose, 0.5 g / L NADP + After the reaction of 30 U / mL GDH at 30°C for 10 hours, HPLC analysis showed a conversion rate of 99.0%.

[0062] 0.9 g of mutant 19 wet bacterial cell lysis solution was resuspended in 30 mL phosphate buffer (pH 7.0, 100 mM), 300 g / L 1-C-(β-D-xylopyranosyl)-acetone, 1.7 M glucose, 0.5 g / L NADP + After the reaction of 30 U / mL GDH at 30°C for 20 hours, HPLC analysis showed a conversion rate of 99.2%.

Claims

1. A mutant protein of carbonyl reductase, characterized in that The mutant protein has only the following mutations based on the amino acid sequence shown in SEQ ID NO: 1: a combined mutation at position 85 and position 87; a combined mutation at position 85, position 87 and position 128; a combined mutation at position 85, position 87 and position 134; a combined mutation at position 85, position 87, position 134 and position 197; or a combined mutation at position 85, position 87, position 134, position 197 and position 219; The phenylalanine at position 85 mutates to cysteine; the phenylalanine at position 87 mutates to tryptophan; the tyrosine at position 128 mutates to alanine; the phenylalanine at position 134 mutates to valine; the valine at position 197 mutates to threonine; and the tyrosine at position 219 mutates to isoleucine.

2. A nucleic acid encoding the mutant protein according to claim 1.

3. An expression vector containing the nucleic acid according to claim 2.

4. A recombinant host bacteria containing the nucleic acid according to claim 2 or the expression vector according to claim 3.

5. Use of the mutant protein as claimed in claim 1 in the synthesis of S-bosone.

6. A method for reducing 1-C-(β-D-xylopyranosyl)-acetone, characterized in that: Includes steps: The mutant protein of claim 1 is contacted with a reaction substrate 1-C-(β-D-xylopyranosyl)-acetone, and a coenzyme circulation system is added to carry out a catalytic reaction, thereby obtaining S - configuration Bose; after the reaction is completed, the reduced pressure is concentrated, and the S -Configurational Bose.

7. The method according to claim 6, characterized in that The pH of the catalytic reaction system is 6.0-8.0; the substrate concentration is 80-240 g / L; and the reaction time is 10-48 hours.

8. The method according to claim 7, characterized in that The pH of the catalytic reaction system is pH 6.0-7.0; the substrate concentration is 120-200 g / L; and the reaction time is 12-24 hours.

9. The method according to any one of claims 6 to 8, characterized in that The mutant protein is in the form of pure protein, or a mutant crude protein solution obtained by culturing the recombinant host bacteria as claimed in claim 4, or a catalytic reaction is carried out using the recombinant host bacteria as claimed in claim 4 as a biocatalyst.

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