Bosin dehydrogenase with high catalytic activity and high selectivity and application thereof

Abstract

The application discloses a Bose factor dehydrogenase and application thereof. The Bose factor dehydrogenase is a) a sequence as shown in SEQ ID NO: 2 or a sequence as shown in SEQ ID NO: 2; or b) a mutant based on SEQ ID NO: 2 containing one or more than two mutations. The application of the Bose factor dehydrogenase as a catalyst in the production of L-Bose factor can obtain L-Bose factor with chemical purity and optical purity of more than 99%, the reaction condition is mild, the environment is friendly, the operation is simple, the industrial amplification is easy, and the Bose factor dehydrogenase has a good industrial application development prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bioengineering, and particularly relates to a Bose factor dehydrogenase with high catalysis and high selectivity and application thereof. BACKGROUND

[0002] Bose factor, chemical name is hydroxypropyl tetrahydro pyran triol, molecular formula C8H 18 O5, molecular weight is 192.21. Bose factor is a xylose derivative with cosmetic anti-wrinkle effect discovered by L'Oreal for many years, and has wide biological activity. Studies have shown that it can rebuild cell structure by promoting the formation of intercellular mucopolysaccharide, enhance the connectivity of dermal layer and epidermal layer, and improve the binding capacity of dermal layer and epidermal layer, so as to achieve the effect of cosmetic anti-wrinkle. It can increase the tightness of cells and skin, and also help maintain the elasticity of dermis and prevent skin aging. In addition, Bose factor is easy to biodegrade, does not accumulate in the body, has no toxicity, and has increasingly wide application in the fields of biology, medicine, cosmetics and the like.

[0003] Bose factor is a xylose derivative, and it has multiple stereoisomers. Different isomer characteristics have an impact on biological activity. For example, it is mentioned in Synthesis of Pro-Xylane™ : A new biologically active C-glycoside in aqueous media that the importance of β-glycosidic bond to maintain the biological activity of Bose factor is much greater than that of α-glycosidic bond, and the L-hydroxyl on the chiral carbon atom at position 7 of hydroxypropyl tetrahydro pyran triol is the dominant configuration, that is, the Bose factor with both β-glycosidic bond and 7L-hydroxyl has better biological activity.

[0004] In 2002, the international patent application WO002 / 051828A1 of L'Oreal Company first disclosed a Bose factor preparation method. The method takes D-xylose and acetylacetone as raw materials, sodium bicarbonate as base, and water as solvent, and stirs the reaction at 90℃ for 6 hours. Acetylacetone is condensed, cyclized and then cleaved with xylose, and β-ketone xyloside can be obtained in a relatively high yield. Then, the ketone carbonyl of β-ketone xyloside is reduced with sodium borohydride using methanol as solvent, and the hydroxylated product β-ketone xyloside alcohol, i.e. Bose factor, is obtained in a high yield. However, the method has the disadvantages of low reduction selectivity, pollution caused by sodium borohydride, and difficulty in purifying the product.

[0005] The existing synthetic routes for Bosein mainly consist of two steps. The first step, the preparation of xyloside from D-xylose, is relatively straightforward. The challenge lies in the second step: the selective reduction of xyloside. First, the stereoselectivity of chemically reducing the ketone carbonyl group is poor, yielding a pair of enantiomers that are difficult to separate. Second, existing routes often use hazardous reagents such as sodium borohydride and lithium aluminum hydride for catalytic hydrogenation, introducing significant amounts of pollutants. Third, the byproducts generated by these reagents make product separation and purification extremely difficult. For example, the aforementioned route uses sodium borohydride as a reducing agent to reduce the ketone carbonyl group in β-acetone xyloside, producing a large amount of boric acid as a byproduct. Boric acid readily binds to the polyhydroxyl-containing Bosein hinge, requiring column chromatography for purification and hindering mass production.

[0006] Chinese patent application CN111876452A discloses a one-pot method for preparing Bosein using biological enzymes; however, this method uses enzymes of unknown origin, such as isopropanol dehydrogenase, Bosein synthase, and carbonyl reductase, making it unreliable. Chinese patent application CN113717997A discloses an enzyme composition and a chemical enzymatic method for synthesizing Bosein, finding that sorbitol dehydrogenase RDH has the activity to reduce β-pyruvate xyloside; however, the source of this enzyme is unclear, and the substrate concentration is relatively low. Chinese patent application CN115896199A discloses an asymmetric reduction reaction of β-pyruvate xyloside using a short-chain alcohol dehydrogenase from *Lactobacillus kefiri*, which can achieve a high yield but requires the additional introduction of isopropanol dehydrogenase, increasing the reaction cost.

[0007] In summary, the enzymatic reduction of β-pyruvate to synthesize L-Bosein remains challenging, with limitations including low substrate concentration, complex enzyme systems, and high costs. Summary of the Invention

[0008] In view of the above-mentioned technical problems, this application provides a bosine dehydrogenase with high efficiency and high selectivity and its application, wherein the bosine dehydrogenase has high catalytic activity and stereoselectivity.

[0009] The specific technical solution of this application is as follows:

[0010] 1. A bosine dehydrogenase, wherein the bosine dehydrogenase comprises:

[0011] a) Contains a sequence as shown in SEQ ID NO:2 or a sequence as shown in SEQ ID NO:2; or

[0012] b) A mutant based on SEQ ID NO:2 containing one or more mutations.

[0013] 2. The bosine dehydrogenase according to claim 1, wherein the amino acid sequence of the mutant contains an amino acid mutation corresponding to at least one of the sites V71, H90 and S283 of SEQ ID NO:2, preferably containing an amino acid mutation corresponding to the sites V71, H90 and S283 of SEQ ID NO:2.

[0014] 3. A bosine dehydrogenase comprising an amino acid sequence as shown in SEQ ID NO:2 or an amino acid sequence as shown in any one of SEQ ID NO:11-17.

[0015] 4. A nucleic acid molecule encoding any one of items 1-3, namely, bosine dehydrogenase.

[0016] 5. The nucleic acid molecule according to claim 4, wherein the nucleic acid molecule comprises the sequence shown in any one of SEQ ID NO:1 or SEQ ID NO:18-24.

[0017] 6. An expression vector comprising the nucleic acid molecule described in claim 4 or 5;

[0018] Preferably, the expression vector is a plasmid, granule, bacteriophage, or viral vector.

[0019] 7. A host cell comprising the expression vector described in item 6;

[0020] Preferably, the host cell is a bacterium, fungus, plant cell, or animal cell.

[0021] 8. The use of any one of the Bosein dehydrogenases described in items 1-3, the nucleic acid molecule described in item 4 or 5, the expression vector described in item 6, or the host cell described in item 7 in the production of L-Bosein.

[0022] 9. A method for producing L-Bosone, comprising:

[0023] L-Bosein is obtained by reducing β-acetone xyloside with the bosine dehydrogenase according to any one of items 1-3.

[0024] 10. The method according to claim 9, wherein the reduction reaction temperature is 20-60°C, preferably 30-50°C; and / or

[0025] The reduction occurs at a pH of 6.0-10.0, preferably 7.0-8.0; and / or

[0026] The concentration of β-acetone xyloside is 50-1000 mM.

[0027] The effects of the invention

[0028] The Bosein dehydrogenase described in this application has high catalytic activity and good stereoselectivity. When using the Bosein reductase to reduce β-acetone xyloside to synthesize L-Bosein, the reaction conditions are mild, the operation is simple, and it is easy to scale up. Furthermore, the L-Bosein prepared by this reaction has advantages such as high product concentration and high optical and chemical purity, and has good prospects for industrial application. Detailed Implementation

[0029] The present application will now be described in detail. While specific embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0030] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0031] This application provides a bosine dehydrogenase, wherein the bosine dehydrogenase is:

[0032] a) Contains a sequence as shown in SEQ ID NO:2 or a sequence as shown in SEQ ID NO:2; or

[0033] b) A mutant based on SEQ ID NO:2 containing one or more mutations.

[0034] Stereoselectivity refers to the chemical property in which one stereoisomer takes precedence over another in a chemical reaction. In this application, the stereoselective Bosein dehydrogenase refers to the Bosein dehydrogenase that preferentially acts on a specific stereoisomer, such as β-acetone xyloside, thereby enabling the asymmetric synthesis of L-Bosein.

[0035] The amino acid sequence of SEQ ID NO:2 is as follows:

[0036] MKAAVFHEYGKPPLQIEEVPTPEPGPGEVLVKVKACGVCHSDLHIMEGEFPFPPKLPLILGHEVAGEVVEVGEGVTGFKVGDRVVVYFYHSCGQCRYCRKGRENLCENHKWLGFNTDGGYAEYMVVPARNVVKLPDGLDPEEAAPLACAGVTAYHAIKKRAKVRPGETVV VIGCGGLGLMAIQIAKAMGARVIAVDVNEEKLELARQLGADHVINSEDEDAVEQVRELTDGRGVDVVIDFVGSPETFQQALEMLRRGGRLVIVGLSSENLPLNLAQLVLKEISIIGSYAGTRQELREVLDLAARGKIKPVITKRYPLEEVNEAFEKLRNGKITGRVVITPN

[0037] The mutant refers to a mutation relative to the amino acid sequence of SEQ ID NO:2, which includes changes at one or more positions, namely substitution, insertion and / or deletion mutations, and still retains its activity.

[0038] The Bosein dehydrogenase described in this application can be a Bosein dehydrogenase ancestor enzyme constructed using the ancestor sequence reconstruction method, a Bosein dehydrogenase obtained by artificially synthesizing the complete amino acid sequence, and a Bosein dehydrogenase obtained by cloning and expressing it through genetic engineering methods. In this application, the Bosein dehydrogenase uses the amino acid sequence of an alcohol dehydrogenase derived from Rhodococcus as a probe, obtains its ancestor enzyme sequence through ancestor sequence reconstruction, selects root alcohol dehydrogenases and measures their reducing activity, thus obtaining a Bosein dehydrogenase ancestor enzyme with high catalytic activity and stereoselectivity.

[0039] In this application, the ancestral sequence reconstruction (ASR) described is discussed in Randall et al. (Nat. Commun. 7: 12847 doi: 10.1038 / ncomms 12847 (2016)). The authors define ASR as "the process of analyzing modern sequences in an evolutionary / phylogenetic context to infer ancestral sequences at specific nodes in a tree." Ancestor sequence reconstruction (ASR) is used in molecular evolutionary studies. Unlike traditional methods of studying protein evolution by horizontally comparing related protein homologs at the ends of different branches of a phylogenetic tree, ASR probes statistically inferred ancestral proteins within tree nodes in a vertical manner. A phylogenetic tree is a branching graph showing evolutionary relationships between multiple biological species or other entities based on similarities and differences in their physical or genetic characteristics. In a rooted phylogenetic tree, each node with offspring represents the inferred most recent common ancestor of those offspring. In ASR, multiple related homologs of the target protein are selected and aligned using multiple sequence alignment (MSA) to construct a phylogenetic tree with statistically inferred sequences at the nodes of the branches. These sequences are known as "ancestors." The process of synthesizing the corresponding DNA, converting it into cells, and producing proteins is called "reconstruction."

[0040] Ancestor sequences are typically calculated using maximum likelihood, although the Bayesian method can also be performed. Since ancestors are inferred from phylogeny, the topology and composition of the phylogeny play a major role in the output ASR sequence. An ASR does not claim to reconstruct the actual sequence of an ancient protein / DNA, but rather a sequence likely similar to the sequence at that node. Maximum likelihood (ML) works by generating sequences where residues at each position are predicted to be most likely to occupy that position using the inference method used. Typically, this is a score matrix calculated from existing sequences (similar to those used in BLAST or MSA). Alternative methods include maximum parsimony (MP), which constructs sequences based on sequence evolution models, where the concept of the minimum number of nucleotide sequence changes typically represents the most efficient and most probable evolutionary pathway. MP is often considered the least reliable reconstruction method because it can oversimplify evolution to a degree unsuitable for a billion-year scale. Other methods include the Bayesian method, which involves considering residue uncertainties. Such methods are sometimes used to supplement ML methods, but they typically produce more ambiguous sequences (i.e., sequences containing residue positions for which definitive substitutions cannot be predicted). In such cases, multiple ASR sequences covering most of the ambiguity are usually generated and compared with each other. In some implementations, ancestor sequence reconstruction is performed using the online software FireProt-ASR (FireProt-ASR (muni.cz)).

[0041] The bosonic dehydrogenase described in this application is derived from Rhodococcus. The bosonic dehydrogenase is obtained by ancestral sequence reconstruction. The ancestral enzyme of bosonic dehydrogenase is constructed, and the bosonic dehydrogenase obtained by artificially synthesizing the full amino acid sequence is cloned and expressed by genetic engineering methods.

[0042] In this process, after constructing the progenitor enzyme of bosoxane dehydrogenase, the cloned enzymes were repeatedly compared and screened by measuring and comparing the activities of the hydrolases, and finally the progenitor enzyme of bosoxane dehydrogenase with the best catalytic performance was obtained, whose amino acid sequence is shown in SEQ ID NO:2.

[0043] After obtaining the optimal progenitor enzyme of bosonic dehydrogenase, the full-length gene sequence was obtained through codon optimization based on the corresponding amino acid sequence. This gene sequence was then delivered to a gene synthesis company for artificial synthesis. After obtaining the gene, it was amplified using PCR, and the sequence was ligated into pET28a. The primers used are as follows:

[0044] Upstream primer:

[0045] 5'-gtgccgcgcggcagccatatg ATGAAAGCCGCGGTTTTTC-3' (SEQ ID NO:3)

[0046] Downstream primer:

[0047] 5'-acggagctcgaattc ggatcc TTAGTTTGGCGTAATAACCACACG-3' (SEQ ID NO:4)

[0048] The underlined portion of the upstream primer nucleotide sequence is... Nde I restriction site, the underlined part of the downstream primer is... BamH I. Restriction site. Then, using the artificially synthesized gene as a template, the gene was amplified by polymerase chain reaction (PCR) to obtain the complete full-length Bosein dehydrogenase gene DNA fragment. The full-length Bosein dehydrogenase gene (nucleotide sequence as shown in SEQ ID NO.1 in the sequence listing) is named... phd The gene is 1026 nucleotides in length. Its coding sequence, from the first base to the 1026th base, has the start codon ATG and the stop codon TAA. This sequence contains no introns. The amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO.2 of the sequence listing.

[0049] The sequence of SEQ ID NO:1 is as follows:

[0050]

[0051] Due to codon degeneracy, nucleic acid molecules encoding the aforementioned bosonic dehydrogenase (amino acid sequence as shown in SEQ ID NO.2) are not limited to those with sequences as shown in SEQ ID NO.1. A polynucleotide homologue can also be provided by appropriately introducing substitutions, deletions, alterations, insertions, or additions of nucleotides.

[0052] In some embodiments, the amino acid sequence of the mutant contains an amino acid mutation corresponding to at least one of the sites V71, H90 and S283 of SEQ ID NO:2, preferably containing an amino acid mutation corresponding to the sites V71, H90 and S283 of SEQ ID NO:2.

[0053] In this application, the positions mentioned above are counted starting from the N-terminus. For example, V71 refers to the 71st amino acid mutation starting from the N-terminus of SEQ ID NO:2.

[0054] The term "corresponds" has the meaning commonly understood by those skilled in the art. Specifically, "corresponds" means the position in one sequence that corresponds to a specified position in another sequence after two sequences have been aligned for homology or sequence identity.

[0055] In this application, the valine V at position 71 can be mutated to threonine T, the histidine H at position 90 can be mutated to alanine A, or the serine S at position 283 can be mutated to threonine T; preferably, the V at position 71 can be mutated to T, the H at position 90 can be mutated to A, and the S at position 283 can be mutated to T.

[0056] In this application, V71T refers to the mutation of valine V at position 71 to threonine T, and H90A and S283T have a similar meaning;

[0057] V71T\H90A refers to the mutation of valine V at position 71 to threonine T and histidine H at position 90 to alanine A. Similarly, V71T\S283T and H90A\S283T have similar meanings.

[0058] V71T\H90A\S283T refers to mutating the 71st bit V to T, the 90th bit H to A, and the 283rd bit S to T.

[0059] The sequence in which valine V at position 71 is mutated to threonine T is shown in SEQ ID NO:11, and the corresponding nucleic acid sequence is shown in SEQ ID NO:18.

[0060] The sequence of mutating histidine H at position 90 to alanine A is shown in SEQ ID NO:12, and the corresponding nucleic acid sequence is shown in SEQ ID NO:19.

[0061] The sequence of mutating serine S at position 283 to threonine T is shown in SEQ ID NO:13, and the corresponding nucleic acid sequence is shown in SEQ ID NO:20.

[0062] The sequence of mutating valine V at position 71 to threonine T and histidine H at position 90 to alanine A is shown in SEQ ID NO:14, and the corresponding nucleic acid sequence is shown in SEQ ID NO:21.

[0063] The sequence of mutating valine V at position 71 to threonine T and serine S at position 283 to threonine T is shown in SEQ ID NO:15, and the corresponding nucleic acid sequence is shown in SEQ ID NO:22.

[0064] The sequence of mutating histidine H at position 90 to alanine A and serine S at position 283 to threonine T is shown in SEQ ID NO:16, and the corresponding nucleic acid sequence is shown in SEQ ID NO:23.

[0065] The sequence of mutating V at position 71 to T, H at position 90 to A, and S at position 283 to T is shown in SEQ ID NO:17, and the corresponding nucleic acid sequence is shown in SEQ ID NO:24.

[0066] The amino acid sequence of SEQ ID NO:11 is as follows:

[0067] MKAAVFHEYGKPPLQIEEVPTPEPGPGEVLVKVKACGVCHSDLHIMEGEFPFPPKLPLILGHEVAGEVVETGEGVTGFKVGDRVVVYFYHSCGQCRYCRKGRENLCENHKWLGFNTDGGYAEYMVVPARNVVKLPDGLDPEEAAPLACAGVTAYHAIKKRAKVRPGETVV VIGCGGLGLMAIQIAKAMGARVIAVDVNEEKLELARQLGADHVINSEDEDAVEQVRELTDGRGVDVVIDFVGSPETFQQALEMLRRGGRLVIVGLSSENLPLNLAQLVLKEISIIGSYAGTRQELREVLDLAARGKIKPVITKRYPLEEVNEAFEKLRNGKITGRVVITPN

[0068] The sequence of SEQ ID NO:12 is as follows:

[0069] MKAAVFHEYGKPPLQIEEVPTPEPGPGEVLVKVKACGVCHSDLHIMEGEFPFPPKLPLILGHEVAGEVVEVGEGVTGFKVGDRVVVYFYASCGQCRYCRKGRENLCENHKWLGFNTDGGYAEYMVVPARNVVKLPDGLDPEEAAPLACAGVTAYHAIKKRAKVRPGETVVVIGCGGLGLMAIQIAKAMGARVIAVDVNEEKLELARQLGADHVINSEDEDAVEQVRELTDGRGVDVVIDFVGSPETFQQALEMLRRGGRLVIVGLSSENLPLNLAQLVLKEISIIGSYAGTRQELREVLDLAARGKIKPVITKRYPLEEVNEAFEKLRNGKITGRVVITPN

[0070] The sequence of SEQ ID NO:13 is as follows:

[0071] MKAAVFHEYGKPPLQIEEVPTPEPGPGEVLVKVKACGVCHSDLHIMEGEFPFPPKLPLILGHEVAGEVVEVGEGVTGFKVGDRVVVYFYHSCGQCRYCRKGRENLCENHKWLGFNTDGGYAEYMVVPARNVVKLPDGLDPEEAAPLACAGVTAYHAIKKRAKVRPGETVVVIGCGGLGLMAIQIAKAMGARVIAVDVNEEKLELARQLGADHVINSEDEDAVEQVRELTDGRGVDVVIDFVGSPETFQQALEMLRRGGRLVIVGLSSENLPLNLAQLVLKEITIIGSYAGTRQELREVLDLAARGKIKPVITKRYPLEEVNEAFEKLRNGKITGRVVITPN

[0072] The sequence of SEQ ID NO:14 is as follows:

[0073] MKAAVFHEYGKPPLQIEEVPTPEPGPGEVLVKVKACGVCHSDLHIMEGEFPFPPKLPLILGHEVAGEVVETGEGVTGFKVGDRVVVYFYASCGQCRYCRKGRENLCENHKWLGFNTDGGYAEYMVVPARNVVKLPDGLDPEEAAPLACAGVTAYHAIKKRAKVRPGETVVVIGCGGLGLMAIQIAKAMGARVIAVDVNEEKLELARQLGADHVINSEDEDAVEQVRELTDGRGVDVVIDFVGSPETFQQALEMLRRGGRLVIVGLSSENLPLNLAQLVLKEISIIGSYAGTRQELREVLDLAARGKIKPVITKRYPLEEVNEAFEKLRNGKITGRVVITPN

[0074] The sequence of SEQ ID NO:15 is as follows:

[0075] MKAAVFHEYGKPPLQIEEVPTPEPGPGEVLVKVKACGVCHSDLHIMEGEFPFPPKLPLILGHEVAGEVVETGEGVTGFKVGDRVVVYFYHSCGQCRYCRKGRENLCENHKWLGFNTDGGYAEYMVVPARNVVKLPDGLDPEEAAPLACAGVTAYHAIKKRAKVRPGETVVVIGCGGLGLMAIQIAKAMGARVIAVDVNEEKLELARQLGADHVINSEDEDAVEQVRELTDGRGVDVVIDFVGSPETFQQALEMLRRGGRLVIVGLSSENLPLNLAQLVLKEITIIGSYAGTRQELREVLDLAARGKIKPVITKRYPLEEVNEAFEKLRNGKITGRVVITPN

[0076] The sequence of SEQ ID NO:16 is as follows:

[0077] MKAAVFHEYGKPPLQIEEVPTPEPGPGEVLVKVKACGVCHSDLHIMEGEFPFPPKLPLILGHEVAGEVVEVGEGVTGFKVGDRVVVYFYASCGQCRYCRKGRENLCENHKWLGFNTDGGYAEYMVVPARNVVKLPDGLDPEEAAPLACAGVTAYHAIKKRAKVRPGETVVVIGCGGLGLMAIQIAKAMGARVIAVDVNEEKLELARQLGADHVINSEDEDAVEQVRELTDGRGVDVVIDFVGSPETFQQALEMLRRGGRLVIVGLSSENLPLNLAQLVLKEITIIGSYAGTRQELREVLDLAARGKIKPVITKRYPLEEVNEAFEKLRNGKITGRVVITPN

[0078] The sequence of SEQ ID NO:17 is as follows:

[0079] MKAAVFHEYGKPPLQIEEVPTPEPGPGEVLVKVKACGVCHSDLHIMEGEFPFPPKLPLILGHEVAGEVVETGEGVTGFKVGDRVVVYFYASCGQCRYCRKGRENLCENHKWLGFNTDGGYAEYMVVPARNVVKLPDGLDPEEAAPLACAGVTAYHAIKKRAKVRPGETVVVIGCGGLGLMAIQIAKAMGARVIAVDVNEEKLELARQLGADHVINSEDEDAVEQVRELTDGRGVDVVIDFVGSPETFQQALEMLRRGGRLVIVGLSSENLPLNLAQLVLKEITIIGSYAGTRQELREVLDLAARGKIKPVITKRYPLEEVNEAFEKLRNGKITGRVVITPN

[0080] The sequence of SEQ ID NO:18 is as follows:

[0081]

[0082] The sequence of SEQ ID NO:19 is as follows:

[0083]

[0084] The sequence of SEQ ID NO:20 is as follows:

[0085]

[0086] The sequence of SEQ ID NO:21 is as follows:

[0087]

[0088] The sequence of SEQ ID NO:22 is as follows:

[0089]

[0090] The sequence of SEQ ID NO:23 is as follows:

[0091]

[0092] The sequence of SEQ ID NO:24 is as follows:

[0093]

[0094] This application does not impose any limitations on the mutation method; mutations can be performed according to conventional methods in the art, such as directed mutagenesis, random mutagenesis, or the construction of synthetic oligonucleotides. The mutated DNA sequence is then expressed in a host cell to obtain mutants with amino acid substitutions, insertions, and / or deletions. In this application, directed mutagenesis is used. For example, to mutate V at position 71 to T, the primer sequence used is:

[0095] Upstream primer: GAAGTCGTGGAGACCGGTGAAGGAGTA (SEQ ID NO:5)

[0096] Downstream primer: TACTCCTTCACCGGTCTCCACGACTTC (SEQ ID NO:6)

[0097] The primer sequence used to mutate the 90th position H to A is:

[0098] Upstream primer: GTCTATTTCTATGCATCTTGTGGCCAA (SEQ ID NO:7)

[0099] Downstream primer: TTGGCCACAAGATGCATAGAAATAGAC (SEQ ID NO:8)

[0100] The primer sequence used to mutate the S at position 283 to T is:

[0101] Upstream primer: TTGAAGGAAATTACCATCATCGGTTCT (SEQ ID NO:9)

[0102] Downstream primer: AGAACCGATGATGGTAATTTCCTTCAA (SEQ ID NO:10)

[0103] The Bosein dehydrogenase obtained in this application has high enzyme activity, which improves the industrial application potential of the enzyme.

[0104] The mutant described in this application has more than 90% homology with SEQ ID NO:2.

[0105] This application provides a nucleic acid molecule encoding the aforementioned bosine dehydrogenase. In some embodiments, the nucleic acid molecule comprises the sequence shown in SEQ ID NO:1 or any one of SEQ ID NO:18-24.

[0106] This application provides an expression vector comprising the nucleic acid molecules described above.

[0107] In this application, the expression vector is constructed by cloning the above-mentioned bosine dehydrogenase gene into the expression vector using conventional methods in the art. The expression vector includes various conventional vectors in the art, such as commercially available plasmids, granules, bacteriophages or viral vectors, etc., preferably pET-28a plasmid.

[0108] In this application, the term "clay particle" refers to a sticky particle.

[0109] For example, expression vectors can be prepared using the following methods:

[0110] The Bosein dehydrogenase gene product obtained by PCR amplification was digested with restriction endonucleases NdeI and BamHI. Simultaneously, the expression vector, such as pET-28a, was also digested with NdeI and BamHI to form complementary sticky ends. The digested Bosein dehydrogenase gene product and the digested expression vector, such as pET-28a, were recovered and ligated using T4 DNA ligase to construct an expression vector, such as pET28a-, containing the Bosein dehydrogenase gene. phd .

[0111] This application provides a host cell that includes the expression vector described above.

[0112] In this application, the host cell is a conventional host cell in the art, as long as the expression vector can stably replicate itself and the bosine dehydrogenase gene it carries can be effectively expressed. The host cell can be, for example, bacteria, fungi, plant cells, animal cells, etc.

[0113] The bacteria are preferably Escherichia coli, and more preferably Escherichia coli. E. coli BL21(DE3) or Escherichia coli E. coli DH5α.

[0114] In this application, the expression vector, such as pET28a- phd Transformed into host cells such as Escherichia coli E. coli The host cell, Escherichia coli, can be obtained from BL21(DE3). E. coli BL21(DE3) / pET28a- phd .

[0115] This application provides a method for preparing bosine dehydrogenase, which includes inoculating the host cells described above into a culture medium for fermentation to obtain a fermentation broth, centrifuging the fermentation broth to collect the bacterial cells, and breaking the bacterial cells to obtain bosine dehydrogenase.

[0116] The culture medium can be any culture medium in the art that can grow the transformant and produce bosine dehydrogenase. For example, the culture medium can be LB medium, preferably, the components of which include: 5-15 g / L peptone, 1-10 g / L yeast extract, 5-15 g / L NaCl, and pH 6.0-8.0.

[0117] In this application, there are no special restrictions on culture methods and conditions. Appropriate selections can be made according to general knowledge in the art, based on factors such as host cell type and culture method, as long as the transformant can grow and produce bosine dehydrogenase. Other specific operations for culturing transformants can be performed according to conventional procedures in the art.

[0118] For example, the strain culture method includes: culturing the host cells (e.g. E. coli BL21(DE3) was inoculated into LB medium containing kanamycin and cultured. When the optical density OD of the culture medium reached a certain level... 600 When the concentration reaches 0.6-0.8 (preferably 0.6), bosine dehydrogenase can be efficiently expressed under the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 0.1-1.0 mmol / L (preferably 0.2 mmol / L).

[0119] This application provides the use of the above-described bosoxane dehydrogenase, the above-described nucleic acid molecule, the above-described expression vector, or the above-described host cell in the production of L-bosoxane.

[0120] The bosine dehydrogenase described in this application has stereoselectivity, which allows for the asymmetric synthesis of L-bosine.

[0121] This application provides a method for producing L-Bosonic, comprising:

[0122] L-Bosein is obtained by reducing β-acetone xyloside using the Bosein dehydrogenase described above. In some embodiments, the reduction reaction temperature is 20-60°C, preferably 30-50°C.

[0123] For example, the temperature of the reduction reaction can be 20℃, 25℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 55℃, 60℃, etc.

[0124] In some embodiments, the reduction reaction is carried out at a pH of 6.0-10.0, preferably 7.0-8.0.

[0125] For example, the pH of the reduction reaction can be 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, etc.

[0126] In some embodiments, L-Bosein is obtained by reducing β-acetone xyloside with the bosine dehydrogenase described above in a buffer system. Preferably, the buffer is sodium phosphate buffer, Tris-HCl buffer, or glycine-NaOH buffer.

[0127] In some embodiments, the aforementioned Bosein dehydrogenase is dissolved in a buffer solution, and β-acetone xyloside is added to a final concentration of 50-1000 mM. The reaction is carried out at 20-60°C with mechanical stirring until the substrate conversion is close to 99%. After the reaction, L-Bosein is obtained by enzyme removal, resin separation, activated carbon decolorization, and recrystallization, with both chemical and optical purity >99%.

[0128] Using the aforementioned bosine dehydrogenase for reduction, L-bosine with a chemical purity and optical purity of over 99% can be obtained. Furthermore, the reaction conditions are mild, the operation is relatively simple, and it has excellent prospects for industrial application.

[0129] Example

[0130] This application provides a general and / or specific description of the materials and test methods used in the experiments. In the following examples, unless otherwise specified, % represents wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0131] Example 1: Cloning of the bosonic dehydrogenase gene pdh

[0132] The ancestral enzyme of bosine dehydrogenase, constructed using conventional ancestral sequence reconstruction methods in this field, has the amino acid sequence shown in SEQ ID NO:2. Its encoding gene was obtained through codon optimization, and the full-length sequence was further synthesized artificially by a gene synthesis company. PCR primers were designed as follows:

[0133] Upstream primer:

[0134] 5'-gtgccgcgcggcagc catatg ATGAAGGCAATCCAATACACCC-3'

[0135] Downstream primer:

[0136] 5'-acggagctcgaattc ggatcc CTATAATCCCGGGACCACAACC-3'

[0137] The underlined part of the upstream primer is... Nde I restriction site, the underlined part of the downstream primer is... BamH I restriction site.

[0138] Using the DNA of the artificially synthesized progenitor enzyme of bosine dehydrogenase as a template, PCR amplification was performed. The PCR system consisted of 10 μL of 2×Taq PCR MasterMix, 1 μL each of upstream and downstream primers (0.3 μmol / L), 1 μL (0.1 μg) of DNA template, and 7 μL of ddH2O. The PCR amplification program was as follows: (1) 95℃ pre-denaturation for 3 min; (2) 94℃ denaturation for 30 s; (3) 55℃ annealing for 30 s; (4) 72℃ extension for 2 min; steps (2) to (4) were repeated for 30 cycles; (5) 72℃ extension for 10 min, followed by cooling to 4℃. The PCR product was purified by agarose gel electrophoresis and recovered using an agarose gel DNA recovery kit. A complete full-length gene sequence of bosine dehydrogenase was obtained, which was sequenced to be 1047 bp in length and named as follows. pdh The nucleotide sequence of the gene is shown in SEQ ID NO.1 of the sequence listing.

[0139] Example 2 Preparation of Bosein dehydrogenase expression vector and expression transformant

[0140] The Bosein dehydrogenase gene DNA fragment and pET-28a empty plasmid obtained in Example 1 were subjected to restriction endonuclease at 37°C. Nde I and BamH I Double digestion for 2 h, followed by agarose gel electrophoresis purification. The target fragment was recovered using an agarose gel DNA recovery kit (Jierui Biotechnology Co., Ltd.). The target fragment was ligated overnight at 4°C using T4 DNA ligase to obtain the expression plasmid pET28a- pdh .

[0141] The above expression plasmid was transformed into Escherichia coli. E. coli In DH5α competent cells, positive clones were selected on kanamycin-containing resistant plates, and single clones were picked for colony PCR verification. The cells were cultured, and after plasmid amplification, the plasmid was extracted and re-transformed into *Escherichia coli*. E. coli In BL21(DE3) competent cells, the transformation solution was spread onto LB agar plates containing kanamycin and incubated overnight at 37°C inverted to obtain positive transformants of Escherichia coli. E. coli BL21(DE3) / pET28a- pdh Positive clones were verified by colony PCR and gene sequencing.

[0142] Example 3: Preparation of crude Bosein dehydrogenase solution

[0143] The positive colonies obtained in Example 2 were inoculated into LB medium and cultured for 12 h to obtain a seed culture. The seed culture was then transferred to 100 mL of fresh LB medium and cultured until the OD reached. 600 When the concentration reaches 0.6-0.8, lactose or IPTG is added as an inducer to a final concentration of 0.2 mM, and the cells are cultured at 25°C for 12 h. The culture medium is removed by centrifugation to obtain cells expressing recombinant Bosein dehydrogenase. The cells are then freeze-dried to obtain crude enzyme powder. 0.1 g of crude enzyme powder is weighed and added to 10 mL of phosphate buffer (100 mM, pH 7.0). The cells are then sonicated to release intracellular Bosein dehydrogenase. The supernatant obtained after centrifugation at 4°C and 8000 r / min for 10 min is the crude enzyme solution of Bosein dehydrogenase.

[0144] Example 4: Determination of Bosein dehydrogenase

[0145] The activity assay was performed as follows: A 200 μL reaction system (100 mmol / L sodium phosphate buffer, pH 7.0) containing 2 mmol / L β-pyruvate xyloside and 0.5 mM NADPH was incubated at 30°C for 2 min. Then, an appropriate amount of the crude enzyme solution prepared in Example 3 was added, and the mixture was rapidly mixed. The change in absorbance at 340 nm was monitored. The activity was determined by measuring the OD value of the reaction system within 3 min. 340 The enzyme activity is calculated by the change in the value of the enzyme activity, where enzyme activity (U) is defined as the amount of enzyme required to catalyze the hydrolysis of 1 μmol β-acetone xyloside per minute under the above conditions.

[0146] The specific activity of bosine dehydrogenase (PDH) against β-acetone xyloside was determined to be 8.5 U / mg.

[0147] Example 5: Optimization of the reduction reaction of β-acetone xyloside catalyzed by bosine dehydrogenase

[0148] The effects of reaction temperature and pH on the synthesis of L-BOXOR from β-pyruvate catalyzed by BOXOR dehydrogenase PDH were investigated. The substrate concentration used was 50 mM, the reaction time was 12 h, and the enzyme addition was fixed at 1 g / L.

[0149] First, the reaction pH was fixed at 7.0, and the effect of catalytic reaction under the conditions of 20℃, 30℃, 40℃, 50℃ and 60℃ was investigated. The results are shown in Table 1.

[0150] As can be seen from Table 1, the reaction has a high conversion rate between 30-50℃, and the highest conversion rate of 94% is achieved when the reaction temperature is 30℃ and the pH is 7.0.

[0151] Then, with the reaction temperature fixed at 30℃, the effect of catalytic reaction under pH conditions of 6.0, 7.0, 8.0, 9.0 and 10.0 was investigated, and the results are shown in Table 1.

[0152] As can be seen from Table 1, the highest conversion rate of 94% was achieved at a reaction temperature of 30℃ and a pH of 7.0.

[0153] In summary, the optimal reaction temperature for bosine dehydrogenase is 30℃, and the optimal reaction pH is 7.0.

[0154] Table 1. Effects of different temperatures and pH on the enzymatic hydrolysis of bosine dehydrogenase.

[0155]

[0156] Example 6: Preparation of Bosein dehydrogenase mutant

[0157] A structural model of bosoxane dehydrogenase PDH was constructed using conventional methods in the art. After inserting substrate pairs into the active site, the interaction between bosoxane dehydrogenase PDH and the substrate was analyzed. Furthermore, a mutation was introduced to enhance the interaction between the enzyme and the substrate using conventional methods in the art.

[0158] The full-length gene sequence of the bosonic dehydrogenase PDH obtained in Example 1 (nucleotide sequence as shown in SEQ ID NO. 1) was mutated by three bases. The mutation positions were: V at position 71 of the bosonic dehydrogenase gene coding sequence was mutated to T, H at position 90 was mutated to A, and S at position 283 was mutated to T. Primers SEQ ID NO: 5-10 were used to mutate the sequence, pET28a- pdhUsing DNA as a template, single mutants V71T, H90A, and S283T, double mutants V71T\H90A, V71T\S283T, H90A\S283T, and triple mutants V71T\H90A\S283T were constructed. The PCR system consisted of 10 μL of 2×Taq PCR MasterMix, 1 μL each of upstream and downstream primers (0.3 μmol / L), 1 μL (0.1 μg) of DNA template, and 7 μL of ddH2O. The PCR amplification program was as follows: (1) 95℃ for pre-denaturation for 5 min; (2) 94℃ for denaturation for 30 s; (3) 55℃ for annealing for 30 s; (4) 72℃ for extension for 6 min; steps (2) to (4) were repeated for 20 cycles; (5) 72℃ for further extension for 10 min, followed by cooling to 4℃. The rapid endonuclease DpnI was added to the PCR system to eliminate template interference. Recombinant transformants of the mutant enzyme were constructed according to Example 2. The nucleic acid sequences of mutants V71T, H90A, S283T, V71T\H90A, V71T\S283T, H90A\S283T, and V71T\H90A\S283T are shown in SEQ ID: 18-24, and the amino acid sequences of mutants V71T, H90A, S283T, V71T\H90A, V71T\S283T, H90A\S283T, and V71T\H90A\S283T are shown in SEQ ID: 11-17. After successful mutant construction, crude mutant enzyme solution was prepared according to the methods described in Examples 2-3.

[0159] Example 7: Bosein dehydrogenase mutant catalyzes reduction reactions of different concentrations of β-pyruvate xyloside

[0160] The effect of the Bosein dehydrogenase mutant catalyst prepared in Example 6 on the reduction reaction of β-acetone xyloside to L-Bosein was further investigated. The reaction was carried out for 6 h at a substrate concentration of 50 mM, a catalyst amount of 1 g / L, a reaction temperature of 30 °C, and a pH of 7.0. The conversion results are shown in Table 2.

[0161] Table 2 shows that the transformation rate of the wild type under these conditions was 94%. The transformation rates of the single mutants V71T, H90A, and S283T were higher than those of the wild type, reaching 96%, 95%, and 98%, respectively, demonstrating that the above mutations all made a positive contribution to improving the transformation rate. Further double mutants were constructed, with V71T\H90A, V71T\S283T, and H90A\S283T showing slightly higher transformation rates than the single mutants, reaching 97%, 98%, and 98%, respectively. The triple mutant V71T\H90A\S283T had the highest transformation rate among all mutants, with a transformation rate greater than 99% after 6 hours of reaction.

[0162] Table 2 Comparison of catalytic performance of different Bosein dehydrogenase mutants

[0163]

[0164] Furthermore, the effects of the three mutants V71T\H90A\S283T on the conversion reaction at substrate concentrations of 50 mM, 100 mM, 200 mM, 400 mM and 1000 mM were investigated at 30℃ and pH 7.0. The results are shown in Table 3.

[0165] As shown in Table 3, when the substrate concentration was 50 mM, the conversion rate reached >99% after 6 hours of reaction, higher than the wild type's 94%, indicating that the mutant exhibits better substrate conversion ability. Furthermore, with increasing substrate concentration, the reaction required a longer time to achieve a conversion rate >99%. Nevertheless, even at a substrate concentration of 1000 mM, a conversion rate >99% was still achieved after 24 hours of reaction. This represents the highest level of enzymatic synthesis of L-Bosoxane to date.

[0166] Table 3. Comparison of catalytic performance of Bosein dehydrogenase mutants V71T, H90A, and S283T

[0167]

[0168] Example 8: Preparation of L-Bosoxane Catalyzed by Bosoxane Dehydrogenase Mutant

[0169] The crude enzyme solution of the PDH mutants (V71T, H90A, S283T) prepared in Example 6 was added to 100 mL of sodium phosphate buffer (100 mmol / L, pH 7.0) to a final concentration of 20 g / L, followed by the addition of β-pyruvyl xyloside to a final concentration of 1 mol / L. The reaction was allowed to proceed until the substrate was completely transformed. After the reaction, cells were removed by centrifugation, and the L-Bosein product was separated by molecular sieve resin. L-Bosein was obtained by decolorization with activated carbon and recrystallization, with a product yield of 92% and a chemical purity >99%.

[0170] The above description is merely a preferred embodiment of this application and is not intended to limit the application in any other way. Any person skilled in the art may make changes or modifications to the disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this application without departing from the scope of the technical solution of this application shall still fall within the protection scope of this application.

Claims

1. A Bos taurus somatotropic hormone dehydrogenase, the amino acid sequence of which is shown as SEQ ID NO: 2 or as shown in any one of SEQ ID NO: 11-17. 2.A nucleic acid molecule encoding the Bos taurus somatotropic hormone dehydrogenase of claim 1.

3. The nucleic acid molecule of claim 2, wherein, the sequence of which is shown as SEQ ID NO: 1 or as shown in any one of SEQ ID NO: 18-24. 4.An expression vector comprising the nucleic acid molecule of claim 2 or 3. 5.The expression vector of claim 4, wherein the expression vector is a plasmid. 6.The expression vector of claim 4, wherein the expression vector is a cosmid. 7.The expression vector of claim 4, wherein the expression vector is a bacteriophage. 8.The expression vector of claim 4, wherein the expression vector is a viral vector. 9.A host cell comprising the expression vector of any one of claims 4-8. 10.The host cell of claim 9, wherein the host cell is a bacterium, a fungus, a plant cell or an animal cell. 11.Use of the Bos taurus somatotropic hormone dehydrogenase of claim 1, the nucleic acid molecule of claim 2 or 3, the expression vector of any one of claims 4-8 or the host cell of claim 9 or 10 in the production of L-somatotropin. 12.A method for producing L-somatotropin, comprising: reducing β-ketosylulose to obtain L-somatotropin using the Bos taurus somatotropic hormone dehydrogenase of claim 1.

13. The method of claim 12, wherein, the reaction temperature for the reduction is 20-60℃; and / or the reaction pH for the reduction is 6.0-10.0; and / or the concentration of β-ketosylulose is 50-1000mM. 14.The method of claim 12, wherein the reaction temperature for the reduction is 30-50℃; and / or the reaction pH for the reduction is 7.0-8.0.

Images