A carbonyl reductase mutant and its application in the synthesis of S-configuration bosonicine
By mutating specific amino acids in the carbonyl reductase Gre2, the enzyme activity is enhanced and the coenzyme preference is changed, solving the problems of low carbonyl reductase activity and high coenzyme cost in the existing technology, and realizing the industrial production of S-configuration Bosein with high efficiency.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SHANGHAI ZHONGYI DAILY CHEM CO LTD
- Filing Date
- 2026-03-27
- Publication Date
- 2026-06-02
AI Technical Summary
Existing technologies suffer from problems such as low activity of carbonyl reductases in the synthesis of S-configuration Bosein, dependence on expensive coenzyme NADPH leading to long reaction times and high production costs.
By performing specific amino acid mutations on Gre2, a carbonyl reductase derived from Saccharomyces cerevisiae, especially the F132A-N9E-K36R mutant, the enzyme activity was enhanced and the coenzyme preference was changed from NADPH to the inexpensive NADH. This enabled the construction of a two-enzyme system for one-pot synthesis of S-configuration Bosein.
It achieves efficient utilization of inexpensive coenzyme NADH, significantly improves the conversion rate and production efficiency of S-configuration Bosein, shortens the production cycle, reduces material costs, and is suitable for industrial production.
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Figure CN122128261A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biocatalytic synthesis technology, specifically to a carbonyl reductase mutant and its application in the synthesis of S-configuration Bosein. Background Technology
[0002] Pro-Xylane (hydroxypropyl tetrahydropyranotriol, CAS 439685-79-7) is an important xylose derivative cosmetic ingredient that can effectively promote the synthesis and secretion of glycosaminoglycans and proteoglycans in the epidermis, while also promoting the accumulation of collagen, laminin, and other components in the extracellular matrix of dermal cells. It has broad application value in the cosmetic field. Among them, the S-configuration of Pro-Xylane exhibits significantly higher bioactivity than the R-configuration and is the target product for Pro-Xylane synthesis.
[0003] Chinese invention patent application CN113416756A discloses a method for selectively reducing S-configuration Bosein using β-acetone xyloside as a substrate, isopropanol as a reducing agent, and nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor, under the action of carbonyl reductase and alcohol dehydrogenase. However, this synthetic route has significant technical drawbacks: on the one hand, isopropanol, as a reducing agent, generates acetone as a byproduct during the reaction, which significantly inhibits the activity of carbonyl reductase, resulting in excessively long reaction time and low production efficiency; on the other hand, natural carbonyl reductases tend to use expensive and unstable reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme, and cannot effectively adapt to the cheap and stable reduced nicotinamide adenine dinucleotide (NADH), which greatly increases the material cost of the reaction and is not conducive to large-scale industrial production.
[0004] Therefore, the targeted modification of carbonyl reductase to obtain a carbonyl reductase mutant with high enzyme activity and adaptability to low-cost coenzymes, while optimizing the catalytic synthesis system of S-configuration Bosein, has become the key to solving existing technical problems and promoting the industrial production of S-configuration Bosein. Summary of the Invention
[0005] This invention addresses the shortcomings of existing technologies by providing a carbonyl reductase mutant and its application in the synthesis of S-configuration Bosonicine. The mutant is based on natural carbonyl reductase and undergoes targeted amino acid mutation, resulting in significantly enhanced enzyme activity. Furthermore, it is compatible with the low-cost coenzyme NADH, thus solving the problems of low catalytic efficiency and high coenzyme cost of existing carbonyl reductases.
[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a carbonyl reductase mutant, wherein the carbonyl reductase mutant is a carbonyl reductase that has undergone an amino acid mutation; the amino acid sequence of the carbonyl reductase is shown in SEQ ID No. 1; and the amino acid mutation is one of F132A, V162A, F132A-N9E, F132A-K36R or F132A-N9E-K36R.
[0007] This application successfully solves the technical challenges of low activity, heavy dependence on expensive coenzyme NADPH, long reaction time, and high production cost of wild-type Gre2 enzyme in the synthesis of S-configuration Bosein by providing a specific carbonyl reductase mutant. The core of this technical solution lies in the precise molecular modification of the carbonyl reductase Gre2 (amino acid sequence shown in SEQ ID No. 1) derived from Saccharomyces cerevisiae, introducing synergistic mutations at key sites. In particular, the specific carbonyl reductase triple mutant (F132A-N9E-K36R) significantly enhances enzyme activity and successfully reverses the enzyme's coenzyme dependence, shifting its preference from expensive NADPH to the highly efficient use of low-cost NADH, thus significantly reducing material costs for industrial production. Finally, in the synthesis of S-configuration Bosein, this mutant, combined with a two-enzyme system, can achieve a conversion rate of up to 99.6% within 5 hours, significantly shortening the production cycle. The possible reasons are as follows: The F132A mutation is located at the substrate channel entrance, replacing the large-side-chain phenylalanine with the small-side-chain alanine, effectively expanding the channel for substrate entry and product release, thereby increasing the basic catalytic rate. Both N9E and K36R are located in the coenzyme-binding pocket and interact with the phosphate group of NADPH. The mutation of N9E to negatively charged glutamate breaks the enzyme's dependence on the phosphate-containing NADPH through charge repulsion; while the mutation of K36R to arginine, with its longer side chain, can form a better hydrogen bond network with NADH, optimizing the binding of the enzyme to the inexpensive coenzyme NADH. The synergistic effect of these three factors ultimately creates a high-performance mutant that possesses both ultra-high activity and efficient utilization of the inexpensive coenzyme.
[0008] Preferably, the coding sequence of the carbonyl reductase includes the nucleotide sequence shown in SEQ ID No. 2. SEQ ID No. 2 is the codon-optimized nucleotide sequence of the carbonyl reductase Gre2 from *Saccharomyces cerevisiae*.
[0009] In a second aspect, the present invention provides a nucleic acid molecule that encodes the carbonyl reductase mutant described in the first aspect above.
[0010] Thirdly, the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect above.
[0011] Fourthly, the present invention provides a carbonyl reductase mutant transformant, wherein the carbonyl reductase mutant transformant is a genetically engineered strain expressing the carbonyl reductase mutant described in the first aspect above.
[0012] Preferably, the carbonyl reductase mutant transformant contains the expression vector described in the third aspect above.
[0013] Fifthly, the present invention provides the use of the carbonyl reductase mutant described in the first aspect, or the nucleic acid molecule described in the second aspect, or the expression vector described in the third aspect, or the carbonyl reductase mutant transformant described in the fourth aspect in the synthesis of S-configuration Bosonicine.
[0014] Sixthly, the present invention provides a method for synthesizing S-configuration bosonicine, comprising the following steps: Using β-acetone xyloside as a reaction substrate and isopropanol as a reducing agent, the carbonyl reductase mutant and isopropanol dehydrogenase were used to catalyze a reaction in the presence of a coenzyme to obtain S-configuration bosine.
[0015] Preferably, the amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID No. 13, and the nucleotide sequence is shown in SEQ ID No. 14.
[0016] Preferably, the coenzyme is nicotinamide adenine dinucleotide or reduced nicotinamide adenine dinucleotide.
[0017] Preferably, the pH of the catalytic reaction system is 6-7; the concentration of the reaction substrate is 800-1200 mM; the reaction temperature is 28-32℃; and the reaction time is 4-6 h.
[0018] Beneficial effects 1. The carbonyl reductase mutant of the present invention achieves a significant improvement in enzyme activity and optimization of coenzyme selectivity by directional amino acid mutation at key sites of natural carbonyl reductase. It not only greatly improves the catalytic efficiency of the substrate β-pyruvate, but can also be adapted to low-cost coenzymes NADH / NAD, thus solving the technical problems of low catalytic efficiency and high coenzyme cost of existing carbonyl reductases.
[0019] 2. A dual-enzyme synergistic catalytic system was constructed using a carbonyl reductase mutant and a modified isopropanol dehydrogenase, enabling the one-pot synthesis of S-configuration Bosein. The isopropanol dehydrogenase can recycle the coenzyme and rapidly metabolize the byproduct acetone, further improving reaction efficiency and shortening reaction time.
[0020] 3. The catalytic synthesis conditions of S-configuration Bosein were optimized, and the optimal pH, substrate concentration, reaction temperature and reaction time were determined. Under these conditions, the substrate conversion rate was high, the reaction time was short and the product purity was high.
[0021] 4. The carbonyl reductase mutant of the present invention can be efficiently expressed by engineered strains of Escherichia coli. The culture conditions of Escherichia coli are simple and the growth rate is fast. Moreover, the catalytic reaction system does not require complex equipment. The reaction process is green and environmentally friendly, which greatly reduces the production cost of S-configuration Bosein and has good prospects for large-scale industrial production. Attached Figure Description
[0022] Figure 1 A schematic diagram of Gre2 amino acid residues that interact with NADPH; Figure 2 Electrophoresis results of isopropanol dehydrogenase. Detailed Implementation
[0023] The technical solution of the present invention will be described in detail below with reference to specific embodiments, so that those skilled in the art can better understand the essence of the present invention and its beneficial effects. The following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Any limited modifications made within the scope of the claims of the present invention are still within the protection scope of the present invention.
[0024] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products in the art, and the experimental methods are all conventional methods in the art.
[0025] In this invention, amino acids are represented by single-letter or three-letter codes, with the following meanings: A: Ala (alanine); R: Arg (arginine); N: Asn (asparagine); D: Aspartic acid (aspartic acid); C: Cys (cysteine); Q: Gln (glutamine); E: Glu (glutamic acid); G: Gly (glycine); H: Histidine; I: Ile (isoleucine); L: Leu (leucine); K: Lysine (lysine); M: Met (methionine); F: Phe (phenylalanine); P: Pro (proline); S: Serine (serine); T: Threonine (threonine); W: Tryptophan (tryptophan); Y: Tyrosine (tyrosine); V: Val (valine).
[0026] Example 1: Preparation of carbonyl reductase mutant The wild-type carbonyl reductase Gre2 from *Saccharomyces cerevisiae*, with its amino acid sequence shown in SEQ ID No. 1 and nucleotide sequence shown in SEQ ID No. 2, was selected. After codon optimization for *Escherichia coli*, the entire gene was synthesized and constructed into the pET-28a vector to obtain the expression vector pET28a-Gre2. The expression vector pET28a-Gre2 was transformed into the *Escherichia coli* BL21(DE3) expression host to obtain engineered strains of wild-type Gre2.
[0027] The amino acid sequence of the wild-type carbonyl reductase Gre2 is as follows: MSVFVSGANGFIAQHIVDLLLKEDYKVIGSARSQEKAENLTEAFGNNPKFSMEVVPDISKLDAFDHVFQKHGKDIKIVLHTASPFCFDITDSERDLLIPAVNGVKGILHSIKKYAADSVERVVLTSSYAAVFDMAKENDKSLTFNEESWNPATWESCQSDPVNAYCGSKKFAE KAAWEFLEENRDSVKFELTAVNPVYVFGPQMFDKDVKKHLNTSCELVNSLMHLSPEDKIPELFGGYIDVRDVAKAHLVAFQKRETIGQRLIVSEARFTMQDVLDILNEDFPVLKGNIPVGKPGSGATHNTLGATLDNKKSKKLLGFKFRNLKETIDDTASQILKFEGRI (SEQ ID No.1).
[0028] The nucleotide sequence encoding this wild-type carbonyl reductase Gre2 is as follows:
[0029] The potential substrate entry and exit channels of the Gre2-NADPH complex (PDB: 4PVD) were observed in the PDBsum database, and Phe132, Val162, Asn9, and Lys36 were selected as modification sites. Primers listed in Table 1 were designed, and plasmids for each mutant were constructed using pET28a-Gre2 as a template via overlap extension PCR. These were then transformed into *E. coli* BL21(DE3) expression host to obtain carbonyl reductase mutant engineered bacteria. The culture and enzyme induction expression processes for the mutant engineered bacteria were the same as for the wild type.
[0030] Table 1 Primer Sequences
[0031] Combinatorial mutants were constructed using a stepwise stacking mutation strategy: Using wild-type pET28a-Gre2 as a template, an F132A single mutant was constructed using F132A primers (SEQ ID No. 3 & 4); Using wild-type pET28a-Gre2 as a template, a V162A single mutant was constructed using V162A primers (SEQ ID No. 5 & 6).
[0032] Using the F132A single mutant plasmid as a template, the F132A-N9E double mutant was constructed using N9E primers (SEQ ID No. 7 & 8); Using the F132A single mutant plasmid as a template, the F132A-K36R double mutant was constructed using K36R primers; Using the F132A-N9E double mutant plasmid as a template, the F132A-N9E-K36R triple mutant was constructed using K36R primers (SEQ ID No. 9 & 10).
[0033] Example 2: Induced expression of carbonyl reductase mutant Take the carbonyl reductase mutant engineered bacteria obtained in Example 1, dip it into the stab plate, streak it on an LB agar plate (containing 50 μg / mL kanamycin), and incubate it overnight at 37°C. The next day, pick 3 single colonies of suitable size, inoculate them into 10 mL of LB liquid medium (containing 50 μg / mL kanamycin), and incubate at 37°C and 200 rpm for 6-8 hours until the bacterial solution becomes turbid.
[0034] The above culture was transferred at a 1% inoculum to 500 mL LB liquid medium (containing 50 μg / mL kanamycin) and incubated at 37°C and 200 rpm for 2.5–3.5 h until the bacterial OD reached the target value. 600=0.6~1.0; add isopropyl-β-D-thiogalactoside to the system to a final concentration of 0.1mM, and transfer to 20℃ and 200rpm to induce protein expression for 16~20h.
[0035] After the induction expression is completed, the bacterial culture is placed in an environment of 4-14℃, centrifuged at 5000rpm for 10min, the supernatant is discarded and the bacterial sludge in the shake flask is collected and stored at -20℃ for later use.
[0036] Example 3: Preparation of crude enzyme solution The bacterial cells were resuspended in the disruption buffer at a ratio of 1g of bacterial sludge to 4mL. After ultrasonic disruption, the cells were centrifuged at 4℃ and 12000rpm for 20min. The supernatant was collected, which is the crude enzyme solution of each carbonyl reductase mutant, for later use.
[0037] Example 4: Preparation of IPADH (isopropanol dehydrogenase) The modified isopropanol dehydrogenase IPADH was selected. Its amino acid sequence is shown in SEQ ID No. 13 and its nucleotide sequence is shown in SEQ ID No. 14. The whole gene was synthesized and constructed on the pET-28a vector, and then the plasmid was transformed into the Escherichia coli BL21(DE3) expression host.
[0038] Induced expression was performed according to the method in Example 2, and bacterial sludge was collected; crude enzyme solution was prepared according to the method in Example 3.
[0039] The results of SDS-PAGE protein electrophoresis are shown below. Figure 2 IPADH is mainly expressed in a soluble form in the supernatant.
[0040] Example 5: One-pot synthesis of S-configuration Bosein using a two-enzyme method (1) Preparation of the reaction system Weigh 190.20 g of β-pyruvate xyloside (final concentration 1000 mM, corresponding to a final reaction volume of 1 L) and 0.66 g of NAD (final concentration 1 mM), add them to 900 mL of pure water, and stir to dissolve. Adjust the pH to 7.5 with NaOH solution, and bring the volume to 816 mL. Add 84 mL of isopropanol (final concentration 1100 mM), mix well, and preheat in a 30 °C water bath.
[0041] (2) Enzyme-catalyzed reaction Add 90 mL of the Gre2-F132A-N9E-K36R crude enzyme solution prepared in Example 3 and 10 mL of the IPADH crude enzyme solution prepared in Example 4 to the preheated reaction solution. After mixing, the mixture was shaken at 30°C and 100 rpm. Samples were taken periodically during the reaction, and the residual amount of β-acetone xyloside and the amount of S-configuration Bosein generated were detected by HPLC.
[0042] After 5 hours of reaction, samples were taken for testing. The residue of β-acetone xyloside was 0.32%, and the conversion rate of S-configuration Bosein reached 99.6%.
[0043] (3) Product purification Ceramic membrane filtration: A ceramic membrane with a molecular weight cutoff of 10 kDa is selected to filter and remove bacterial fragments and impurities. Activated carbon decolorization: Add 1% (w / v) activated carbon to the filtrate, stir at 60℃ and 100rpm for 30min, and filter to remove carbon; Ion exchange: The effluent is collected by passing it through a DM-301 cation exchange resin column; Thin-film concentration: Concentrate under reduced pressure at 75℃ to 1 / 4 of the original volume; Ethanol crystallization: Add 2 times the volume of anhydrous ethanol to the concentrate and let it stand at 4°C for 12 hours to crystallize; Drying: Filter and collect the crystals, then vacuum dry at 55°C to constant weight.
[0044] HPLC analysis showed that the purity of the obtained S-configuration Bosein product was 99.82%, and the conductivity was 2.35 μS / cm, which meets the quality requirements for cosmetic raw materials.
[0045] Performance testing 1. Enzyme activity assay Take the crude enzyme solution of each carbonyl reductase mutant, dilute it 10 times with the disruption buffer, add 100 μL to 10 mL of activity test solution, react at 30 °C for 15 min, and use high performance liquid chromatography (HPLC) to detect the amount of S-configuration Bosein generated and calculate the enzyme activity.
[0046] The activity assay solution is formulated as follows: 100mM Na2HPO4 / NaH2PO4 (pH 7.5), 20mM β-acetone xyloside, 20mM NADPH (for determining NADPH-dependent enzyme activity) or 20mM NADH (for determining NADH-dependent enzyme activity).
[0047] Enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 mM S-configuration Bosein per minute at 30°C and pH 7.5.
[0048] Enzyme activity (U / g) = Bosein (mM) × assay volume (mL) × enzyme dilution factor × ultrasonic dilution ratio / [analysis time (min) × enzyme volume (mL)] The assay volume was 10 mL, the enzyme solution dilution factor was 10, the ultrasonic dilution ratio was 5, the assay time was 15 min, and the enzyme addition volume was 0.1 mL.
[0049] Using the crude enzyme solution of wild-type carbonyl reductase Gre2 as a control, the enzyme activity assay results of each mutant are shown in Tables 2 and 3. A schematic diagram of the Gre2 amino acid residues that interact with NADPH is shown below. Figure 1 .
[0050] Table 2 Enzyme activity of each mutant under NADPH conditions
[0051] Table 3 Enzyme activity of each mutant under NADH conditions
[0052] The results showed that the F132A mutation significantly improved the enzyme's basic activity, increasing it by 250% under NADPH conditions; the N9E and K36R mutations altered the enzyme's coenzyme preference, enabling it to efficiently utilize inexpensive NADH; the triple mutant F132A-N9E-K36R achieved an enzyme activity of 1582 U / g under NADH conditions, which was 24.3 times that of the wild type, realizing a synergistic enhancement of coenzyme preference shift and high catalytic activity.
[0053] 2. Permeability test The S-configuration Bosein prepared in Example 5 was used to prepare a 15% (w / v) aqueous solution as a control group. Commercially available ectoine was also used to prepare an aqueous solution containing 15% S-configuration Bosein and 10% ectoine as an experimental group.
[0054] Transdermal absorption was determined using a Franz diffusion cell. Isolated porcine skin was fixed between a donor cell and a receiver cell, with the stratum corneum facing the donor cell. 2 mL of sample solution was added to the donor cell, and pH 7.4 phosphate buffer was added to the receiver cell as the receiving solution. The mixture was circulated in a 37°C water bath with magnetic stirring. After 24 hours, a sample was taken from the receiver cell, and the concentration of S-configuration Bosein was determined by HPLC to calculate the transdermal absorption rate.
[0055] The results showed that the transdermal absorption rate of the control group was 3.0% at 24 hours, while the transdermal absorption rate of the experimental group with added ectoine increased to 10% at 24 hours, indicating that ectoine can significantly promote the transdermal absorption of S-configuration bosonicine, increasing the transdermal absorption rate by 2.33 times.
Claims
1. A carbonyl reductase mutant, characterized in that, The carbonyl reductase mutant is a carbonyl reductase that has undergone an amino acid mutation; the amino acid sequence of the carbonyl reductase is shown in SEQ ID No. 1; the amino acid mutation is one of F132A, V162A, F132A-N9E, F132A-K36R or F132A-N9E-K36R.
2. The carbonyl reductase mutant according to claim 1, characterized in that, The coding sequence of the carbonyl reductase includes the nucleotide sequence shown in SEQ ID No.
2.
3. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the carbonyl reductase mutant as described in claim 1 or 2.
4. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 3.
5. A carbonyl reductase mutant transformant, characterized in that, The carbonyl reductase mutant transformant is a genetically engineered strain expressing the carbonyl reductase mutant as described in claim 1 or 2.
6. The carbonyl reductase mutant transformant according to claim 5, characterized in that, The carbonyl reductase mutant transformant contains the expression vector described in claim 4.
7. The use of a carbonyl reductase mutant as described in claim 1 or 2, a nucleic acid molecule as described in claim 3, an expression vector as described in claim 4, or a carbonyl reductase mutant transformant as described in claim 5 or 6 in the synthesis of S-configuration Bosonicine.
8. A method for synthesizing S-configuration Bosein, characterized in that, Includes the following steps: Using β-acetone xyloside as a reaction substrate and isopropanol as a reducing agent, and in the presence of a coenzyme, the carbonyl reductase mutant as described in claim 1 and isopropanol dehydrogenase were used to catalyze the reaction to obtain S-configuration Bosein.
9. The method according to claim 8, characterized in that, The amino acid sequence of the isopropanol dehydrogenase is shown in SEQ ID No. 13, and the nucleotide sequence is shown in SEQ ID No. 14; the coenzyme is nicotinamide adenine dinucleotide.
10. The method according to claim 8, characterized in that, The catalytic reaction was carried out at a pH of 6-7; the concentration of the reaction substrate was 800-1200 mM; the reaction temperature was 28-32℃; and the reaction time was 4-6 h.