Isopropanol system for synthesis of glassine and application of isopropanol system

Abstract

The invention relates to the technical field of biology, in particular to an isopropanol system for synthesis of glassine and application thereof.In the presence of coenzyme NAD < + >, isopropanol dehydrogenase catalyzes isopropanol to generate acetone, 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 (S)-glassine is improved.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to an isopropanol system for the synthesis of bosonicine and its application. Background Technology

[0002] Pro-Xylane is a compound with important biological activities, widely used in pharmaceuticals, cosmetics, and health products. Its unique biological activity gives it significant effects in anti-oxidation, anti-inflammation, and anti-aging.

[0003] Currently, in the enzymatic synthesis of bosine, carbonyl reductase reduces β-acetone xyloside to bosine. Carbonyl reductase is a type of NADH (Nicotinamide adenine dinucleotide) dependent reductase, which requires NADH for electron transfer. The catalytic activity of carbonyl reductase directly affects the synthesis efficiency of bosine. Summary of the Invention

[0004] In view of the above problems, this application provides an isopropanol system for the synthesis of bosonic acid and its application, so as to solve the above-mentioned technical problems that are not conducive to improving the efficiency of bosonic acid synthesis.

[0005] In a first aspect, embodiments of this application provide an isopropanol system for the synthesis of bosonicine, including NADH or NAD. + Isopropanol, isopropanol 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.

[0006] Optionally, the mass ratio of the carbonyl reductase mutant to the isopropanol dehydrogenase is 1.0 to 3.0:1.0.

[0007] Optionally, the volume percentage of the isopropanol is 8% to 12%.

[0008] Optionally, the NAD + The mass concentration is 0.25 g / L to 0.5 g / L.

[0009] Optionally, the carbonyl reductase mutant is derived from a first recombinant engineered bacterium containing a carbonyl reductase mutant encoding gene, and the isopropanol dehydrogenase is derived from a second recombinant engineered bacterium containing an isopropanol dehydrogenase encoding gene.

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

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

[0012] Optionally, the mass ratio of isopropanol to β-acetone xyloside is 0.63 to 0.94:1.0.

[0013] Optionally, the mass concentration of β-acetone xyloside in the catalytic system is 47 g / L to 52 g / L.

[0014] Optionally, the mass ratio of the carbonyl reductase mutant to the isopropanol dehydrogenase is 2.0:1.0.

[0015] The isopropanol system and its application for the synthesis of Bosein provided in this application are relevant to the coenzyme NAD+. + In the presence of isopropanol dehydrogenase, isopropanol is catalyzed to produce acetone 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 (S)-Bosonic.

[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 isopropanol system for the synthesis of bosine provided in the embodiments of this application is shown.

[0018] Figure 2 The experimental results of the addition ratio of β-acetone xyloside in Examples 40-42 of this application are shown in the figure.

[0019] Figure 3 The experimental results of isopropanol addition ratios in Examples 43-47 of this application are shown in the figure.

[0020] Figure 4 The experimental results of Embodiment 48 of this application are shown in the figure. Detailed Implementation

[0021] The embodiments of this application are described in detail below. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0022] To enable those skilled in the art to better understand the solutions of this application, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this application without creative effort are within the scope of protection of this application.

[0023] In the embodiments of this application, it should be noted that, in this document, relational terms such as first and second are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0024] Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0025] In the description of the embodiments of this application, the words "example" or "for example" are used to indicate exemplification, illustration, or description. Any embodiment or design described as "example" or "for example" in the embodiments of this application is not to be construed as being more preferred or having more advantages than another embodiment or design. The use of the words "example" or "for example" is intended to present relative concepts in a clear manner.

[0026] Furthermore, in the embodiments of this application, "multiple" refers to two or more. Therefore, in the embodiments of this application, "multiple" can also be understood as "at least two". "At least one" can be understood as one or more, such as one, two, or more. For example, including at least one means including one, two, or more, and is not limited to which ones are included. For example, including at least one of A, B, and C, then it could include 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 this application, "and / or" describes the relationship between associated objects, indicating that there can be three relationships. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. In addition, the character " / ", unless otherwise specified, generally indicates that the associated objects before and after it are in an "or" relationship.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] In this article, the term "Bosine" refers to "(S)-Bosine," which is a strictly chiral compound and not a racemic compound.

[0031] In this article, the term "β-acetone xyloside" refers to "1-C-(BD-xylopyranyl)-acetone", whose CAS (Chemical Abstracts Service) registration number is 439685-73-1, and it is a bosine intermediate.

[0032] In this article, the terms "carbonyl reductase" and "carbonyl reductase mutant" refer to enzymes that exhibit carbonyl reduction activity, which can convert β-acetone xyloside into (S)-Bosine.

[0033] The reaction principle for converting β-acetone xyloside to (S)-Bosine using the aforementioned carbonyl reductase or carbonyl reductase mutant is shown below:

[0034]

[0035] The description herein refers to "a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO:". Obviously, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO:, even with some sequence deletions, modifications, substitutions, conserved substitutions, or additions, can also be used in this application, as long as they exhibit the same or corresponding activity as the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO:. For example, it is not excluded to add sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions before or after "the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO:". Furthermore, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO:, when subjected to the addition of the aforementioned sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions, also fall within the scope of this application, as long as they exhibit the same or corresponding activity as the amino acid sequence shown in SEQ ID NO: after the addition of the aforementioned sequences.

[0036] One embodiment of this application provides an isopropanol system for the synthesis of Bosein, which includes NADH or NAD. + Isopropanol, isopropanol dehydrogenase, and carbonyl reductase mutants.

[0037] During the saturation mutation screening of the carbonyl reductase mutant, amino acid sequences as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 39 were constructed, totaling 39 amino acid sequences. That is, the carbonyl reductase mutant possesses any one of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 39.

[0038] Among them, NAD + It is the oxidized state of nicotinamide adenine dinucleotide, which does not have an additional hydride (i.e., a negatively charged hydrogen atom), and therefore presents a positive charge. NAD + In cells, it primarily functions as an electron acceptor, participating in various redox reactions. NADH is the reduced state of nicotinamide adenine dinucleotide, which structurally contains an additional hydride (i.e., a negatively charged hydrogen atom).

[0039] Please see Figure 1 As shown, in coenzyme NAD + In the presence of isopropanol as a substrate and isopropanol dehydrogenase as a catalyst, isopropanol is converted to acetone using the oxidative catalytic activity of isopropanol dehydrogenase. During this conversion, NAD+ is also involved. + It is reduced to the coenzyme NADH.

[0040] Please continue reading. Figure 1As shown, in the presence of coenzyme NADH, β-pyruvate xyloside serves as the substrate, and a carbonyl reductase mutant acts as the catalyst. Utilizing the carbonyl reductase mutant's carbonyl reduction catalytic activity, β-pyruvate xyloside is converted to Bosein. Furthermore, during this conversion, NADH is oxidized to NAD. + .

[0041] This carbonyl reductase mutant produces at least one mutation in the amino acid sequence shown in SEQ ID NO: 40, which enhances the enzyme's catalytic activity for carbonyl reduction. The increased catalytic activity of this carbonyl reductase mutant accelerates the carbonyl reduction reaction of β-acetone xyloside to Bosein, and accelerates the oxidation of NADH to NAD. + This also accelerates the conversion of isopropanol to acetone and the oxidation of NADH to NAD. + The speed of both increases, thereby improving NADH and NAD. + The cycle speed.

[0042] Due to coenzyme NAD + Alternatively, the high cost and poor stability of coenzyme NADH prevent its large-scale addition during the reaction. The increased catalytic activity of this carbonyl reductase mutant facilitates the cyclical progress of the reaction. + Even when the content of coenzyme NADH is low, the rapid synthesis of bosine can still be achieved.

[0043] Among them, the amino acid sequence shown in SEQ ID NO: 40 is derived from Lentilactobacillus kefiri (Caucasian lactobacillus).

[0044] When constructing this carbonyl reductase mutant, several key sites in the amino acid sequence shown in SEQ ID NO: 40 that are related to the binding of the substrate β-pyruvate and to the carbonyl reduction activity can be selected as research objects. These key sites include 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), and A145 (alanine A at position 145). L96 (leucine at position 96), L199 (leucine at position 199), K97 (lysine at position 97), V94 (valine at position 94), G125 (glycine at position 125), V196 (valine at position 196), N157 (asparagine at position 157), Q95 (glutamine at position 95), N131 (asparagine at position 31), L87 (leucine at position 87), L17 (leucine at position 17), and M153 (methionine at position 153).

[0045] Saturation mutation screening was performed on the above key sites to obtain carbonyl reductase mutants with improved carbonyl reduction catalytic activity.

[0046] In one embodiment, in the isopropanol system used for Bosein synthesis, the mass ratio of carbonyl reductase mutant to isopropanol dehydrogenase is 1.0–3.0:1.0. In this embodiment, controlling the mass ratio of carbonyl reductase mutant to isopropanol dehydrogenase within the above range is beneficial to improving the conversion efficiency of β-acetone xyloside substrate, thereby improving the synthesis efficiency of Bosein.

[0047] In one embodiment, the volume percentage of isopropanol in the isopropanol system used for the synthesis of Bosein is 8% to 12%. In this embodiment, controlling the content of isopropanol within the above range is beneficial to improving the conversion efficiency of the β-acetone xyloside substrate, thereby improving the synthesis efficiency of Bosein.

[0048] As one implementation method, in the isopropanol system used for the synthesis of Bosein, NAD... + The mass concentration is 0.25 g / L to 0.5 g / L.

[0049] In one implementation, the carbonyl reductase mutant is derived from a first recombinant engineered bacterium containing a gene encoding the carbonyl reductase mutant.

[0050] Specifically, a first recombinant vector expressing the carbonyl reductase mutant can be constructed. This first recombinant vector contains a polynucleotide encoding the carbonyl reductase mutant, and this polynucleotide includes the nucleotide sequence corresponding to the amino acid sequence of the aforementioned carbonyl reductase mutant. The polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides. The polynucleotide only needs to encode the aforementioned carbonyl reductase mutant, and any nucleotide in the polynucleotide can be chemically modified. For example, the first recombinant vector can be a plasmid.

[0051] The first recombinant vector was transformed into a host cell to obtain the first recombinant engineered bacteria, which enabled the carbonyl reductase mutant to be synthesized in the first recombinant engineered bacteria.

[0052] In some embodiments, the carbonyl reductase mutant is a first wet cell obtained by inducing culture of a first recombinant engineered bacterium, or a crude enzyme solution obtained by breaking down the first wet cell, or an immobilized cell prepared from the first wet cell.

[0053] In one implementation, the isopropanol dehydrogenase is derived from a second recombinant engineered bacterium containing an isopropanol dehydrogenase encoding gene.

[0054] Specifically, a second recombinant vector expressing the isopropanol dehydrogenase can be constructed. This second recombinant vector contains a polynucleotide encoding the isopropanol dehydrogenase, which includes the nucleotide sequence corresponding to the amino acid sequence of the isopropanol dehydrogenase described above. The polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides. The polynucleotide only needs to encode the isopropanol dehydrogenase, and any nucleotide in the polynucleotide can be chemically modified. For example, the second recombinant vector can be a plasmid.

[0055] The second recombinant vector was transformed into the host cell to obtain the second recombinant engineered bacteria, which enabled isopropanol dehydrogenase to be synthesized in the second recombinant engineered bacteria.

[0056] In some embodiments, the isopropanol dehydrogenase is a second wet cell obtained by inducing culture of a second recombinant engineered bacterium, or a crude enzyme solution obtained by breaking down a second wet cell, or an immobilized cell prepared from a second wet cell.

[0057] Exemplary, both the first and second recombinant vectors are DNA preparations. The first recombinant vector contains a nucleic acid sequence of a polynucleotide encoding a carbonyl reductase mutant, and may also contain a control sequence; the second recombinant vector contains a nucleic acid sequence of a polynucleotide encoding isopropanol dehydrogenase, and may also contain a control sequence. In the first recombinant vector, the nucleic acid sequence of the polynucleotide encoding the carbonyl reductase mutant is operatively linked to a suitable control sequence, allowing the carbonyl reductase mutant to be expressed in a suitable host; in the second recombinant vector, the nucleic acid sequence of the polynucleotide encoding isopropanol dehydrogenase is operatively linked to a suitable control sequence, allowing isopropanol dehydrogenase to be expressed in a suitable host. For example, the control sequence may include, but is not limited to, a promoter capable of initiating transcription, any operon sequence for regulating transcription, a suitable mRNA ribosome binding site, and sequences for controlling transcription and translation termination. After transformation into a suitable host cell, the first or second recombinant vector may replicate or function independently of the host genome, or it may integrate into the host cell's genome itself for replication or function.

[0058] For example, the first or second recombinant engineered bacteria may be Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, or Brevibacterium; for example, the host cell may be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or Aspergillus oryzae.

[0059] In one embodiment, the isopropanol dehydrogenase has the amino acid sequence shown in SEQ ID NO: 41, and the mass ratio of the carbonyl reductase mutant to the isopropanol dehydrogenase is 2.0:1.0.

[0060] In this embodiment, controlling the mass ratio of carbonyl reductase mutant to isopropanol dehydrogenase within the above-mentioned range is beneficial to improving the conversion efficiency of β-acetone xyloside substrate, thereby improving the synthesis efficiency of bosine.

[0061] One embodiment of this application provides a method for preparing Bosein, which utilizes the above-described isopropanol system for Bosein synthesis to prepare Bosein. The method includes:

[0062] The isopropanol system described above for the synthesis of bosine was contacted with β-acetone xyloside to convert β-acetone xyloside into bosine.

[0063] In the method for preparing Bosein in this embodiment, isopropanol is converted to acetone using the oxidative catalytic activity of isopropanol dehydrogenase, while NAD+ is also converted. + It is reduced to the coenzyme NADH; utilizing the carbonyl reductase mutant's carbonyl reduction catalytic activity, β-acetone xyloside is converted to Bosorin, while NADH is oxidized to NAD. + .

[0064] In one implementation method, the mass ratio of isopropanol to β-acetone xyloside in the catalytic system is 0.63–0.94:1.0. For example, when the amount of β-acetone xyloside added is 1 g, the amount of isopropanol added can be 0.8 mL–1.2 mL.

[0065] In some embodiments, the mass concentration of β-pyruvate in the catalytic system is 47 g / L to 52 g / L. For example, the mass concentration of β-pyruvate in the catalytic system is 50 g / L.

[0066] In some embodiments, the mass ratio of carbonyl reductase mutant to isopropanol dehydrogenase in the catalytic system is 2.0:1.0.

[0067] In one embodiment, the isopropanol system used for Bosein synthesis reacts with the substrate β-pyruvate at a temperature of 30°C to 40°C. Exemplarily, the catalytic temperature for the substrate β-pyruvate can be 30°C, 35°C, 38°C, or 40°C.

[0068] In some embodiments, the catalytic pH of the substrate β-pyruvate can be 6.5 to 7.5. For example, the catalytic pH of the substrate β-pyruvate can be 7.0.

[0069] Preparation of carbonyl reductase mutants

[0070] The carbonyl reductase mutant and the gene encoding the carbonyl reductase can be called the LKCR gene. The LKCR gene contains the nucleotide sequence corresponding to the amino acid sequence of the carbonyl reductase mutant or the carbonyl reductase mutant. For example, the LKCR gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 39. The LKCR gene is constructed in the pET-28a plasmid to obtain the first recombinant vector.

[0071] 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 the various embodiments and comparative examples in this application is Escherichia coli.

[0072] Expression of carbonyl reductase mutants

[0073] a. Transform pET-28a-LKCR into Escherichia coli (e.g., competent cells E.coli BL21(DE3)); pick a single clone of pET-28a-LKCR or a strain preserved at -80℃ and inoculate it into a small test tube containing 5 mL of LB liquid medium (Kan+, 100 μg / mL), and incubate overnight at 37℃ and 220 rpm to obtain the seed culture.

[0074] b. Transfer the seed culture to 50 mL of LB liquid medium (Kan+, 100 μg / mL) and incubate at 37 °C and 220 rpm on a shaker for reactivation.

[0075] c. Transfer the reactivated bacterial culture to 800 mL of 2YT liquid medium (Kan+, 100 μg / mL) at an inoculation rate of 1%, and incubate at 37 °C and 220 rpm on a shaker until the OD 600 is approximately 0.6-0.8.

[0076] d. Lower the temperature of the shaker to 16℃-18℃. After the temperature of the cultured bacterial solution has decreased, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5mM and induce expression for 14-16h.

[0077] Purification of carbonyl reductase mutant protein

[0078] a. Preparation of crude enzyme solution: The collected bacterial cells were resuspended in buffer and then subjected to high pressure low temperature disruption 3-4 times at 1200 bar and 4°C to fully lyse the cells, thereby releasing the expressed target protein and dissolving it in protein buffer.

[0079] b. Centrifugation: Centrifuge the broken bacterial culture at 8000 rpm for 50 min in a pre-cooled centrifuge at 4℃. Take the precipitate and supernatant after centrifugation, prepare the sample, and collect the supernatant.

[0080] c. Purification: The supernatant was filtered through a 0.45 μm microporous membrane and purified by nickel affinity chromatography. The specific steps are as follows:

[0081] (1) Column equilibration: First wash with dd H2O for 2 column volumes, then equilibrate the Ni affinity chromatography column with 20mM imidazole protein buffer for 1 column volume.

[0082] (2) Sample loading: Pass the supernatant slowly through the nickel affinity chromatography column at a flow rate of 0.5 mL / min, and repeat once more to flow through.

[0083] (3) Elution of contaminating proteins: Wash with protein buffer for one column volume, then use 50 mL of protein buffer containing 50 mM imidazole to elute the contaminating proteins with strong binding. Take the first few drops to flow through the sample and prepare the sample.

[0084] (4) Elution of target protein: Elute the target protein with 20 mL of imidazole protein buffer containing 100 mM, 200 mM and 300 mM respectively. Take the first few drops of the sample to flow through, prepare the sample and detect it with 12% SDS-PAGE.

[0085] d. Concentration and buffer replacement: The collected target protein was concentrated by centrifugation (4℃, 3400 rpm) using a 50 mL Amicon ultrafiltration tube (30 kDa, Millipore) to a final volume of 1 mL. Then, 10 mL of protein buffer was added, and the concentration was repeated to 1 mL. This process was repeated once to ensure the removal of imidazole from the protein, yielding purified protein LKCR.

[0086] Electrophoretic analysis of carbonyl reductase mutant proteins

[0087] a. Protein sample preparation: Add the purified protein solution and 5× loading buffer at a ratio of 1:4 (v / v), heat in boiling water for 10 min, and set aside for later use.

[0088] b. Sample loading and electrophoresis: Place the precast protein gel (Genscript, SurePAGE, 4%–20%) in the electrophoresis tank, and add the protein sample and marker to the sample wells of the protein gel using a pipette.

[0089] c. Staining and destaining: Remove the outer shell of the pre-cast gel after electrophoresis, and automatically destain and stain using a protein staining and destaining instrument for 15 minutes.

[0090] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0091] Preparation of isopropanol dehydrogenase

[0092] The gene encoding isopropanol dehydrogenase can be called the ADH gene. The ADH gene contains the nucleotide sequence corresponding to the amino acid sequence of the isopropanol dehydrogenase mentioned above. For example, the ADH gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 41. The ADH gene is constructed in the pET-28a plasmid to obtain the second recombinant vector.

[0093] The second recombinant vector is a pET-28a plasmid containing the ADH gene, hereinafter referred to as pET-28a-ADH; the host cell used in the various embodiments and comparative examples in this application is Escherichia coli.

[0094] Expression of isopropanol dehydrogenase

[0095] a. Transform pET-28a-ADH into Escherichia coli (e.g., competent cells E.coli BL21(DE3)); pick a single clone of pET-28a-ADH or a strain preserved at -80℃ and inoculate it into a small test tube containing 5 mL of LB liquid medium (Kan+, 100 μg / mL), and incubate overnight at 37℃ and 220 rpm to obtain the seed culture.

[0096] b. Transfer the seed culture to 50 mL of LB liquid medium (Kan+, 100 μg / mL) and incubate at 37 °C and 220 rpm on a shaker for reactivation.

[0097] c. Transfer the reactivated bacterial culture to 800 mL of 2YT liquid medium (Kan+, 100 μg / mL) at an inoculation rate of 1%, and incubate at 37 °C and 220 rpm on a shaker until the OD 600 is approximately 0.6-0.8.

[0098] d. Lower the temperature of the shaker to 16℃-18℃. After the temperature of the cultured bacterial solution has decreased, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5mM and induce expression for 14-16h.

[0099] Purification of isopropanol dehydrogenase protein

[0100] a. Preparation of crude enzyme solution: 1.0 g of collected wet bacterial cells were added to 20 mL of equilibration buffer for resuspending. The resuspended cells were then disrupted using a cell disruptor set to 300 W to prevent excessive temperature from affecting enzyme activity. The disruption program was set to run for 1 second and pause for 3 seconds. The disruption solution was continuously cooled with an ice-water mixture until the suspension became clear and transparent. The disruption solution was then centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected and filtered through a 0.22 μm filter to obtain the crude enzyme solution. All proteins used in this study were unlabeled, and the predicted isoelectric point (PI) was 6.35; therefore, weakly basic anionic groups were selected for purification.

[0101] b. Regeneration and equilibration of ion exchange chromatography column: Protein purification was performed using a DEAE Sepharose Fast Flow anion exchange column. The column was washed with a high-salt buffer (containing 1-2 M NaCl) at a flow rate of 1 mL / min for 3-5 column volumes, then washed with 0.1 M NaOH for 3-5 column volumes, then washed with elution buffer for 3-5 column volumes, and finally washed with equilibration buffer until the detector parameters such as OD280, conductivity, and pH value stabilized.

[0102] c. Loading and elution of crude enzyme solution: Load the prepared crude enzyme solution at a loading rate of 0.5 mL / min, with a loading volume of 20 mL. After loading, wash with equilibration buffer for 3–5 column volumes, then elute using an increasing salt concentration gradient with elution buffer. Collect each fraction and confirm by protein electrophoresis. If the purification effect is unsatisfactory, this step can be repeated, or purification can be performed again using agarose gel G75FF.

[0103] d. Protein concentration: The collected target protein was concentrated using ultrafiltration membrane concentration method. The protein was concentrated using a 10kDa protein concentration tube and centrifuged at 5000rpm for 30min at 4℃.

[0104] e. Protein desalting: Dilute the concentrated protein with an appropriate amount of PBS buffer (20mM, pH 7.0) and place it in a dialysis bag (molecular weight cutoff 8-14kDa). Use 20mM, pH 7.0 PBS dialysate and let it stand overnight at 4°C. The dialysate needs to be changed once during the process.

[0105] f. Preservation of ion exchange chromatography columns: After use, rinse the ion exchange chromatography column with 1M NaOH for 3-5 column volumes, then rinse with 20% ethanol, and store in a refrigerator at 4°C.

[0106] Electrophoretic analysis of isopropanol dehydrogenase protein

[0107] a. Protein sample preparation: Add the purified protein solution and 5× loading buffer at a ratio of 1:4 (v / v), heat in boiling water for 10 min, and set aside for later use.

[0108] b. Sample loading and electrophoresis: Place the precast protein gel (Genscript, SurePAGE, 4%–20%) in the electrophoresis tank, and add the protein sample and marker to the sample wells of the protein gel using a pipette.

[0109] c. Staining and destaining: Remove the outer shell of the pre-cast gel after electrophoresis, and automatically destain and stain using a protein staining and destaining instrument for 15 minutes.

[0110] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0111] Comparative experiment on the activity of carbonyl reductase mutants

[0112] In vitro enzyme catalytic reaction conditions:

[0113] Reaction buffer: 100 mM PBS buffer;

[0114] Reaction pH: pH 7, pH 7.2, pH 7.4;

[0115] The concentrations of β-pyruvyl xyloside were 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, and 200 g / L.

[0116] The volume percentages of isopropanol are 8%, 10%, 12%, 20%, 30%, 40%, and 50%.

[0117] The concentration of the carbonyl reductase mutant or carbonyl reductase is 100 g / L; or, the concentration of the crude enzyme solution of the carbonyl reductase mutant or the crude enzyme solution of carbonyl reductase is 100 g / L.

[0118] The concentration of isopropanol dehydrogenase is 50 g / L; or, the concentration of crude isopropanol dehydrogenase solution is 50 g / L.

[0119] NAD + The concentrations were 0.2 g / L, 0.25 g / L, 0.3 g / L, 0.4 g / L, and 0.5 g / L.

[0120] Reaction temperatures: 30℃, 35℃, 40℃;

[0121] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, or 24h;

[0122] The total reaction volume is 10 mL.

[0123] Table 1. Parameters of Examples 1 to 39 and Comparative Example 1

[0124]

[0125]

[0126]

[0127]

[0128]

[0129] As shown in Table 1, when the carbonyl reductase mutant has 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, the catalytic activity of the carbonyl reductase mutant is higher than that of the wild-type carbonyl reductase in Comparative Example 1.

[0130] Experiment on the addition ratio of β-acetone xyloside

[0131] 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 isopropanol dehydrogenase was the induced culture wet cell of the second recombinant engineered bacteria (the isopropanol dehydrogenase is shown in SEQ ID NO: 41).

[0132] The reaction conditions are as follows:

[0133] Reaction buffer: 100 mM PBS buffer;

[0134] Reaction pH: pH 7;

[0135] The concentration of carbonyl reductase mutant or carbonyl reductase is 100 g / L;

[0136] The concentration of isopropanol dehydrogenase was 50 g / L;

[0137] NAD + The concentration was 0.3 g / L;

[0138] Reaction temperature: 40℃;

[0139] Reaction time: 24 hours;

[0140] The total reaction volume is 10 mL.

[0141] Table 2 Comparison of the amount of β-pyruvone xyloside and isopropanol added

[0142]

[0143]

[0144] Please see Figure 2 As shown, when the mass concentration of β-pyruvate was 50 g / L (Example 40), the remaining amount of β-pyruvate after 24 hours of reaction was 10 g / L; when the mass concentration of β-pyruvate was 100 g / L (Example 41), the remaining amount of β-pyruvate after 24 hours of reaction was 36 g / L; and when the mass concentration of β-pyruvate was 200 g / L (Example 42), the remaining amount of β-pyruvate after 24 hours of reaction was 80 g / L.

[0145] Isopropanol addition ratio experiment

[0146] 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 isopropanol dehydrogenase was the induced culture wet cell of the second recombinant engineered bacteria (the isopropanol dehydrogenase is shown in SEQ ID NO: 41).

[0147] The reaction conditions are as follows:

[0148] Reaction buffer: 100 mM PBS buffer;

[0149] Reaction pH: pH 7;

[0150] The concentration of β-acetone xyloside was 100 g / L;

[0151] The concentration of isopropanol is 150 g / L;

[0152] The concentration of the carbonyl reductase mutant was 50 g / L;

[0153] The concentration of isopropanol dehydrogenase was 50 g / L;

[0154] NAD + The concentration was 0.3 g / L;

[0155] Reaction time: 24 hours;

[0156] The total reaction volume is 10 mL.

[0157] Table 3 Comparison of Isopropanol Addition Amount

[0158]

[0159]

[0160] Please see Figure 3 As shown, the synthesis efficiency of bosine is high when the amount of isopropanol added is 1 mL (volume percentage of 10%).

[0161] Example 48

[0162] The reaction conditions are as follows:

[0163] Reaction buffer: 100 mM PBS buffer;

[0164] Reaction pH: pH 7;

[0165] The concentration of the carbonyl reductase mutant was 150 g / L;

[0166] The concentration of isopropanol dehydrogenase was 50 g / L;

[0167] The amount of isopropanol added is 1 mL;

[0168] The concentration of β-acetone xyloside was 100 g / L;

[0169] NAD + The concentration was 0.3 g / L;

[0170] Reaction time: 40 hours;

[0171] The reaction temperature is 35℃;

[0172] The total reaction volume is 10 mL.

[0173] Please see Figure 4 As shown, the concentration changes of bosine and β-pyruvate in Example 48 are shown. With the extension of reaction time, compared with Example 43, Example 48 increases the concentration of carbonyl reductase mutant, and the substrate β-pyruvate is still not completely consumed.

[0174] The above description is merely an embodiment of this application. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of this application, but these improvements all fall within the protection scope of this application.

Claims

1. An isopropanol system for the synthesis of Bosein, characterized in that, Including NAD + Isopropanol, isopropanol 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 isopropanol system for the synthesis of Bosein according to claim 1, characterized in that, The mass ratio of the carbonyl reductase mutant to the isopropanol dehydrogenase is 1.0–3.0:1.

0.

3. The isopropanol system for the synthesis of Bosein according to claim 2, characterized in that, The volume percentage of isopropanol is 8% to 12%.

4. The isopropanol system for the synthesis of Bosein according to claim 3, characterized in that, The NAD + The mass concentration is 0.25 g / L to 0.5 g / L.

5. The isopropanol system for the synthesis of Bosein according to claim 4, characterized in that, The carbonyl reductase mutant is derived from a first recombinant engineered bacterium containing a gene encoding the carbonyl reductase mutant, and the isopropanol dehydrogenase is derived from a second recombinant engineered bacterium containing a gene encoding the isopropanol dehydrogenase.

6. The isopropanol system for the synthesis of Bosein according to claim 5, characterized in that, The isopropanol dehydrogenase has the amino acid sequence shown in SEQ ID NO: 41, and the mass ratio of the carbonyl reductase mutant to the isopropanol dehydrogenase is 2.0:1.

0.

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

8. The method for preparing Bosein according to claim 7, characterized in that, The mass ratio of isopropanol to β-acetone xyloside is 0.63–0.94:1.

0.

9. The method for preparing Bosein according to claim 7, characterized in that, The mass concentration of β-acetone xyloside in the catalytic system is 47 g / L to 52 g / L.

10. The method for preparing Bosein according to claim 7, characterized in that, The mass ratio of the carbonyl reductase mutant to the isopropanol dehydrogenase is 2.0:1.0.

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