Carbonyl reductase mutant and method for catalyzing the synthesis of high-concentration bosogenin

Abstract

The present invention discloses a carbonyl reductase mutant and a method for catalyzing the synthesis of high-concentration bosogenin, belonging to the field of biocatalysis. The present invention adopts a one-pot method to catalyze β-acetone xyloside to synthesize high-concentration bosogenin, uses an enzyme encoded by carbonyl reductase CnMDR, rationally designs multiple key binding sites of the substrate pocket, combines beneficial mutations, uses a recombinant bacterium with an advantageous mutant enzyme as a catalyst, uses β-acetone xyloside as a substrate, and simultaneously introduces formate dehydrogenase variants and NAD + Kinase, drives NADP + ‑NADPH coenzyme cycle, the yield of synthesized bosone is 200g / L, the conversion rate is>99%, and the product ee value is>99%, which are all the highest levels publicly reported so far. The present invention provides a one-pot method for synthesizing bosone with high efficiency and low cost, which only requires the addition of a trace amount (0.3mM) of coenzyme, and the by-products in the reaction system are easy to separate, which provides great convenience for industrial scale-up production.

Description

Technical Field

[0001] The invention relates to a carbonyl reductase mutant and a method for catalyzing the synthesis of high-concentration bosogenin and belongs to the field of biocatalysis. Background Art

[0002] Pro-Xylane TM , chemical name is hydroxypropyl tetrahydropyrantriol (CAS: 439685-79-7), a xylose derivative first discovered by L'Oreal that has the function of delaying aging. Bosin can prevent the reduction and degradation of glycosaminoglycans and proteoglycans in vivo, promote their synthesis in epidermal cells, increase the skin's ability to bind water and improve the skin's biological defense ability. Bosin is easily biodegradable, does not accumulate in the body, and is non-toxic. This feature further increases the popularity of bosin in the fields of biology, medicine, cosmetics, etc.

[0003] Early synthesis methods of bosaiin mainly focused on chemical methods. For example, the patent "Nouveaux dérivés C-glycosides et utilization (New carbon sugar anhydride and its application)" (patent number WO02 / 051828Al) first disclosed the original research and preparation method of bosaiin. This method uses D-xylose as the raw material, dissolves it in water, and then adds acetylacetone and sodium bicarbonate. Xylose and acetylacetone undergo Knoevenagel condensation in an alkaline aqueous solution to obtain β-acetone xyloside, and then the keto group is reduced by sodium borohydride to obtain a tetrahydroxy compound β-acetone xyloside alcohol, namely bosaiin. Among them, the selective reduction of β-acetone xyloside is a difficult point. The patent "A method for preparing C-glycoside derivatives" (application publication number CN 102040575 A) proposes the use of Raney nickel catalysts; the patent "Improved synthesis method of cosmetic active ingredient Bose" (application publication number CN113773291 A) uses a precious metal Ru / C catalyst to reduce the intermediate β-acetone xyloside to avoid impurities and toxic substances brought by sodium borohydride. However, these conventional chemical reduction reactions have the following problems: low stereoselectivity; high emissions of three wastes, serious environmental pollution; the reaction requires high-pressure hydrogen, the equipment required is complex, and there are safety hazards. In particular, some reactions using transition metal catalysis increase the difficulty of purification, and there will be metal residues in the product, which is particularly unfavorable for pharmaceutical and cosmetic active substances.

[0004] The bioenzymatic synthesis of bosyltransferase is safer, more efficient, low-cost, stereoselective and more environmentally friendly. The key to the bioenzymatic synthesis of bosyltransferase is the reduction of the ketocarbonyl group at position 7 to a hydroxyl group. The enzymes reported for the biosynthesis of bosyltransferase are mainly alcohol dehydrogenase ADHs (EC 1.1.1.1). Shenzhen Redlin Biotechnology Co., Ltd. used carbonyl reductase to reduce 34g / L intermediate β-acetone xyloside in the patent "An enzyme composition and a method for synthesizing bosyltransferase by chemical enzymatic method" (application publication number CN 113717997 A) to synthesize bosyltransferase with a yield of 93%. The short-chain carbonyl reductase LkCR reported in "A one-pot method for synthesizing high-concentration (S)-configuration bosyltransferase" (application publication number CN 115896199 A) catalyzes 190g / L substrate, but the total reaction time required is relatively long, which is 36h. The dehydrogenase KPADH mutant used in the patent "A dehydrogenase mutant and its application in the synthesis of S-bosone" (application publication number CN 116926028 A) can catalyze 100g / L of substrate to produce bosone with a conversion rate of more than 99% in about 4-6 hours. However, since the biocatalyst uses a reduced coenzyme (NADH or NADPH) to catalyze the hydrogenation of the keto group of β-acetone xyloside to produce hydroxypropyl tetrahydropyrantriol and an oxidized coenzyme (NAD + or NADP + ), the coenzyme cycle mediated by glucose dehydrogenase pMGDH will produce a large amount of by-products, namely soluble gluconic acid, which greatly increases the difficulty of separation and purification of the target product.

[0005] Therefore, screening for a more efficient and low-cost bosogenin synthase system and reducing the difficulty of by-product separation are of great significance for the industrial production of bosogenin. Summary of the invention

[0006] The present invention uses the carbonyl reductase from Canariomyces notabilis as a template enzyme, and obtains an excellent recombinant bosyl synthase strain through rational design. The enzymatic properties of the bosyl synthase and the conditions for the optimal catalytic reaction are characterized in detail. At the same time, the recombinant bosyl synthase is transformed by protein engineering technology, and the whole cell catalytic synthesis of bosyl synthase is carried out, and the conversion reaction conditions are optimized, which significantly improves the product Pro-Xylane TM The synthesis efficiency is high. Under the scale of 50L, Bose is efficiently synthesized. The separation and purification of the product by this method is simple and efficient, laying a foundation for industrial and efficient preparation.

[0007] The present invention provides a carbonyl reductase mutant, wherein the mutant is obtained by mutating at least one of positions 37, 87, 98, 200, 208 and 295 of the carbonyl reductase; the carbonyl reductase is a carbonyl reductase having an amino acid sequence as shown in SEQ ID NO.1, or a carbonyl reductase having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the sequence as shown in SEQ ID NO.1.

[0008] In one embodiment of the present invention, the carbonyl reductase mutant comprises one or more amino acid substitutions at positions 37, 87, 98, 200, 208, and 295.

[0009] In one embodiment of the present invention, the catalytic efficiency of the carbonyl reductase mutant is measured by using the first intermediate β-acetone xyloside as a substrate to carry out a whole-cell reaction and detecting the product peak area by HPLC.

[0010] The present invention provides a carbonyl reductase mutant, wherein the carbonyl reductase mutant is obtained by mutating one or more amino acids at positions 37, 87, 98, 200, 208 and 295 of the carbonyl reductase of the amino acid sequence as shown in SEQ ID NO.1.

[0011] The amino acid sequence shown in SEQ ID NO.1 is the carbonyl reductase from C. notabilis discovered in our laboratory.

[0012] SEQ ID NO.1 sequence is:

[0013] MTATEAPGAHSKGKVLLTGGTGFVASHVLDCLLDHGFEVVVTARSEEKGQRIIQSIDTPLKKNVSYVVVGNVADEGAFDEAVKSNPPFDYVVHTASPYQFTWNDPVKECLDPAIKGTTGILKSIKAFAPSVKRVVITSSSAAILSPPNHPTKVYNESFWCDLTWEQALNPVHTYRAS KGSPHQKFAEKAAWSFVETEKPNFDLATINNTYTFGPIPRSLDSLSSESINASNKRIYDLVAGNMREGIEPTQPVFTFVDVRDVALAHVRAMTVPGAGGKRFYVVGGFFSNPRLAAIIRDRFPQLEDRLPPADTPDDFPDDHFECDVSRSREVLGLEYTSLEKSVADTVESILRFEGR

[0014] In one embodiment of the present invention, the parent nucleotide sequence encoding the carbonyl reductase is shown as SEQ ID NO.2.

[0015] SEQ ID NO.2

[0016]

[0017] The invention provides a carbonyl reductase mutant. The carbonyl reductase mutant is a mutant obtained by mutating a carbonyl reductase having an amino acid sequence as shown in SEQ ID NO.1: F37A, F37C, F37D, P87S, P87A, P87F, Y98K, N200S, N200T, N208Q, I295C, I295Q, F37C / P87A, F37C / I295C, Y98K / N208Q, P87A / I295C or F37C / P87A / I295C.

[0018] In one embodiment of the present invention, the mutant is obtained by mutating the phenylalanine at position 37 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to alanine, and is named F37A;

[0019] Or the mutant is obtained by mutating the phenylalanine at position 37 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to cysteine, and is named F37C;

[0020] Or the mutant is obtained by mutating the phenylalanine at position 37 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to aspartic acid, and is named F37D;

[0021] Or the mutant is obtained by mutating the proline at position 87 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to serine, and is named P87S;

[0022] Or the mutant is obtained by mutating the proline at position 87 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to alanine, and is named P87A;

[0023] Or the mutant is obtained by mutating the proline at position 87 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to phenylalanine, and is named P87F;

[0024] Or the mutant is a mutant in which the tyrosine at position 98 of the carbonyl reductase in the amino acid sequence as shown in SEQ ID NO.1 is mutated to lysine, and is named Y98K;

[0025] Or the mutant is obtained by mutating the asparagine at position 200 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to serine, and is named N200S;

[0026] Or the mutant is obtained by mutating the asparagine at position 200 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to threonine, and is named N200T;

[0027] Or the mutant is obtained by mutating the asparagine at position 208 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to glutamic acid, and is named N208Q;

[0028] Or the mutant is obtained by mutating the isoleucine at position 295 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to cysteine, and is named I295C;

[0029] Or the mutant is obtained by mutating the isoleucine at position 295 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to glutamine, and is named I295Q;

[0030] Or the mutant is obtained by mutating the phenylalanine at position 37 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to cysteine, and mutating the proline at position 87 to alanine, and is named F37C / P87A;

[0031] Or the mutant is obtained by mutating the phenylalanine at position 37 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to cysteine, and mutating the isoleucine at position 295 to cysteine, and is named F37C / I295C;

[0032] Or the mutant is obtained by mutating the proline at position 87 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to alanine, and mutating the isoleucine at position 295 to cysteine, and is named P87A / I295C;

[0033] Or the mutant is obtained by mutating the tyrosine at position 98 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to lysine, and mutating the asparagine at position 208 to glutamic acid, and is named Y98K / N208Q;

[0034] Or the mutant is obtained by mutating the phenylalanine at position 37 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to cysteine, mutating the proline at position 87 to alanine, and mutating the isoleucine at position 295 to cysteine, and is named F37C / P87A / I295C;

[0035] The present invention also provides a gene encoding the mutant.

[0036] The present invention also provides a recombinant vector carrying the mutant or the gene.

[0037] In one embodiment of the present invention, the recombinant vector is an expression vector using pETDuet series vectors such as pETDuet-1, pETDuet S2, pACYDuet-1, pET series vectors such as pET-28a plasmid, pET-21a plasmid, pRSF series vectors such as pRSF-Duet1 plasmid, and pGEX series vectors such as pGEX-6p-1 plasmid.

[0038] In one embodiment of the present invention, the recombinant vector uses pETDuet-1 plasmid, pET28a plasmid, pRSF-Duet1 plasmid, pET21a plasmid or pGEX-6P-1 plasmid as an expression vector.

[0039] The present invention also provides a recombinant cell expressing the mutant, carrying the gene, or carrying the recombinant vector.

[0040] In one embodiment of the present invention, the recombinant cell uses bacteria or fungi as expression hosts.

[0041] In one embodiment of the present invention, the recombinant cell uses Escherichia coli as an expression host.

[0042] The invention provides a recombinant vector carrying the carbonyl reductase.

[0043] In one embodiment of the present invention, the recombinant vector uses pETDuet-1, pET21a, pRSF-Duet1, pACY-Duet1, pET21a or pGEX-6P-1 as an expression vector.

[0044] The present invention also provides enzymatic properties of the engineered carbonyl reductase, including optimum temperature, pH, temperature stability, and pH stability.

[0045] The present invention also provides a recombinant enzyme catalyst containing the above carbonyl reductase mutant sequence, which is any one of the following forms:

[0046] (1) culturing the recombinant expression transformant and isolating transformant cells containing the recombinase;

[0047] (2) culturing the recombinant expression transformant, isolating transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid;

[0048] (3) culturing the recombinant expression transformant, isolating transformant cells containing the recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant enzyme to obtain a lyophilized enzyme powder.

[0049] The present invention provides a recombinant Escherichia coli, wherein the recombinant Escherichia coli simultaneously overexpresses the above-mentioned bosera synthase, a formate dehydrogenase variant derived from Azospirillum palustre and NAD from Corynebacterium glutamicum. + Kinase.

[0050] In one embodiment of the present invention, the recombinant Escherichia coli uses pET-21a as an expression vector and simultaneously overexpresses the above-mentioned bosera synthase, formate dehydrogenase from A. palustre and NAD from C. glutamicum. + Kinase.

[0051] In one embodiment of the present invention, the amino acid sequence of the formate dehydrogenase is shown as SEQ ID NO.3, and the nucleotide sequence encoding the formate dehydrogenase is shown as SEQ ID NO.4.

[0052] In one embodiment of the present invention, the NAD + The amino acid sequence of the kinase is shown in SEQ ID NO.5, encoding the NAD + The nucleotide sequence of the kinase is shown in SEQ ID NO.6.

[0053] The present invention also provides a method for improving the diastereomeric selectivity and / or conversion rate and / or yield of a product synthesized by carbonyl reductase, the method comprising: mutating the phenylalanine at position 37 of the carbonyl reductase with an amino acid sequence such as that shown in SEQ ID NO.1 to alanine or cysteine; or mutating the proline at position 87 of the carbonyl reductase with an amino acid sequence such as that shown in SEQ ID NO.1 to serine, alanine or phenylalanine; or mutating the tyrosine at position 98 of the carbonyl reductase with an amino acid sequence such as that shown in SEQ ID NO.1 to lysine; or mutating the asparagine at position 200 of the carbonyl reductase with an amino acid sequence such as that shown in SEQ ID NO.1 to serine or threonine; or mutating the asparagine at position 208 of the carbonyl reductase with an amino acid sequence such as that shown in SEQ ID NO.1 to glutamic acid; or mutating the isoleucine at position 295 of the carbonyl reductase with an amino acid sequence such as that shown in SEQ ID NO.1 to cysteine ​​or glutamine; or mutating the tyrosine at position 98 of the carbonyl reductase with an amino acid sequence such as that shown in SEQ ID NO.1 to lysine; The phenylalanine at position 37 of the carbonyl reductase shown in NO.1 is mutated to cysteine, and the proline at position 87 is mutated to alanine; or the phenylalanine at position 37 of the carbonyl reductase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to cysteine, and the isoleucine at position 295 is mutated to cysteine; or the proline at position 87 of the carbonyl reductase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine, and the isoleucine at position 295 is mutated to cysteine; or the tyrosine at position 98 of the carbonyl reductase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to lysine, and the asparagine at position 208 is mutated to glutamate; or the phenylalanine at position 37 of the carbonyl reductase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to cysteine, the proline at position 87 is mutated to alanine, and the isoleucine at position 295 is mutated to cysteine.

[0054] The present invention also provides a method for preparing bosense, which comprises using β-acetone xyloside as a substrate, and then using a carbonyl reductase with an amino acid sequence as shown in SEQ ID NO.1 or the above mutant or the above recombinant cell or the above recombinant Escherichia coli whole cell, and using ammonium formate buffer as a reaction medium to form a reaction system, and transforming and preparing bosense.

[0055] In one embodiment of the present invention, the β-acetone xyloside can be purchased from Shanghai Adamas.

[0056] The method of the present invention is achieved through the following technical solutions:

[0057] The buffer solution, β-acetone xyloside purchased from Shanghai Adamas, catalyst, coenzyme, auxiliary catalyst and cosubstrate are mixed in the buffer solution, and the mixture is converted at 25-35°C for 6-10 hours. After the conversion, acetonitrile is added to terminate the reaction, and the organic phases are extracted and combined, dried by adding anhydrous sodium sulfate, and then evaporated under reduced pressure to remove the solvent to obtain the bosogenin.

[0058] In one embodiment of the present invention, the bosone is obtained by whole-cell biocatalysis of recombinant engineered carbonyl reductase using β-acetone xyloside as a substrate.

[0059] The reaction formula is shown below:

[0060]

[0061] In one embodiment of the present invention, the catalyst is wet bacteria obtained by fermentation culture of genetically engineered bacteria obtained by introducing a gene encoding a wild type or mutant of dehydrogenase CnMDR into cells or Escherichia coli, or a broken liquid, or an enzyme preparation.

[0062] In one embodiment of the present invention, the reaction system further contains formate dehydrogenase or a recombinant cell expressing formate dehydrogenase; the amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO.3.

[0063] In one embodiment of the present invention, the nucleotide sequence encoding formate dehydrogenase is shown as SEQ ID NO.4.

[0064] In one embodiment of the present invention, the reaction system further contains ammonium formate, NAD + .

[0065] In one embodiment of the present invention, the concentration of ammonium formate is 150 mM-1260 mM.

[0066] In one embodiment of the present invention, the concentration of the β-acetone xyloside is 100 mM-1052 mM.

[0067] In one embodiment of the present invention, the NAD + The concentration is 0.1mM-0.5mM.

[0068] In one embodiment of the present invention, the reaction conditions are pH 6.0-8.0 and temperature 20°C-40°C.

[0069] In one embodiment of the present invention, the whole cell catalytic system of the carbonyl reductase includes but is not limited to 1052 mM β-acetone xyloside, NAD +0.3mM, whole cells 40mg / mL. The reaction buffer is 1260mM ammonium formate solution (pH7.5), the reaction time is 8 hours, and the reaction temperature is 25-35°C, such as 30°C.

[0070] In one embodiment of the present invention, the reaction system further contains ammonium formate, NADP + ; Preferably, the concentration of ammonium formate is 150mM-1260mM; Preferably, the concentration of β-acetone xyloside is 100mM-1052mM; Preferably, the NADP + The concentration is 0.1mM-0.3mM; preferably, the reaction conditions are pH 6.0-8.0; the temperature is 20°C-40°C; preferably, the whole cell catalytic system of the carbonyl reductase includes but is not limited to β-acetone xyloside 1052mM, NADP + 0.3 mM, whole cells 40 mg / mL; preferably, the reaction buffer is 1260 mM ammonium formate solution (pH 7.5), the reaction time is 8 hours, and the reaction temperature is 25-35°C, such as 30°C.

[0071] In one embodiment of the present invention, the reaction system further contains sodium formate, NAD + ; Preferably, the concentration of sodium formate is 150mM-1260mM; Preferably, the concentration of β-acetone xyloside is 100mM-1052mM; Preferably, the NAD + The concentration is 0.1mM-0.3mM; preferably, the reaction conditions are pH 6.0-8.0; the temperature is 20°C-40°C; preferably, the whole cell catalytic system of the carbonyl reductase includes but is not limited to 1052mM β-acetone xyloside, NAD + 0.3 mM, whole cells 40 mg / mL; preferably, the reaction buffer is 1260 mM sodium formate solution (pH 7.5), the reaction time is 8 hours, and the reaction temperature is 25-35°C, such as 30°C.

[0072] In one embodiment of the present invention, the reaction system further contains sodium formate, NADP + ; Preferably, the concentration of sodium formate is 150mM-1260mM; Preferably, the concentration of β-acetone xyloside is 100mM-1052mM; Preferably, the NADP + The concentration is 0.1mM-0.3mM; preferably, the reaction conditions are pH 6.0-8.0; the temperature is 20°C-40°C; preferably, the whole cell catalytic system of the carbonyl reductase includes but is not limited to β-acetone xyloside 1052mM, NADP +0.3mM, whole cells 40mg / mL. The reaction buffer is 1260mM sodium formate solution (pH 7.5), the reaction time is 8 hours, and the reaction temperature is 25-35°C, such as 30°C.

[0073] In one embodiment of the present invention, the reaction system further contains ammonium formate, NADP + ; Preferably, the concentration of ammonium formate is 50mM; Preferably, the concentration of β-acetone xyloside is 100mM-1052mM; Preferably, the NADP + The concentration is 0.1mM-0.3mM; preferably, the reaction conditions are pH 6.0-8.0; the temperature is 20°C-40°C; preferably, the buffer in step (2) is selected from potassium phosphate buffer, PBS buffer, ammonium formate buffer, sodium formate buffer, and Tris-hydrochloric acid buffer, with a pH of 7.0-8.0. Preferably, it is formate buffer, the reaction temperature is 30°C, and the reaction time is 8h.

[0074] In one embodiment of the present invention, NAD + Kinases can convert intracellular NAD + Convert to NADP + , thereby reducing NADP + Added.

[0075] In one embodiment of the present invention, after the conversion in step (2) is completed, 200 μL of the reaction solution is sampled and centrifuged at 12,000×g for 3 min, 100 μL of the supernatant is added to 400 μL of dd H2O, filtered with a 0.22 μm filter membrane, and the reaction progress is detected by HPLC. When the conversion rate reaches 90-99%, the pH of the reaction system is adjusted to 2.0-3.0, filtered with diatomaceous earth, and ethyl acetate is added to the filtrate for multiple extractions. The solvent is removed by rotary evaporation to obtain hydroxypropyl tetrahydropyrantriol.

[0076] The present invention also provides a two-step reaction system coupled with a coenzyme regeneration system, wherein the coenzyme circulation system is realized by coupling formate dehydrogenase. The formate dehydrogenase is mainly responsible for the cofactor NADPH / NADP + The general structural formula of the reaction is as follows:

[0077]

[0078] In one embodiment of the present invention, in step (2), the auxiliary catalyst and the cosubstrate act on the coenzyme cycle, and the auxiliary catalyst is formate dehydrogenase ApFDH (the cosubstrate is formate).

[0079] The present invention also provides a scaled-up 50L reaction system, which comprises but is not limited to 200 g / L β-acetone xyloside, 0.3 mM NADP + , 100 mg / mL ammonium formate, 40 mg / mL whole cells. The reaction mixture is reacted in a 100 L reactor at a speed of 300 rpm for 8 hours at a temperature of 25-35°C, such as 30°C.

[0080] The present invention also provides the use of the above-mentioned carbonyl reductase mutant or the above-mentioned gene or recombinant vector, or the above-mentioned recombinant cell, or the above-mentioned recombinant enzyme catalyst, or the above-mentioned recombinant Escherichia coli, or the above-mentioned method in the preparation of bosone or a product containing the above-mentioned bosone.

[0081] In one embodiment of the present invention, the product is a chemical.

[0082] In one embodiment of the present invention, the products include but are not limited to skin care products, makeup products, soap products, bath products, shampoo products, and hair care products.

[0083] Beneficial Effects

[0084] (1) Since the present invention uses β-acetone xyloside and ammonium formate as substrates, the screened dehydrogenase as a catalyst, and NADP + The high-value cosmetic raw material bosine was prepared in two steps for the coenzyme. The biocatalytic system is applicable to 1052mM substrate β-acetone xyloside (200g / L), the catalytic efficiency of the substrate is >99% for 8h, and the carbon dioxide generated by the oxidation of formate by formate hydrogenase ApFDH during the coenzyme cycle can be directly separated from the product. This method has the advantages of high catalytic efficiency, high applicable substrate concentration and easy separation and purification of the product, and is suitable for industrial scale-up production.

[0085] (2) The present invention uses the mined carbonyl reductase sequence as a template to mutate the relevant mutation sites and obtain a carbonyl reductase mutant with significantly improved catalytic efficiency.

[0086] (3) The present invention introduces a coenzyme circulation system to construct a two-step reaction system. After optimizing the conditions of the multi-enzyme catalytic reaction, the multi-enzyme reaction in a 50L scale reaction system improves the yield of bosera catalytic synthesis in one step.

[0087] (4) The yield of bosylamine obtained by constructing a two-step reaction system using the method of the present invention reaches 1050 mM (a total of 200 g / L), and the diastereomeric selectivity (ee value) is greater than 99%. This yield is the highest yield reported for enzyme catalysis so far. The whole-cell biocatalytic reaction is completed in one step, does not require cumbersome steps, does not generate unnecessary by-products, has mild reaction conditions, is environmentally friendly, and is a green and efficient method for biosynthesizing bosylamine. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The present invention is further described in detail below in conjunction with the accompanying drawings.

[0089] Figure 1 This is a high performance liquid chromatography detection diagram of benzophenone; wherein, a is benzophenone and substrate β-acetone xyloside standard; b is the detection of benzophenone in the conversion solution.

[0090] Figure 2 These are the Bose standard curves: a is the S configuration standard curve, and b is the R configuration standard curve.

[0091] Figure 3 The present invention relates to an SDS-PAGE diagram of protein expression of an enzyme; wherein a and b are crude enzyme expression diagrams of CnMDR mutants: wherein 1-17 represent mutants F37A, F37C, F37D, P87S, P87A, P87F, Y98K, N200S, N200T, N208Q, I295C, I295Q, F37C / P87A, F37C / I295C, P87A / I295C, Y98K / N208Q and F37C / P87A / I295C, respectively; in the diagram c, 1 represents FDH crude enzyme, 2 represents FDH pure enzyme, 3 represents CnMDR_WT crude enzyme, and 4 represents CnMDR_WT pure enzyme.

[0092] Figure 4 This is an investigation of the enzymatic properties of the bosera synthase CnMDR mutant Y98K / N208Q; where a is the optimum temperature; b is the optimum pH; c is the pH stability; and d is the temperature stability.

[0093] Figure 5 The active center of the homology modeling of the CnMDR mutant Y98K / N208Q of bosera synthase, and the docking results of the substrate and coenzyme NADPH. DETAILED DESCRIPTION

[0094] Technical terms:

[0095] C-Xyloside: Trade name Pro-Xylane TM , chemical name is hydroxypropyl tetrahydropyrantriol (CAS: 439685-79-7), a xylose derivative first discovered by L'Oreal to have anti-aging function. Bosine can prevent the reduction and degradation of glycosaminoglycans (GAGs) and proteoglycans (PGs) in vivo, promote their synthesis in epidermal cells, increase the skin's ability to bind water and improve the skin's biological defense ability.

[0096] Expression: The term "expression" includes any step involved in the production of bosera synthase CnMDR including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0097] Expression vector: The term "expression vector" means a linear or circular DNA molecule comprising a polynucleotide encoding the bosone synthase CnMDR of the present invention and operably linked to a control sequence providing for its expression.

[0098] Fragment: The term "fragment" means a polypeptide that lacks one or more (e.g., several) amino acids at the amino and / or carboxyl terminus of the polypeptide; wherein the fragment has bosera synthase activity. In one aspect, the fragment contains at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, but less than 100% of the number of amino acids 1 to 440 (i.e., excluding the length of the zymogen region sequence) shown in SEQ ID NO.1.

[0099] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.

[0100] The host cell can be any cell useful in the recombinant production of bosera synthase, such as a prokaryotic cell or a eukaryotic cell.

[0101] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanic Bacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Mycobacterium, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0102] The host cell may also be a eukaryotic organism, such as a mammalian, insect, plant, or fungal cell.

[0103] The culture medium involved in the following examples is as follows:

[0104] LB liquid medium (natural pH) contained 10% peptone, 5% yeast powder, and 10% sodium chloride.

[0105] TB medium (pH 7.2) contains peptone (1.2%), yeast extract (2.4%), glycerol (0.8%), dipotassium hydrogen phosphate (0.94%) and potassium dihydrogen phosphate (0.22%)

[0106] The detection methods involved in the following embodiments are as follows:

[0107] The content of bosine was determined by high performance liquid chromatography (HPLC), and the specific method is as follows:

[0108] Sample treatment: Take 200 μL of the reaction solution and centrifuge at 12,000×g for 3 min, take 100 μL of the supernatant and add it to 400 μL of ddH2O, and filter it with a 0.22 μm filter membrane. Chromatographic column: Aminex HPX-87H, 300×7.8 mm; detector: differential detector, detector temperature 35°C; mobile phase: 5 mM dilute sulfuric acid; flow rate: 0.6 mL / min; column temperature: 60°C; injection volume: 10 μL; running time: 30 min.

[0109] like Figure 1 As shown in Figure 2, the elution time of β-acetone xyloside is 5.386 min, and the elution time of phosphene is 5.935 min. According to the peak area of ​​phosphene and the standard curve ( Figure 2 ) Calculate the yield of boson and then determine the substrate conversion rate.

[0110] Sample treatment: Take 200μL of the reaction solution and centrifuge at 12,000×g for 3min, take 100μL of the supernatant and add it to 400μL ddH2O, filter it with a 0.22μm filter membrane and then detect it. Chromatographic column: Aminex HPX-87H, 300×7.8mm Detector: differential detector, detector temperature 35℃; mobile phase: 5mM dilute sulfuric acid; flow rate: 0.6mL / min; column temperature: 60℃; injection volume: 10μL; running time: 30min.

[0111] Example 1: Obtaining genes encoding biocatalysts and auxiliary biocatalysts

[0112] The specific steps are as follows:

[0113] (1) Acquisition of the gene encoding bosera synthase (carbonyl reductase) CnMDR

[0114] The gene encoding carbonyl reductase CnMDR from Gluconobacter oxydans was artificially synthesized, and the nucleotide sequence and amino acid sequence were shown in SEQ ID NO.2 and SEQ ID NO.1.

[0115] (2) Obtaining the gene encoding formate dehydrogenase FDH

[0116] The disclosed formate dehydrogenase ApFDH encoding gene from Azospirillum palustre was artificially synthesized, and the nucleotide sequence and amino acid sequence were shown in SEQ ID NO.4 and SEQ ID NO.3 respectively.

[0117] Example 2: Construction of recombinant bacteria

[0118] The specific steps are as follows:

[0119] 1. Construction of pET21a-CnMDR

[0120] Construction of genetically engineered bacteria expressing CnMDR: After the codon sequence of the carbonyl reductase CnMDR gene was optimized, the sequence was sent to Sangon Biotech for whole gene synthesis, and then double-digested with BamH I / Xho I enzymes and subcloned into the pET21a expression vector, named pET21a-CnMDR.

[0121] 2. Construction of recombinant vector containing mutants

[0122] (1) First, pET21a-CnMDR was used as a template and the following primer sequences were used for amplification to prepare a recombinant vector containing single point mutants: F37A, F37H, F37T, F37N, F37C, F37D, P87S, P87A, P87F, N200S, N200T, I295C, Y98K, N208Q, I295C, I295Q;

[0123] The primer sequences involved are as follows:

[0124] F37A-F: GCgcgGAAGTTGTTGTTACCGCCCGTTCCGAA;

[0125] F37A-R: AACAACAACTTCcgcGCCGTGATTCCAGCAGACAGT;

[0126] F37C-F: TCACGGCtgtGAAGTTGTTGTTACCGCCCGTT;

[0127] F37C-R: CAACTTCacaGCCGTGATCCAGCAGACAGTCC;

[0128] F37D-F: TCACGGCgatGAAGTTGTTGTTACCGCCCGTT;

[0129] F37D-R: CAACTTCatcGCCGTGATTCCAGCAGACAGTCC;

[0130] P87S-F: GTCTAACCCAtccTTTGATTACGTGGTTCACACCG;

[0131] P87S-R: CAAAggaTGGGTTAGACTTCACAGCTTCGTCA;

[0132] P87A-F: CTAACCCAgccTTTGATTACGTGGTTTCACACCG;

[0133] P87A-R: ATCAAAggcTGGGTTAGACTTCACAGCTTCGT;

[0134] P87F-F: GTCTAACCCAttTTTGATTACGTGGTTCACACCG;

[0135] P87F-R: CAAAaaaTGGGTTAGACTTCACAGCTTCGTCA;

[0136] Y98K-F: CGaagAGTTCACGTGGAACGATCCGGTTAAAG

[0137] Y98K-R: TCCACGTGAACTcttCGGGGACGCGGTGTGAAC

[0138] N200S-F: GAAACCGtctTTCGATCTGGCTACGATTAACAAC;

[0139] N200S-R: GATCGAAagaCGGTTTCTCGGTTTCTACAAAGG;

[0140] N200T-F: GAAACCGaccTTCGATCTGGCTACGATTAACAAC;

[0141] N200T-R: GATCGAAggtCGGTTTCTCGGTTTCTACAAAGG;

[0142] N208Q-F: CGATTAACcagACCTACACTTTTGGTCCGATTCC

[0143] N208Q-R: GTAGGTctgGTTAATCGTAGCCAGATCGAAGTTC

[0144] I295C-F: GGCTATCtgtCGTGATCGTTTCCCACAACTGG;

[0145] I295C-R: GATCACGacaGATAGCCGCCAGACGCGGATTG;

[0146] I295Q-F: GGCTATCcaaCGTGATCGTTTCCCACAACTGG;

[0147] I295Q-R: GATCACGttgGATAGCCGCCAGACGCGGATTG.

[0148] (2) Construction of double mutants or triple mutants:

[0149] Taking the preparation of a recombinant vector containing mutant F37C / P87A as an example, the preparation method is to use the F37C mutant as a template and amplify with primers P87A-F and P87A-R to obtain a recombinant vector containing mutant F37C / P87A.

[0150] According to the above method, recombinant vectors containing mutants F37C / I295C, P87A / I295C or F37C / P87A / I295C were constructed respectively.

[0151] 3. Construction of FDH recombinant vector

[0152] The formate dehydrogenase ApFDH encoding gene was inserted between the BamH I and Xho I sites on the plasmid pET21a to obtain the recombinant plasmid pET21a-ApFDH. The recombinant plasmid was introduced into E. coli BL21 (DE3) competent cells to obtain the engineered bacteria E. coli BL21 / pET21a-ApFDH.

[0153] The gene encoding formate dehydrogenase ApFDH was fully synthesized by Sangon Biotechnology Co., Ltd., and was double-digested with BamH I / Xho I enzymes and then subcloned into the pET21a expression vector, named pET21a-ApFDH.

[0154] 4. Construction of recombinant strains

[0155] The recombinant vectors obtained in step 2 above were sequenced and then transformed into E. coli BL21 (DE3) competent cells for subsequent whole-cell catalysis verification.

[0156] The recombinant strains were prepared: E. coli BL21 / pET21a-CnMDR_WT, E. coli BL21 / pET21a-F37A, E. coli BL21 / pET21a-F37C, E. coli BL21 / pET21a-F37D, E. coli BL21 / pET21a-P87S, E. coli BL21 / pET21a-P87A, E. coli BL21 / pET21a-P87F, E. coli BL21 / pET21a-Y98K, E. coli BL21 / pET21a-N200S, E. coli BL21 / pET21a-N200T, E. coli BL21 / pET21a-N208Q, E. coli BL21 / pET21a-I295C,E.coli BL21 / pET21a-I295Q,E.coli BL21 / pET21a-F37C / P87A,E.coli BL21 / pET21a-F37C / I295C,E.coli BL21 / pET21a-P87A / I295C,E.coli BL21 / pET21a-Y98K / N208Q,E.coli BL21 / pET21a-F37C / P87A / I295C.

[0157] The recombinant vectors obtained in step 3 above were sequenced and then transformed into E. coli BL21 (DE3) competent cells to prepare a recombinant strain: E. coli BL21 / pET21a-ApFDH.

[0158] Example 3: Protein expression and catalyst acquisition

[0159] The specific steps are as follows:

[0160] 1. Protein expression in shake flasks

[0161] After the recombinant bacteria were constructed according to the method of Example 2, the constructed engineered bacteria were plated to obtain positive monoclonal strains; the single colony was transferred into 5 mL of LB culture medium containing 50 μg / mL ampicillin, and cultured at 37°C, 200 rpm shaking for 8-10 h, and then 1 mL of the bacterial solution was inoculated into a 500 mL shaking flask containing 100 mL of LB liquid culture medium (containing 50 μg / mL ampicillin), and the culture was continued at 37°C, 200 rpm shaking until the cell OD 600 When the pH value was 0.6-0.8, the temperature was rapidly lowered, IPTG was added to a final concentration of 0.3 mM, and the culture was continued at 28°C and 200 rpm to induce protein expression for 12 h.

[0162] The composition of the protein expression LB liquid culture medium is: 10 g / L tryptone, 5 g / L yeast powder, and 10 g / L NaCl.

[0163] 2. Expression in 5L fermenter

[0164] The strain constructed in Example 2 was activated on a plate, and a single colony was picked and transferred into 5 ml of LB liquid culture medium containing 50 μg / mL ampicillin (37°C) for cultivation. After culturing for 8 hours, a culture solution was obtained, and the obtained culture solution was inoculated into 250 ml of TB liquid culture medium containing 50 μg / mL ampicillin at an inoculum volume of 1%, and cultured for 8 hours to prepare a seed solution; the prepared seed solution was transferred into a 5L culture fermentation tank (culture medium: TB culture medium (pH 7.2) containing peptone (1.2%), yeast extract (2.4%), glycerol (0.8%), dipotassium hydrogen phosphate (0.94%) and potassium dihydrogen phosphate (0.22%)) at an inoculum volume of 1% (v / v), and cultured at 37°C. When the cell OD 600 When the value was 20, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM, protein expression was induced at 28°C, and fermentation was continued for about 16 hours.

[0165] 3. Preparation of catalyst wet bacteria and crude enzyme solution

[0166] After protein induction expression is completed, centrifuge at 8,000×g for 10 min, discard the supernatant, collect the wet cells at the bottom, and store them in a -20℃ refrigerator. The obtained wet cells can be used as biocatalysts or auxiliary biocatalysts during whole cell catalysis;

[0167] Suspend the wet cells with 200 mg of wet cells and 5 mL of potassium phosphate buffer (50 mM), and crush the suspension in an ultrasonic crusher with a program of working for 1 second, resting for 3 seconds, and a power of 450 W for 5 minutes. Centrifuge at 4°C and 12,000×g for 10 minutes, and collect the supernatant, which is the crude enzyme solution of the biocatalyst or auxiliary biocatalyst.

[0168] Wet bacterial cells and crude enzyme solutions of F37A, F37C, F37D, P87S, P87A, P87F, Y98K, N200S, N200T, N208Q, I295C, I295Q, F37C / P87A, F37C / I295C, P87A / I295C, Y98K / N208Q, and F37C / P87A / I295C were prepared respectively. Figure 3 ).

[0169] 4. Detection of enzyme activity

[0170] The crude enzyme obtained in step 3 was purified by nickel column chromatography, and the final imidazole washing concentration of the target protein was 500 mM. The enzyme activity of the purified CnMDR wild type and its mutants was determined. The determination method is as follows:

[0171] The enzyme activity of CnMDR wild type and its mutants in reducing β-acetone xyloside was measured at 30°C by measuring the absorbance change rate of NADH or NADPH at 340nm. Enzyme activity assay system: 250μL reaction volume, 0.25mM NADH and 50mM β-acetone xyloside were added to 50mM PBS buffer (pH7.0), incubated in a 30°C metal bath for 3min, 50μL of pure enzyme of appropriate concentration was added, and the absorbance change at 340nm was scanned using the Bio-Tek Cytation 5 Cell Imaging Multifunctional Detection System.

[0172] Enzyme activity is defined as: under the above conditions, catalyzing the oxidation of 1 μmol NADH or NADPH to NAD per minute. + The amount of enzyme is defined as one unit U.

[0173] The calculation formula of enzyme activity is: enzyme activity (U) = EW × V × 10 3 / (6220×0.625);

[0174] The calculation formula of specific activity is: specific activity (U·mg -1 ) = enzyme activity (U) / protein amount (mg);

[0175] Where, EW: change in absorbance at 340 nm within 1 min; V: volume of reaction solution (mL); 6220: molar extinction coefficient L·mol -1 cm -1 ); 0.5: optical path distance (cm). The enzyme activity data of the mutants are shown in Table 1 below.

[0176] Table 1: Enzyme activities of mutants

[0177] Mutant strain Enzyme activity (U / mg) Mutant strain Enzyme activity (U / mg) F37A 11.92 P87S 12.09 F37T 5.27 P87A 25.86 F37N 2.41 P87F 7.75 F37C 13.51 Y98K 18.41 F37D 6.04 N200S 12.63 N200T 12.96 I295C 30.24 I295Q 12.82 N208Q 27.45 F37C / I295C 5.27 F37C / P87A 3.44 Y98K / N208Q 60.11 P87A / I295C 35.27 F37C / P87A / I295C 51.69

[0178] The activity of the wild-type enzyme is 12.77 U / mg, and the activity of the dominant double mutant Y98K / N208Q is 60.11 U / mg (enzyme performance studies such as Figure 4 , the active center of homology modeling is as follows Figure 5 ), which was 3.7 times higher than that of the wild type.

[0179] Example 4: Synthesis of Hydroxypropyl Tetrahydropyrantriol

[0180] 40 g / L of wet bacteria obtained by fermentation culture of the engineered bacteria carrying the wild type and mutants of the carbonyl reductase CnMDR encoding gene of C. notabilis prepared above was used as a catalyst (the preparation method of the biocatalyst was the same as steps 1 to 3 of Example 3), and 40 g / L of wet bacteria of formate dehydrogenase ApFDH from A. palustre was added as an auxiliary catalyst (the preparation method of the auxiliary biocatalyst was the same as steps 1 to 3 of Example 3). The specific reaction system was as follows:

[0181] The 10 ml reaction system contained 40 g / L biocatalyst (wet bacteria carrying wild-type or mutant carbonyl reductase), 40 g / L auxiliary biocatalyst (wet bacteria carrying formate dehydrogenase), 200 g / L β-acetone xyloside as substrate, 0.3 mM coenzyme NADP + Ammonium formate was added as a cosubstrate to drive the coenzyme cycle, the molar concentration ratio of ammonium formate cosubstrate to substrate was 1.5:1, potassium phosphate buffer (pH 7.0) was used as the reaction medium to form a reaction system, and the reaction was carried out at a temperature of 30°C and 200rpm for 8h to obtain hydroxypropyltetrahydropyrantriol, i.e., bosine.

[0182] After the reaction was completed, 200 μL of the reaction solution was sampled and centrifuged at 12,000×g for 3 min. 100 μL of the supernatant was added to 400 μL of ddH2O and filtered with a 0.22 μm filter membrane for liquid phase analysis.

[0183] The calculation method of ee value and conversion rate is as follows: According to the peak area obtained by liquid phase analysis and the standard curve of boson, the concentration of S and R configurations produced are calculated respectively, and the formula ee value = ([S]-[R] / [R]+[S])*100% is used to obtain the enantiomeric excess value of S-configuration boson. The concentration of S-configuration boson is divided by the concentration of the product that should be generated theoretically with 200g / L substrate. The results are shown in Table 2.

[0184] Table 2: Product yields and diastereomeric selectivity of carbonyl reductase mutants

[0185]

[0186] The results show:

[0187] As shown in Table 1, the yield and diastereomeric selectivity of the mutated carbonyl reductase for the synthesis of bosylamine are improved. The single mutants Y98K and N208Q at the two sites have higher enantioselectivity and conversion rate for β-acetone xyloside. Therefore, the two sites were randomly combined and mutated to obtain the double mutant Y98K / N208Q, which has further enhanced the synthesis ability. The whole cell catalysis is 8 hours, the product yield is 99.2%, and the ee value is 99.0%. The superior mutant Y98K / N208Q will be studied in the future.

[0188] Example 5: Optimization of buffer for the synthesis of high concentration bosogenin catalyzed by carbonyl reductase mutants

[0189] The benchmark reaction system with optimized conditions is obtained in Example 4:

[0190] The engineered bacteria E. coli BL21 / pET21a-Y98K / N208Q obtained by fermentation culture, 40 g / L wet bacteria as a catalyst (the preparation method of the catalyst is the same as steps 1 to 3 of Example 3) are added to 10 ml of the reaction system, and then 40 g / L of formate dehydrogenase ApFDH wet bacteria as an auxiliary catalyst (the preparation method of the auxiliary catalyst is the same as steps 1 to 3 of Example 3) are added. The specific reaction system is as follows:

[0191] In a 10 ml reaction system, add 40 g / L biocatalyst (wet bacteria carrying carbonyl reductase mutant Y98K / N208Q), 40 g / L auxiliary biocatalyst (wet bacteria carrying formate dehydrogenase), 200 g / L β-acetone xyloside as substrate, and 0.3 mM NADP + , add 100g / L ammonium formate as a cosubstrate to drive the coenzyme cycle.

[0192] In this example, different buffer solutions (pH 7.0) were used as reaction media, and the reaction was carried out at 30° C. and 200 rpm for 8 h to obtain hydroxypropyl tetrahydropyrantriol, i.e., bosylamine.

[0193] After the reaction was completed, 200 μL of the reaction solution was sampled and centrifuged at 12,000×g for 3 min. 100 μL of the supernatant was added to 400 μL of ddH2O and filtered with a 0.22 μm filter membrane for liquid phase analysis.

[0194] The buffer (pH 7.0) in this example is: potassium phosphate buffer, PBS buffer, ammonium formate buffer, sodium formate buffer, Tris-hydrochloric acid buffer. The results are shown in Table 3 below.

[0195] Table 3: Buffer optimization

[0196]

[0197] Example 6: Optimization of the concentration of coenzyme in the synthesis of high-concentration bosera catalyzed by carbonyl reductase mutants

[0198] The specific steps are as follows:

[0199] A small amount of NADP needs to be added at the beginning of the reaction + It is used to start the reaction and is crucial in the synthesis of bosone catalyzed by carbonyl reductase mutants. + The concentration of the added product was set to 6 concentration gradients: 0, 0.1, 0.2, 0.3, 0.4 and 0.5 mM. The reaction solution was subjected to liquid phase treatment, and its yield was analyzed to explore the conversion rate under different concentration addition amounts.

[0200] Reaction system based on the optimized conditions in Example 6:

[0201] In a 10 ml reaction system, 40 g / L of biocatalyst (wet bacteria carrying carbonyl reductase mutant Y98K / N208Q), 40 g / L of auxiliary biocatalyst (wet bacteria carrying formate dehydrogenase), 200 g / L of crude β-acetone xyloside as substrate, and different concentrations of NADP were added. + (0, 0.1, 0.2, 0.3, 0.4 and 0.5 mM), 100 g / L ammonium formate was added as a cosubstrate to drive the coenzyme cycle, and ammonium formate buffer (pH 7.0) was used as the reaction medium. The reaction was carried out at 30°C and 200 rpm for 8 hours to obtain hydroxypropyltetrahydropyrantriol, i.e., bosine.

[0202] After the reaction, 200 μL of the reaction solution was sampled and centrifuged at 12,000×g for 3 min. 100 μL of the supernatant was added to 400 μL of ddH2O and filtered with a 0.22 μm filter membrane for liquid phase analysis.

[0203] The liquid phase detection method of the conversion liquid is according to the method shown in Example 4:

[0204] Sample treatment: reaction solution, centrifuge, collect supernatant, filter with 0.22μm filter membrane, and perform HPLC detection. Chromatographic column: Aminex HPX-87H, 300×7.8mm Detector: differential detector, detector temperature 35℃; mobile phase: 5mM dilute sulfuric acid; flow rate: 0.6mL / min; column temperature: 60℃; injection volume: 10μL; running time: 30min.

[0205] The results are shown in the table below. A better conversion rate can be achieved when the concentration is 0.3 mM.

[0206] Table 4: Conversion rate at different coenzyme concentrations

[0207]

[0208] Example 7: pH optimization for the synthesis of high concentration bosogenin catalyzed by carbonyl reductase mutants

[0209] The specific method is as follows:

[0210] 1. Too low pH (too acidic) or too high pH (too alkaline) can denature the enzyme protein and inactivate it. Changes in pH can affect the degree of dissociation of the essential groups on the enzyme active center, and can also affect the degree of dissociation of the substrate and coenzyme, thereby affecting the binding and catalysis of the enzyme molecule to the substrate molecule. In the reaction, four ammonium formate buffers with different pH values ​​(5.0, 6.0, 7.0, and 8.0) were selected for the conversion reaction, and the changes in conversion rate were recorded.

[0211] Reaction system based on the optimized conditions in Example 7:

[0212] In a 10 ml reaction system, add 40 g / L biocatalyst (wet bacteria carrying carbonyl reductase mutant Y98K / N208Q), 40 g / L auxiliary biocatalyst (wet bacteria carrying formate dehydrogenase), 200 g / L β-acetone xyloside as substrate, and 0.3 mM NADP + , 100 g / L ammonium formate was added as a cosubstrate to drive the coenzyme cycle, and ammonium formate buffer with pH values ​​of 5.0, 6.0, 7.0, and 8.0 were used as reaction media, respectively. The reaction was carried out at 30°C and 200 rpm for 8 hours to obtain hydroxypropyltetrahydropyrantriol, i.e., bosine.

[0213] After the reaction, 200 μL of the reaction solution was sampled and centrifuged at 12,000×g for 3 min. 100 μL of the supernatant was added to 400 μL of ddH2O and filtered with a 0.22 μm filter membrane for liquid phase analysis.

[0214] The results are shown in Table 5:

[0215] Table 5: Conversion rate at different pH

[0216]

[0217] 2. High temperature will cause the enzyme to denature and lose its catalytic ability, while low temperature will inhibit the activity of the enzyme and affect the conversion of the enzyme molecule to the substrate molecule. According to the pH optimization results, ammonium formate buffers at different temperatures (20-40°C) were selected for the conversion reaction and the changes in conversion rate were recorded.

[0218] In a 10 ml reaction system, add 40 g / L biocatalyst (wet bacteria carrying carbonyl reductase mutant Y98K / N208Q), 40 g / L auxiliary biocatalyst (wet bacteria carrying formate dehydrogenase), 200 g / L β-acetone xyloside as substrate, and 0.3 mM NADP +, 100 g / L ammonium formate was added as a cosubstrate to drive the coenzyme cycle, and ammonium formate buffer was used as the reaction medium. At pH 7.0, the reaction temperatures were: 20°C, 25°C, 30°C, 35°C, and 40°C; the reaction conditions were: 200 rpm for 8 hours to obtain hydroxypropyltetrahydropyrantriol, i.e., bosine.

[0219] After the reaction, 200 μL of the reaction solution was sampled and centrifuged at 12,000×g for 3 min. 100 μL of the supernatant was added to 400 μL of ddH2O and filtered with a 0.22 μm filter membrane for liquid phase analysis.

[0220] The results are shown in Table 6 below. When the pH is 7.0 and the temperature is 30°C, the conversion rate of the carbonyl reductase mutant to synthesize bosone is as high as 99.6%.

[0221] Table 6: Conversion rate at different temperatures

[0222]

[0223] Example 8: Scale-up reaction at 1 L scale

[0224] First, the engineered bacteria E. coli BL21 / pET21a-Y98K / N208Q and E. coli BL21 / pET21a-ApFDH were fermented at high density in 5L fermenters, and the cell density in the obtained 3L fermentation liquid was 220g / L. The collected wet cells were reacted in a 1L reaction system.

[0225] Based on the optimized reaction conditions:

[0226] 1L reaction system, including 200g / L β-acetone xyloside, 0.3mM NADP + , 100 g / L ammonium formate, 40 g / L engineered bacteria E. coli BL21 / pET21a-Y98K / N208Q whole cells as catalyst, 40 g / L engineered bacteria E. coliBL21 / pET21a-ApFDH as auxiliary catalyst. In an aqueous medium, the reaction was carried out at 30°C, pH 7.0, and a rotation speed of 200 rpm for 8 hours to obtain the product bosogenin.

[0227] The reaction results are:

[0228] The recombinant bacteria catalyzed the conversion of 200 g / L substrate for 8 hours, with a conversion rate of over 99% and an ee value of 99.42%. At this time, the bosamine production was 200 g / L.

[0229] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A carbonyl reductase mutant, characterized in that: The mutants are: The amino acid sequence of the carbonyl reductase shown in SEQ ID NO. 1 was obtained by mutating the tyrosine at position 98 to lysine, and the result was named Y98K; or the one obtained by mutating the asparagine at position 208 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to glutamine, named N208Q; or the one obtained by mutating the isoleucine at position 295 of the carbonyl reductase shown in the amino acid sequence of SEQ ID NO.1 to cysteine, which is named I295C; Or the amino acid sequence of the carbonyl reductase shown in SEQ ID NO.1 is obtained by mutating the tyrosine at position 98 to lysine and the asparagine at position 208 to glutamine, which is named Y98K / N208Q; Or the amino acid sequence of the carbonyl reductase shown in SEQ ID NO.1 is obtained by mutating the phenylalanine at position 37 to cysteine, mutating the proline at position 87 to alanine, and mutating the isoleucine at position 295 to cysteine, and the result is named F37C / P87A / I295C.

2. A gene encoding the mutant according to claim 1 or a recombinant vector carrying the gene.

3. A recombinant cell expressing the mutant according to claim 1 or carrying the gene or the recombinant vector according to claim 2.

4. The recombinant cell according to claim 3, characterized in that The recombinant cell uses bacteria or fungi as expression hosts.

5. A recombinant enzyme catalyst comprising the carbonyl reductase mutant sequence of claim 1, characterized in that: The recombinase catalyst is any one of the following forms: (1) culturing recombinant expression transformants and isolating transformant cells containing the recombinase; (2) culturing the recombinant expression transformant, isolating the transformant cells containing the recombinase, and disrupting the transformant cells containing the recombinase to obtain a cell disrupted liquid; (3) Cultivating the recombinant expression transformant, isolating transformant cells containing the recombinant enzyme, disrupting the transformant cells containing the recombinant enzyme, obtaining a cell disrupted liquid, and freeze-drying the cell disrupted liquid of the recombinant enzyme to obtain a lyophilized enzyme powder.

6. A method for improving the diastereomeric selectivity and / or substrate conversion rate and / or yield of carbonyl reductase-catalyzed synthesis of bosone, characterized in that: The method is: The tyrosine at position 98 of the carbonyl reductase of the amino acid sequence shown in SEQ ID NO.1 is mutated to lysine; Or the asparagine at position 208 of the carbonyl reductase of the amino acid sequence as shown in SEQ ID NO.1 is mutated to glutamine; or mutating the isoleucine at position 295 of the carbonyl reductase whose amino acid sequence is shown in SEQ ID NO.1 to cysteine; Or the tyrosine at position 98 of the carbonyl reductase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to lysine, and the asparagine at position 208 is mutated to glutamine; Or the phenylalanine at position 37 of the carbonyl reductase with the amino acid sequence as shown in SEQ ID NO.1 is mutated to cysteine, the proline at position 87 is mutated to alanine, and the isoleucine at position 295 is mutated to cysteine.

7. A method for preparing boson, characterized in that: The method comprises: using β-acetone xyloside as a substrate, using the carbonyl reductase mutant according to claim 1, or the recombinant cell according to claim 3 or 4, or the recombinant enzyme catalyst according to claim 5, and using a buffer as a reaction medium to form a reaction system, and converting and preparing bosogen; The reaction system also contains formate dehydrogenase or a recombinant cell expressing formate dehydrogenase, ammonium formate, NADP + .

8. The method according to claim 7, characterized in that The buffer is potassium phosphate buffer, PBS buffer, ammonium formate buffer, sodium formate buffer or Tris-hydrochloric acid buffer.

9. The method according to claim 8, characterized in that The reaction conditions of the reaction system are: temperature of 25-35° C., reaction at 150-300 rpm for 6-10 h.

10. The method according to claim 7, characterized in that The amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO.

3.

11. The method according to claim 7, characterized in that The concentration of the ammonium formate is 150 mM to 1260 mM, and the concentration of the β-acetone xyloside is 100 mM to 1052 mM.

12. The method according to claim 7, characterized in that The added amount of the recombinant cells is: 20 g / L ~60 g / L.

13. The method according to claim 7, characterized in that The added enzyme activity of the mutant enzyme or recombinant enzyme catalyst is 2-65 U / mg.

14. Use of the mutant according to claim 1, or the gene or recombinant vector according to claim 2, or the recombinant cell according to claim 3 or 4, or the recombinase catalyst according to claim 5, or the method according to any one of claims 7 to 13 in the preparation of bosogen or a product containing bosogen.

15. The use according to claim 14, characterized in that: The products are skin care products, makeup products, soap products, bath products, shampoo products, and hair care products.

Images