Petonin synthesis system and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a bose synthesis system and application thereof.In the presence of coenzyme NAD < + >, glucose dehydrogenase catalyzes glucose to generate gluconic acid, and NAD < + > is reduced into coenzyme NADH; in the presence of a coenzyme NADH (Nicotinamide Adenine Dinucleotide), the carbonyl reductase mutant performs carbonyl reduction catalysis on beta-acetone xyloside to generate a bose, the NADH is oxidized into NAD < + >, and the carbonyl reductase mutant generates mutation which is favorable for improving the reducibility of carbonyl in the beta-acetone xyloside at at least one key site in an amino acid sequence shown as SEQ ID NO: 40. Excellent carbonyl reduction catalytic activity is shown, so that the circulating speed of coenzymes NADH and NAD < + > is increased, and the synthesis efficiency of the bose is improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology, in particular to a Pro-Xylane synthesis system and application. BACKGROUND

[0002] Pro-Xylane is a compound with important biological activity, widely used in medicine, cosmetics and health products and other fields. The unique biological activity of Pro-Xylane makes it show significant effect in antioxidant, anti-inflammatory and anti-aging, etc.

[0003] At present, in the process of enzyme-catalyzed synthesis of Pro-Xylane, the carbonyl reductase reduces beta-ketoxyl glycoside to Pro-Xylane. It is a class of NADH (Nicotinamide adenine dinucleotide) dependent reductase, which needs to rely on NADH for electron transfer. The catalytic activity of carbonyl reductase directly affects the synthesis efficiency of Pro-Xylane. SUMMARY

[0004] In view of the above problems, the embodiments of the present application provide a Pro-Xylane synthesis system and application to solve the technical problem that the above is not conducive to the improvement of the synthesis efficiency of Pro-Xylane.

[0005] In a first aspect, the embodiments of the present application provide a Pro-Xylane synthesis system, comprising NADH or NAD + , glucose, glucose dehydrogenase and a carbonyl reductase mutant, wherein the carbonyl reductase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 or SEQ ID NO: 8 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 17 or SEQ ID NO: 21-SEQ ID NO: 38.

[0006] Optionally, the carbonyl reductase mutant is derived from a first recombinant engineering bacterium containing a carbonyl reductase mutant encoding gene.

[0007] Optionally, the carbonyl reductase mutant is a first wet bacterium obtained by induced culture of the first recombinant engineering bacterium, a crude enzyme liquid obtained by crushing the first wet bacterium, or an immobilized cell prepared from the first wet bacterium.

[0008] Optionally, the glucose dehydrogenase is derived from a second recombinant engineering bacterium containing a glucose dehydrogenase encoding gene.

[0009] Optionally, the glucose dehydrogenase is a second wet bacterium obtained by induced culture of the second recombinant engineering bacterium, a crude enzyme liquid obtained by crushing the second wet bacterium, or an immobilized cell prepared from the second wet bacterium.

[0010] Optionally, the glucose dehydrogenase has the amino acid sequence shown in SEQ ID NO: 41, and the mass ratio of the carbonyl reductase mutant to the glucose dehydrogenase is 3.0-4.0:1.0-2.0.

[0011] Secondly, embodiments of this application provide a method for preparing bosine using the above-described bosine synthesis system, comprising: contacting the bosine synthesis system with β-acetone xyloside to convert the β-acetone xyloside into bosine.

[0012] Optionally, the molar ratio of glucose to β-acetone xyloside is 1.1 to 1.5:1.0.

[0013] Optionally, the reaction temperature between the bosine synthesis system and the β-acetone xyloside is 30°C to 40°C.

[0014] Optionally, the mass ratio of the carbonyl reductase mutant to the glucose dehydrogenase is 3.5:1.5.

[0015] The Bosein synthesis system and its application provided in this application's embodiments are relevant to the coenzyme NAD+. + In the presence of glucose dehydrogenase, glucose dehydrogenase catalyzes the conversion of glucose into gluconic acid and NAD+. + It is reduced to the coenzyme NADH; in the presence of coenzyme NADH, the carbonyl reductase mutant catalyzes the carbonyl reduction of β-acetone xyloside to generate Bosein, and NADH is oxidized to NAD. + This carbonyl reductase mutant produces a mutation at at least one key site in the amino acid sequence shown in SEQ ID NO: 40 that enhances the carbonyl reducing activity in β-acetone xyloside, exhibiting excellent carbonyl reduction catalytic activity, thereby improving the carbonyl reducing activity of coenzymes NADH and NAD. + The faster cycle speed is beneficial to improving the synthesis efficiency of bosonicine.

[0016] These or other aspects of this application will become more apparent in the following description of the embodiments. Attached Figure Description

[0017] Figure 1 A schematic diagram of the reaction principle of the Bosein synthesis system provided in the embodiments of this application is shown.

[0018] Figure 2 The experimental results of the addition ratio of recombinant engineered bacteria in Examples 40-44 of this application are shown in the figure.

[0019] Figure 3 The experimental results of reaction temperature in Examples 45-49 of this application are shown in the figure.

[0020] Figure 4Figures showing experimental results of Example 50 of the present application are shown. DETAILED DESCRIPTION

[0021] Embodiments of the present application are described below in detail, examples of which are shown in the accompanying drawings, wherein the same or similar notations represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below are exemplary only, and are not intended to limit the present application, which can be embodied in various forms.

[0022] In order to better understand the scheme of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0023] In the embodiments of the present application, it should be noted that, in this document, relational terms such as first and second and the like are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between or among the entities or actions.

[0024] Moreover, the terms "comprising", "comprises", "including", "includes" or any other variant thereof are intended to mean non-exclusive inclusion, such that processes, methods, articles or apparatuses that comprise a list of elements are not limited to those elements, but can include other elements not expressly listed or inherent to such processes, methods, articles or apparatuses. Without further limitation, an element preceded by "comprises a" does not, without more constraints, foreclose the existence of additional identical elements in the process, method, article or apparatus that includes the recited element.

[0025] In the description of the embodiments of the present application, the words "example" or "for example" are used to mean serving as an example, instance, or illustration, and not to imply any preference or superiority. The use of the words "example" or "for example" is intended to present clear conceptual examples, and is not intended to limit the scope of the application.

[0026] In addition, "multiple" in the embodiments of the present application refers to two or more, and therefore, "multiple" in the embodiments of the present application can also be understood as "at least two". "At least one" can be understood as one or more, for example, understood as one, two or more. For example, "including at least one" refers to including one, two or more, and does not limit which ones are included, for example, including at least one of A, B and C, and the included can be A, B, C, A and B, A and C, B and C, or A and B and C.

[0027] It should be noted that, in the embodiments of the present application, the association relationship of the associated objects described by "and / or" indicates that there can be three relationships, for example, A and / or B can represent the following three cases: A exists alone, A and B exist together, and B exists alone. In addition, the character " / ", unless otherwise specified, generally represents an "or" relationship between the associated objects before and after it.

[0028] The experimental methods in the following examples are all conventional methods, unless otherwise specified.

[0029] The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0030] The term "Boscalid" in this paper refers to "(S)-Boscalid", which is a strictly chiral compound, not a racemic compound.

[0031] The term "beta-ketoxylulopyranoside" in this paper is "1-C-(B-D- pyranosyl)-propanone", which has a CAS (Chemical Abstracts Service) registration number of 439685-73-1, and is an intermediate of Boscalid.

[0032] The terms "carbonyl reductase" and "carbonyl reductase mutant" in this paper are enzymes that exhibit carbonyl reduction activity, which can convert beta-ketoxylulopyranoside into (S)-Boscalid.

[0033] The reaction principle formula for converting beta-ketoxylulopyranoside into Boscalid by using the above-mentioned carbonyl reductase or carbonyl reductase mutant is as follows:

[0034]

[0035] The polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO: described herein, obviously, the polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO: in which some sequences are deleted, modified, substituted, conservatively substituted or added, can also be used in the present application, as long as it can exhibit the same or corresponding activity as the polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:. For example, it is not excluded that a sequence which does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution thereof is added before or after the "polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:"; and the polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO: when it is added with the above-mentioned sequence which does not change the function of the protein, naturally occurring mutation, silent mutation or conservative substitution thereof, also falls within the scope of the present application, as long as it exhibits the same or corresponding activity as the amino acid sequence as shown in SEQ ID NO: after being added with the above-mentioned sequence.

[0036] An embodiment of the present application provides a biosynthesis system of bovine spongiform encephalopathy, which comprises NADH or NAD + , glucose, glucose dehydrogenase and a carbonyl reductase mutant.

[0037] In the process of screening the saturated mutation of the carbonyl reductase mutant, 39 amino acid sequences as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 39 are constructed. That is, the carbonyl reductase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 39.

[0038] wherein NAD + is the oxidized form of nicotinamide adenine dinucleotide, which does not have an extra hydride (i.e. a hydrogen atom with a negative charge), thus presenting a positive charge. NAD + In cells, it mainly acts as an electron acceptor and participates in various redox reactions. NADH is the reduced form of nicotinamide adenine dinucleotide, which contains an extra hydride (i.e. a hydrogen atom with a negative charge) in structure.

[0039] Referring to Figure 1 , in the presence of coenzyme NAD + , glucose is used as a substrate, glucose dehydrogenase is used as a catalyst, and glucose is converted into gluconic acid by using the oxidative catalytic activity of glucose dehydrogenase, and in the process of converting glucose into gluconic acid, NAD + is reduced into coenzyme NADH.

[0040] Please continue to refer to Figure 1As shown, in the presence of coenzyme NADH, β-ketoxylate is used as a substrate, and the carbonyl reductase mutant is used as a catalyst. The carbonyl reductase mutant catalyzes the conversion of β-ketoxylate into Bosein by using the carbonyl reduction catalytic activity of the carbonyl reductase mutant. During the conversion of β-ketoxylate into Bosein, NADH is oxidized into NAD + .

[0041] The carbonyl reductase mutant has at least one mutation in the amino acid sequence shown in SEQ ID NO: 40, which can improve the catalytic activity of the enzyme for carbonyl reduction. The catalytic activity of the carbonyl reductase mutant is improved, the speed of the carbonyl reduction reaction of β-ketoxylate to generate Bosein is accelerated, and the oxidation of NADH to NAD + is also accelerated, thereby accelerating the speed of the conversion of glucose into gluconic acid and the oxidation of NADH to NAD + , thereby improving the recycling speed of NADH and NAD + .

[0042] Since coenzyme NAD + or coenzyme NADH has high cost and poor stability, it cannot be added in large amounts during the reaction. The improvement of the catalytic activity of the carbonyl reductase mutant is beneficial to the recycling of the reaction, and when the content of coenzyme NAD + or coenzyme NADH is low, the rapid synthesis of Bosein can also be achieved.

[0043] The amino acid sequence shown in SEQ ID NO: 40 is derived from Lentilactobacillus kefiri (Lactobacillus kefiri).

[0044] In constructing the carbonyl reductase mutant, the amino acid sequence shown in SEQ ID NO: 40 can be selected as the research object of multiple key sites related to the binding of the substrate β-ketoxysaccharoside and the exertion of carbonyl reduction activity, including I147 (isoleucine I at position 147), A152 (alanine A at position 152), G154 (glycine G at position 154), A155 (alanine A at position 155), G201 (glycine G at position 201), F202 (phenylalanine F at position 202), G190 (glycine G at position 190), A145 (alanine A at position 145), L96 (leucine L at position 96), L199 (leucine L at position 199), K97 (lysine K at position 97), V94 (valine V at position 94), G125 (glycine G at position 125), V196 (valine V at position 196), N157 (asparagine N at position 157), Q95 (glutamine Q at position 95), N131 (asparagine N at position 31), L87 (leucine L at position 87), L17 (leucine L at position 17), and M153 (methionine M at position 153).

[0045] The above-mentioned key sites are subjected to saturation mutation screening to obtain a carbonyl reductase mutant with improved carbonyl reduction catalytic activity.

[0046] As an embodiment, the carbonyl reductase mutant is derived from a first recombinant engineering bacterium containing a gene encoding the carbonyl reductase mutant.

[0047] Specifically, a first recombinant vector expressing the carbonyl reductase mutant can be constructed, and the first recombinant vector contains a polynucleotide encoding the carbonyl reductase mutant, which includes a nucleotide sequence corresponding to the amino acid sequence of the above-mentioned carbonyl reductase mutant. The polynucleotide is a DNA chain or an RNA chain formed by the polymerization of a plurality of nucleotides. The polynucleotide can be chemically modified as long as it can encode the above-mentioned carbonyl reductase mutant. Exemplarily, the first recombinant vector can be a plasmid.

[0048] The first recombinant vector is transformed into a host cell to obtain a first recombinant engineering bacterium, so that the carbonyl reductase mutant can be synthesized in the first recombinant engineering bacterium.

[0049] In some embodiments, the carbonyl reductase mutant is a first wet bacterium obtained by induced culture of the first recombinant engineering bacterium, a crude enzyme solution obtained by crushing the first wet bacterium, or an immobilized cell prepared from the first wet bacterium.

[0050] As an embodiment, the glucose dehydrogenase is derived from a second recombinant engineering bacterium containing a gene encoding the glucose dehydrogenase.

[0051] Specifically, a second recombinant vector expressing the glucose dehydrogenase can be constructed, the second recombinant vector comprising a polynucleotide encoding the glucose dehydrogenase, the polynucleotide comprising a nucleotide sequence corresponding to the amino acid sequence of the glucose dehydrogenase described above. The polynucleotide is a DNA strand or an RNA strand formed by a plurality of nucleotides. The polynucleotide can be chemically modified as long as it can encode the glucose dehydrogenase described above. Exemplarily, the second recombinant vector can be a plasmid.

[0052] The second recombinant vector is transformed into a host cell to obtain a second recombinant engineered bacterium, so that the glucose dehydrogenase can be synthesized in the second recombinant engineered bacterium.

[0053] In some embodiments, the glucose dehydrogenase is a second wet bacterium obtained by induced culture of the second recombinant engineered bacterium, or a crude enzyme solution obtained by crushing the second wet bacterium, or an immobilized cell prepared from the second wet bacterium.

[0054] Exemplarily, the first recombinant vector and the second recombinant vector are both DNA preparations, the first recombinant vector containing a nucleic acid sequence of a polynucleotide encoding the carbonyl reductase mutant, which can also contain a control sequence; the second recombinant vector containing a nucleic acid sequence of a polynucleotide encoding the glucose dehydrogenase, which can also contain a control sequence. The nucleic acid sequence of the polynucleotide encoding the carbonyl reductase mutant in the first recombinant vector is operably linked to a suitable control sequence, so that the carbonyl reductase mutant can be expressed in a suitable host; the nucleic acid sequence of the polynucleotide encoding the glucose dehydrogenase in the second recombinant vector is operably linked to a suitable control sequence, so that the glucose dehydrogenase can be expressed in a suitable host. For example, the control sequence can include but is not limited to a promoter capable of initiating transcription, any operator sequence for regulating transcription, a suitable mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation. After being transformed into a suitable host cell, the first recombinant vector or the second recombinant vector can be replicated or function independently of the host genome, or can be integrated into the genome of the host cell itself for replication or function.

[0055] Exemplarily, the first or second recombinant engineering bacteria can be Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, or Brevibacterium; and exemplarily, the host cell can be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or Aspergillus oryzae.

[0056] As an implementation form, the glucose dehydrogenase has an amino acid sequence as shown in SEQ ID NO: 41, and the mass ratio of the carbonyl reductase mutant to the glucose dehydrogenase is 3.0-4.0:1.0-2.0.

[0057] In the present embodiment, the mass ratio of the carbonyl reductase mutant to the glucose dehydrogenase is controlled within the above range, which is beneficial to improve the conversion efficiency of the β-ketoxylucoside substrate and further improve the synthesis efficiency of the Bose factor.

[0058] An embodiment of the present application provides a method for preparing a Bose factor, which utilizes the Bose factor synthesis system described above to prepare the Bose factor, and the method comprises the following steps:

[0059] The Bose factor synthesis system described above is contacted with the β-ketoxylucoside to convert the β-ketoxylucoside into the Bose factor.

[0060] In the method for preparing the Bose factor in the present embodiment, the glucose is converted into gluconic acid by utilizing the oxidative catalytic activity of the glucose dehydrogenase, and NAD + is reduced into coenzyme NADH; the β-ketoxylucoside is converted into the Bose factor by utilizing the carbonyl reduction catalytic activity of the carbonyl reductase mutant, and NADH is oxidized into NAD + .

[0061] As an implementation form, the molar ratio of the glucose to the β-ketoxylucoside can be 1.1-1.5:1.0. Exemplarily, the molar ratio of the glucose to the β-ketoxylucoside can be 1.1:1, 1.2:1, 1.3:1, 1.4:1, or 1.5:1.

[0062] As an implementation form, the reaction temperature of the Bose factor synthesis system and the substrate β-ketoxylucoside is 30-40°C. Exemplarily, the catalytic temperature of the substrate β-ketoxylucoside can be 30°C, 35°C, 38°C, or 40°C.

[0063] As an embodiment, the glucose dehydrogenase has an amino acid sequence as shown in SEQ ID NO: 41, and the mass ratio of the carbonyl reductase mutant to the glucose dehydrogenase is 3.0-4.0: 1.0-2.0.

[0064] In some embodiments, the mass ratio of the carbonyl reductase mutant to the glucose dehydrogenase can be 3.5: 1.5.

[0065] In some embodiments, the molar ratio of glucose to β-ketoxysaccharoside can be 1.1-1.2. Illustratively, the molar ratio of glucose to β-ketoxysaccharoside can be 1.1 or 1.2.

[0066] In some embodiments, the catalytic pH value of the substrate β-ketoxysaccharoside can be 6.5-7.5. Illustratively, the catalytic pH value of the substrate β-ketoxysaccharoside can be 7.0.

[0067] Preparation of carbonyl reductase mutants

[0068] The carbonyl reductase mutant and the gene encoding the carbonyl reductase can be referred to as an LKCR gene containing the nucleotide sequence corresponding to the amino acid sequence of the above-mentioned carbonyl reductase mutant or carbonyl reductase mutant, for example, the LKCR gene contains the nucleotide sequence corresponding to the amino acid sequence as shown in SEQ ID NO: 1-SEQ ID NO: 39, and the first recombinant vector is constructed by constructing the LKCR gene in the pET-28a plasmid.

[0069] The first recombinant vector is a pET-28a plasmid containing the LKCR gene, hereinafter referred to as pET-28a-LKCR; the host cell used in each example and comparative example in the present application is Escherichia coli.

[0070] Expression of carbonyl reductase mutants

[0071] a. Transform pET-28a-LKCR into Escherichia coli (e.g., competent cells E. coli BL21(DE3)); pick a single colony of pET-28a-LKCR or a strain stored at -80°C and inoculate it into a small test tube containing 5 mL of LB liquid medium (Kan+, 100 μg / mL), and incubate it at 37°C, 220 rpm overnight as a seed liquid.

[0072] b. Transfer the seed liquid into 50 mL of LB liquid medium (Kan+, 100 μg / mL), and incubate it at 37°C, 220 rpm, and 220 rpm on a shaker for reactivation.

[0073] c. After reactivation, the bacteria solution was transferred into 800 mL 2YT liquid medium (Kan+, 100 μg / mL) at an inoculation amount of 1%, and cultured at 37°C, 220 rpm, until the OD600 was about 0.6-0.8.

[0074] d. The temperature of the shaker was reduced to 16-18°C, and after the temperature of the cultured bacteria solution was reduced, isopropylthio-β-D-galactoside (IPTG) was added to a final concentration of 0.5 mM, and expression was induced for 14-16 h.

[0075] Purification of carbonyl reductase mutant proteins

[0076] a. Preparation of crude enzyme solution: the collected bacteria were resuspended using Buffer, and then broken by a high-pressure low-temperature disrupter at a pressure of 1200 bar and a temperature of 4°C for 3-4 times, so that the expressed target protein was released and dissolved in the protein buffer.

[0077] b. Centrifugation: the broken bacteria solution was centrifuged at 8000 rpm for 50 min in a pre-cooled 4°C centrifuge, and the precipitate and supernatant after centrifugation were taken, sample was prepared, and the supernatant was collected.

[0078] c. Purification: the supernatant was filtered by a 0.45 μm microporous filter, and nickel affinity chromatography purification was performed, and the specific steps were as follows:

[0079] (1) Column equilibration: first wash with dd H2O for 2 column volumes, and then equilibrate the Ni affinity chromatography column with 20 mM imidazole protein buffer for 1 column volume.

[0080] (2) Sample loading: slowly pass the supernatant through the nickel affinity chromatography column at a flow rate of 0.5 mL / min, and repeat once.

[0081] (3) Elution of impurities: wash with protein buffer for 1 column volume, and then elute the strongly bound impurities with 50 mL of protein buffer containing 50 mM imidazole, and take the first few drops of flow-through sample for sample preparation.

[0082] (4) Elution of target protein: elute the target protein with 20 mL of protein buffer containing 100 mM, 200 mM, and 300 mM imidazole, respectively, and take the first few drops of flow-through sample for sample preparation, and detect by 12% SDS-PAGE.

[0083] d. Concentration: the collected target protein was concentrated by centrifugation (4℃, 3400r / min) using an Amicon ultrafiltration tube (30kDa, Millipore) to 1mL. 10mL of protein buffer was added again, and the concentration was repeated to 1mL, and the process was repeated once to ensure the removal of imidazole in the protein, and the purified protein LKCR was obtained.

[0084] Electrophoretic analysis of carbonyl reductase mutant proteins

[0085] a. Protein sample treatment: add the purified protein solution and 5x loading buffer at a ratio of 1:4 (v / v), heat in boiling water for 10min, and store for later use.

[0086] b. Loading and electrophoresis: place the protein precast gel (Genscript, SurePAGE, 4%-20%) in the electrophoresis tank, and add the protein sample and Marker to the protein gel loading well by pipette.

[0087] c. Staining and destaining: remove the shell of the precast gel after electrophoresis, and automatically destain and stain by the protein staining and destaining instrument, with a staining time of 15min.

[0088] d. Protein gel image analysis: take a photo of the protein gel after staining and destaining by the gel imaging instrument.

[0089] Preparation of glucose dehydrogenase

[0090] The coding gene of glucose dehydrogenase can be referred to as a GDH gene, which contains the nucleotide sequence corresponding to the amino acid sequence of the above-mentioned glucose dehydrogenase, for example, the GDH gene contains the nucleotide sequence corresponding to the amino acid sequence as shown in SEQ ID NO: 41, and the GDH gene is constructed in the pET-28a plasmid to obtain a second recombinant vector.

[0091] The second recombinant vector is a pET-28a plasmid containing a GDH gene, hereinafter referred to as pET-28a-GDH; the host cell used in the examples and comparative examples in the present application is Escherichia coli.

[0092] Expression of glucose dehydrogenase

[0093] a. Transform pET-28a-GDH into Escherichia coli (for example, competent cell E. coli BL21 (DE3)); pick a single colony of pET-28a-GDH or a strain stored at -80℃ and inoculate it into a small test tube containing 5mL of LB liquid medium (Kan+, 100μg / mL), and incubate it at 37℃, 220rpm overnight as a seed liquid.

[0094] b. The seed liquid is transferred into 50 mL of LB liquid medium (Kan+, 100 μg / mL) for reactivation at 37°C, 220 rpm, and shaking.

[0095] c. The reactivated bacterial liquid is transferred into 800 mL of 2YT liquid medium (Kan+, 100 μg / mL) at an inoculation amount of 1%, and is cultured at 37°C, 220 rpm, and shaking until the OD600 is about 0.6-0.8.

[0096] d. The shaking temperature is reduced to 16-18°C, and after the temperature of the cultured bacterial liquid is reduced, isopropylthio-β-D-galactoside (IPTG) is added to a final concentration of 0.5 mM for induction for 14-16 h.

[0097] Purification of glucose dehydrogenase protein

[0098] a. Preparation of crude enzyme liquid: 1.0 g of wet bacterial cells is added to 20 mL of equilibration buffer for resuspension, and the resuspended cells are crushed by a cell crusher with parameters set as 300 W to prevent excessive temperature from affecting enzyme activity, a working time of 1 s and a pause time of 3 s, and the crushing liquid is cooled with an ice water mixture at all times. When the suspension is clear and transparent, the crushing is stopped. The crushing liquid is centrifuged at 4°C, 12000 rpm for 10 min, and the supernatant is collected and filtered through a 0.22 μm filter to obtain the crude enzyme liquid. The proteins used in the present study are all untagged, and the isoelectric point of the proteins is predicted to be PI 6.35, so a weak alkaline anion group is selected for purification.

[0099] b. Regeneration and equilibration of ion exchange chromatography column: DEAE Sepharose Fast Flow anion exchange column is used for protein purification, and a high-salt-concentration buffer (containing 1-2 M NaCl) is used to flush at a flow rate of 1 mL / min for 3-5 column volumes. Then, 0.1 M NaOH is used to flush for 3-5 column volumes, followed by elution buffer for 3-5 column volumes, and then equilibration buffer for 3-5 column volumes until the parameters such as OD280, conductivity, and pH value of the detector are stable.

[0100] c. Crude enzyme liquid is loaded and eluted: The prepared crude enzyme liquid is loaded at a loading speed of 0.5 mL / min, and the loading amount is 20 mL. After loading is completed, the equilibration buffer is used to flush for 3-5 column volumes, and then the gradient elution method is used to elute with elution buffer, and the components are collected and confirmed by protein electrophoresis. If the purification effect is not good, the step can be repeated, or Sephadex G75 FF is used for secondary purification.

[0101] d. Protein concentration: The collected target protein was concentrated by ultrafiltration membrane concentration method, and a 10 kDa protein concentration tube was used for concentration, 4℃, 5000 rpm centrifugation for 30 min.

[0102] e. Protein desalination: The concentrated protein was diluted with appropriate amount of PBS buffer (20 mM, pH 7.0), and was placed in a dialysis bag (molecular weight cut-off 8-14 kDa), and was dialyzed with 20 mM, pH 7.0 PBS buffer solution, 4℃ refrigerator overnight, and the dialysis solution was replaced once in the middle.

[0103] f. Preservation of ion exchange chromatography column: The used ion exchange chromatography column was washed with 1M NaOH for 3-5 column volumes, and then washed with 20% ethanol, and then stored in a 4℃ refrigerator.

[0104] Electrophoretic analysis of glucose dehydrogenase protein

[0105] a. Protein sample treatment: The purified protein solution and 5x loading buffer were added in a ratio of 1:4 (v / v), boiled for 10 min, and then stored for use.

[0106] b. Sample loading and electrophoresis: The protein precast gel (Genscript, SurePAGE, 4%-20%) was placed in the electrophoresis tank, and the protein sample and Marker were added to the protein gel loading well by a pipette.

[0107] c. Staining and destaining: The precast gel after electrophoresis was removed from the shell, and was automatically destained and stained by a protein staining and destaining instrument, with a staining and destaining time of 15 min.

[0108] d. Protein gel image analysis: The protein gel after staining and destaining was photographed and saved by a gel imaging instrument.

[0109] Carbonyl reductase mutant activity comparison experiment

[0110] In vitro enzyme catalytic reaction conditions:

[0111] Reaction buffer: PBS buffer with a concentration of 100 mM, 200 mM, 300 mM;

[0112] Reaction pH: pH 7, pH 7.2, pH 7.4;

[0113] The concentration of β-ketoxylucoside was 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L;

[0114] The concentration of glucose was 75 g / L, 90 g / L, 105 g / L, 120 g / L, 135 g / L, 150 g / L;

[0115] The concentration of the carbonyl reductase mutant or the carbonyl reductase is 35 g / L; or, the concentration of the crude enzyme solution of the carbonyl reductase mutant or the crude enzyme solution of the carbonyl reductase is 35 g / L.

[0116] The concentration of the glucose dehydrogenase is 15 g / L; or, the concentration of the crude enzyme solution of the glucose dehydrogenase is 15 g / L.

[0117] The concentration of NAD + is 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L.

[0118] The reaction temperature is 30°C, 35°C, 40°C.

[0119] The reaction time is 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, or 24 h; and the total reaction system is 100 mL.

[0120] Table 1 Parameters of Examples 1-39 and Comparative Example 1

[0121]

[0122]

[0123]

[0124]

[0125] As can be seen from Table 1, when the carbonyl reductase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1, or SEQ ID NO: 8, or SEQ ID NO: 10, or SEQ ID NO: 11, or SEQ ID NO: 17, or SEQ ID NO: 21-38, the catalytic activity of the carbonyl reductase mutant is higher than that of the wild-type carbonyl reductase of Comparative Example 1.

[0126] Recombinant engineering bacteria addition ratio experiment

[0127] The carbonyl reductase mutant is the induced culture wet bacteria of the first recombinant engineering bacteria (the carbonyl reductase mutant is shown in SEQ ID NO: 10), and the glucose dehydrogenase is the induced culture wet bacteria of the second recombinant engineering bacteria (the glucose dehydrogenase is shown in SEQ ID NO: 41).

[0128] The reaction conditions are as follows:

[0129] The buffer solution for the reaction is a PBS buffer solution with a concentration of 100 mM.

[0130] The reaction pH is pH 7.

[0131] The concentration of β-acetone xyloside was 100 g / L;

[0132] The glucose concentration is 150 g / L;

[0133] NAD + The concentrations are 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L;

[0134] Reaction temperatures: 30℃, 35℃, 40℃;

[0135] Reaction time: 10 hours;

[0136] The total reaction volume is 10 mL.

[0137] Table 2. Mass ratio of carbonyl reductase mutant and glucose dehydrogenase

[0138] Carbonyl reductase mutant addition amount Glucose dehydrogenase addition amount Example 40 0.4g 0.1g Example 41 0.35g 0.15g Example 42 0.3g 0.2g Example 43 0.2g 0.3g Example 44 0.1g 0.4g

[0139] Please see Figure 2 As shown, the synthesis efficiency of bosoxane was highest when the mass ratio of carbonyl reductase mutant to glucose dehydrogenase was 7:3 (Example 41); the synthesis efficiency of bosoxane was relatively high when the mass ratio of carbonyl reductase mutant to glucose dehydrogenase was 4:1 (Example 40); and the synthesis efficiency of bosoxane was relatively high when the mass ratio of carbonyl reductase mutant to glucose dehydrogenase was 3:2 (Example 42).

[0140] Reaction temperature comparison experiment

[0141] The carbonyl reductase mutant was the induced culture wet cell of the first recombinant engineered bacteria (the carbonyl reductase mutant is shown in SEQ ID NO: 10), and the glucose dehydrogenase was the induced culture wet cell of the second recombinant engineered bacteria (the glucose dehydrogenase is shown in SEQ ID NO: 41).

[0142] The reaction conditions are as follows:

[0143] Reaction buffer: 100 mM PBS buffer;

[0144] Reaction pH: pH 7;

[0145] The concentration of β-acetone xyloside was 100 g / L;

[0146] The glucose concentration is 150 g / L;

[0147] The concentration of the carbonyl reductase mutant was 35 g / L;

[0148] The concentration of glucose dehydrogenase was 15 g / L;

[0149] NAD+ The concentrations are 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L;

[0150] Reaction time: 10 hours;

[0151] The total reaction volume is 10 mL.

[0152] Table 3 Comparison of different reaction temperatures

[0153]

[0154]

[0155] Please see Figure 3 As shown, the synthesis efficiency of bosine is higher when the reaction temperature is 30℃ and 35℃.

[0156] Two substrates addition ratio experiment

[0157] The reaction conditions are as follows:

[0158] Reaction buffer: 100 mM PBS buffer;

[0159] Reaction pH: pH 7;

[0160] The concentration of the carbonyl reductase mutant was 70 g / L;

[0161] The concentration of glucose dehydrogenase was 30 g / L;

[0162] NAD + The concentrations are 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L;

[0163] Reaction time: 25 hours;

[0164] The reaction temperature is 35℃;

[0165] The total reaction volume is 50 mL.

[0166] Table 4. Molar ratio of β-pyruvyl xyloside and glucose

[0167]

[0168] In Examples 50 to 56, the glucose substrate from Examples 50 and 51 was completely consumed, while glucose remained in Examples 52 to 56. Therefore, controlling the molar ratio of glucose to β-acetone xyloside within 1.1 to 1.2 is beneficial for the complete reaction of both substrates.

[0169] Please see Figure 4The concentration of glucose was also measured. The results are shown in Figure 6. As can be seen, the concentration of glucose decreased with time, and was completely consumed after 24 hours.

[0170] The above merely illustrates the embodiments of the present application, and it should be pointed out that, for those skilled in the art, improvements can be made without departing from the inventive concept of the present application, and these improvements shall fall within the protection scope of the present application.

Claims

1. A bosonicine synthesis system, characterized in that, Including NADH or NAD + Glucose, glucose dehydrogenase, and carbonyl reductase mutants, wherein the carbonyl reductase mutants have an amino acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 17, or any of SEQ ID NO: 21 to SEQ ID NO:

38.

2. The Bosein synthesis system according to claim 1, characterized in that, The carbonyl reductase mutant was derived from a first recombinant engineered bacterium containing a gene encoding the carbonyl reductase mutant.

3. The Bosein synthesis system according to claim 2, characterized in that, The carbonyl reductase mutant is the first wet cell obtained by inducing and culturing the first recombinant engineered bacteria, or the crude enzyme solution obtained by breaking the first wet cell, or the immobilized cell prepared from the first wet cell.

4. The Bosein synthesis system according to claim 1, characterized in that, The glucose dehydrogenase is derived from a second recombinant engineered bacterium containing a glucose dehydrogenase encoding gene.

5. The Bosein synthesis system according to claim 4, characterized in that, The glucose dehydrogenase is the second wet cell obtained by inducing and culturing the second recombinant engineered bacteria, or the crude enzyme solution obtained by breaking the second wet cell, or the immobilized cell prepared from the second wet cell.

6. The Bosein synthesis system according to claim 1, characterized in that, The glucose dehydrogenase has the amino acid sequence shown in SEQ ID NO: 41, and the mass ratio of the carbonyl reductase mutant to the glucose dehydrogenase is 3.0–4.0:1.0–2.

0.

7. A method for preparing bosonic acid using the bosonic acid synthesis system as described in any one of claims 1 to 6, characterized in that, include: The Bosein synthesis system is contacted with β-acetone xyloside to convert the β-acetone xyloside into Bosein.

8. The method for preparing Bosein according to claim 7, characterized in that, The molar ratio of glucose to β-acetone xyloside is 1.1 to 1.5:1.

0.

9. The method for preparing Bosein according to claim 7, characterized in that, The reaction temperature between the Bosein synthesis system and the β-acetone xyloside is 30℃~40℃.

10. The method for preparing Bosein according to claim 7, characterized in that, The mass ratio of the carbonyl reductase mutant to the glucose dehydrogenase is 3.5:1.5.

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