CN116240183A - Dehydrogenase, gene, engineering bacteria and application - Google Patents

Dehydrogenase, gene, engineering bacteria and application Download PDF

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CN116240183A
CN116240183A CN202310014700.5A CN202310014700A CN116240183A CN 116240183 A CN116240183 A CN 116240183A CN 202310014700 A CN202310014700 A CN 202310014700A CN 116240183 A CN116240183 A CN 116240183A
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丁波
孙超
刘会川
孙浩
罗梃楷
曾春玲
刘喜荣
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Hunan Xinhexin Biological Medicine Co ltd
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Abstract

The invention belongs to the technical field of steroid medicine biocatalysis synthesis, and in particular relates to dehydrogenase, gene, engineering bacteria and application, wherein the amino acid sequence of the dehydrogenase is SEQ ID NO.1 or SEQ ID NO. 2; the constructed engineering bacteria can efficiently secrete the dehydrogenase, effectively improve the conversion efficiency of a substrate, and particularly has low cost and obvious industrialization potential for the substrate 6-A format (11 beta, 17 alpha-dihydroxyl-6 alpha-methyl pregna-1, 4-diene-3, 20-dione); the dehydrogenase mutant has definite physicochemical properties, omits complicated steps of enzyme purification by a whole-cell catalysis method, reduces the cost and is simple and convenient to operate.

Description

Dehydrogenase, gene, engineering bacteria and application
Technical Field
The invention belongs to the technical field of steroid medicine biocatalysis synthesis, and particularly relates to dehydrogenase, gene, engineering bacteria and application thereof.
Background
Steroid compounds are also called steroids, and have important roles in regulating metabolism, protein synthesis, reproductive function and the like of human bodies, and mainly comprise plant sterols, bile acids, insect allergies, cardiac glycosides, steroid saponins, steroid alkaloids and the like. Steroid drugs are the second most important class of drugs that are clinically used next to antibiotics.
At present, the synthesis of steroid drugs by a chemical method often has the defects of complicated steps, high cost, residual reaction solvents and the like. Compared with chemical synthesis, the biological transformation of the steroid medicine has the advantages of simple steps, strong specificity, mild reaction conditions, no pollution and the like, so the biological synthesis has become an important way for industrially synthesizing the steroid medicine.
Methylprednisolone (11 beta, 17 alpha, 21-trihydroxy-6 alpha-methylprednisolone-1, 4-diene-3, 20-dione) and derivatives thereof are high-efficiency adrenocorticoid steroids for resisting immune stress reaction, and are widely applied to organ transplantation and clinical application for resisting immune stress reaction. The medicine is an artificially synthesized glucocorticoid, and has typical glucocorticoid action such as antiinflammatory effect, and immune response inhibiting effect. Compared to the glucocorticoid representative hydrocortisone, it has an anti-inflammatory efficacy 4 times that of hydrocortisone. The anti-inflammatory effect of the steroid mother nucleus C1 and 2 can be multiplied after double bonds are introduced by dehydrogenation, and the method is an important reaction process in the industrial production of corticoids. For example, the anti-inflammatory effect of the acetate dehydrocortisone generated after C1,2 dehydrogenation is improved by 3-4 times compared with that of the acetate dehydrocortisone generated before. The chemical method usually adopts a selenium dioxide method to dehydrogenate C1 and C2, but has obvious side effects, so that selenium harmful to human bodies is often contained in the product. Therefore, biological dehydrogenation gradually becomes a key step for synthesizing steroidal anti-inflammatory hormone drugs due to the advantages of green, environmental protection, strong specificity and the like.
In recent years, research progress on biological dehydrogenation of methylprednisolone in China is not remarkable. CN1978457a discloses a method for biological dehydrogenation by using arthrobacter in the process of synthesizing methylprednisolone, but the biological dehydrogenation reaction process is longer (more than 48 h), and the conversion rate is not high (about 85%), so that the industrial production is difficult. The catalytic activity of the 3-ketosteroid-delta 1-dehydrogenase (KstD) on steroid substrates reported in the current literature is low, and in addition, the substrate solubility is low, so that the substrate concentration in practical application is low, and the requirement of industrial production cannot be met. Meanwhile, steroid C1,2 dehydrogenase with higher catalytic activity is not found for certain substrates with higher economic value, such as methylprednisolone. The above problems severely restrict the development of the enzyme, and therefore, it is important to find novel steroid C1,2 dehydrogenase having high activity and good thermostability.
Disclosure of Invention
The technical problem to be solved by the invention is to provide dehydrogenase, gene, engineering bacteria and application, so that the conversion rate of converting steroid hormone is improved, and the impurity content is reduced.
One embodiment of the invention includes a dehydrogenase having an amino acid sequence of SEQ ID NO.1 or SEQ ID NO.2.
SEQ ID NO.1 sequence is:
MDWAEEYDVL VAGSGAGGMA GTYTAAREGL SVCLVEAGDK FGGTTAYSGG CGAWFPANPVLLRAGTDDTI EDALEYYRAV VGDRTPADLQ ETYVRGGAGL VAYLEEDDHF SFESYPWPDYFGDAPKARRD GQRHIIPTPL PVPSAPELRE VVRGPLDNDR LGTPQPDDLF IGGRALVARFLTALATYPHA TLVRETALAE LVVEDGVVVG AIVETDGVRR AIRARRGVLL AAGGFEANDELRQKYGVPGV ARDTMGPPTN VGAAHQAAIA VGADTDLMGE AWWSPGLTHP DGRSAFALWFTGGIFVDGAG RRFVNESAPY DRLGRAVIDH LTEGGVTPRY WMVYDHKEGS IPPVRATNVSMVDEEQYVAA GLWHTADTLP ELAALIGVPA DALVATVARF NELVADGYDA DFGRGGEAYDRFFSGGEPPL VSIDEGPFHA AAFGISDLGT KGGLRTDTSA RVLTADGTPI GGLYAAGNTMAAPSGTTYPG GGNPIGTSML FSHLAVRHMG TEDAR。
SEQ ID NO.2 sequence is:
MDWAEEYDVL VAGSGAGGMA GTYTAAREGL SVCLVEAGDK FGGTTAYSGG GGAWFPANPVLLRAGTDDTI EDALEYYRAV VGDRTPADLQ ETYVRGGAGL VAYLEEDDHF SFESYPWPDYFGDAPKARRD GQRHIIPTPL PVPSAPELRE VVRGPLDNDR LGTPQPDDLF IGGRALVARFLTALATYPHA TLVRETALAE LVVEDGVVVG AIVETDGVRR AIRARRGVLL NAGGFEANDELRQKYGVPGV ARDTMGPPTN VGAAHQAAIA VGADTDLMGE AWWSPGLTHP DGRSAFALWFTGGIFVDGAG RRFVNESAPY DRLGRAVIDH LTEGGVTPRY WMVYDHKEGS IPPVRATNVSMVDEEQYVAA GLWHTADTLP ELAALIGVPA DALVATVARF NELVADGYDA DFGRGGEAYDRFFSGGEPPL VSIDEGPFHA AAFGISDLGT KGGLRTDTSA RVLTADGTPI GGLYAAGNTMAAPSGTTYPG GGNPIGTSML FSHLAVRHMG TEDAR。
the inventor screens from a plurality of reductase with stable property and broad substrate spectrum which are derived from plants and microorganisms, and reforms the 2 dehydrogenases. The main difference between the two dehydrogenases is that the 51 st site of SEQ ID NO.1 is C and the 231 st site is A; the 51 st site of SEQ ID NO.2 is G, and the 231 st site is N.
The present invention provides a gene encoding the dehydrogenase. The nucleotide sequence of the gene can be SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6.
The dehydrogenase sequences corresponding to the genes with the nucleotide sequences of SEQ ID NO.3 and SEQ ID NO.4 are SEQ ID NO.1, and the dehydrogenase sequences corresponding to the genes with the nucleotide sequences of SEQ ID NO.5 and SEQ ID NO.6 are SEQ ID NO.2.
SEQ ID NO.3 sequence is:
ATGGACTGGG CAGAGGAGTA CGACGTACTG GTGGCGGGCT CCGGCGCCGG CGGCATGGCC GGGACCTACA CCGCGGCCCG CGAGGGGCTC AGCGTGTGCC TGGTCGAGGC CGGGGACAAG
TTCGGCGGGA CGACCGCCTA CTCCGGCGGC TGTGGGGCCT GGTTCCCCGC GAACCCGGTGCTGCTGCGGG CGGGCACCGA CGACACGATC GAGGACGCTC TCGAGTACTA CCGAGCGGTCGTCGGCGACC GCACCCCCGC GGACCTGCAG GAGACCTACG TCCGCGGCGG CGCCGGCCTGGTCGCCTACC TCGAGGAGGA CGACCACTTC TCCTTCGAGT CCTACCCGTG GCCGGACTACTTCGGCGACG CCCCCAAGGC CCGTCGCGAC GGCCAGCGGC ACATCATCCC GACGCCGCTGCCGGTGCCCT CCGCACCCGA GCTGCGCGAG GTGGTCCGCG GGCCGCTCGA CAACGACCGGCTCGGCACGC CGCAGCCCGA CGACCTGTTC ATCGGCGGAC GGGCGCTCGT CGCCCGCTTCCTGACCGCGC TCGCGACCTA CCCCCACGCC ACGCTCGTGC GCGAGACCGC ACTGGCCGAGCTCGTCGTCG AGGACGGCGT CGTGGTCGGC GCGATCGTCG AGACCGACGG CGTCCGCCGCGCGATCCGGG CCCGCCGCGG CGTCCTCCTG GCCGCGGGCG GCTTCGAGGC CAATGACGAGCTCCGCCAGA AGTACGGCGT CCCCGGCGTC GCGCGCGACA CGATGGGCCC GCCGACCAACGTCGGCGCCG CGCACCAGGC CGCGATCGCG GTCGGCGCCG ACACCGACCT GATGGGCGAGGCCTGGTGGT CCCCCGGGCT GACCCACCCC GACGGACGAT CGGCGTTCGC GCTCTGGTTCACCGGCGGCA TCTTCGTCGA CGGCGCCGGC CGGCGCTTCG TCAACGAGTC GGCGCCGTACGACCGGCTCG GCCGCGCCGT CATCGACCAC CTCACCGAGG GCGGCGTCAC CCCGCGGTACTGGATGGTCT ACGACCACAA GGAGGGCTCG ATCCCCCCGG TGCGCGCCAC CAACGTCTCGATGGTCGACG AGGAGCAGTA CGTCGCCGCG GGCCTGTGGC ACACCGCCGA CACGCTGCCCGAGCTGGCCG CGCTGATCGG CGTCCCCGCC GACGCGCTGG TCGCCACGGT CGCGCGCTTCAACGAGCTCG TCGCCGACGG GTACGACGCG GACTTCGGCC GCGGCGGCGA GGCCTACGACCGGTTCTTCT CCGGCGGCGA GCCGCCGCTG GTGAGCATCG ACGAGGGGCC GTTCCACGCGGCCGCCTTCG GCATCTCCGA CCTCGGCACC AAGGGCGGGC TGCGCACCGA CACGTCCGCGCGCGTGCTGA CCGCGGACGG CACGCCGATC GGGGGCCTCT ACGCAGCCGG CAATACGATGGCGGCGCCGA GCGGCACCAC CTACCCGGGC GGTGGCAACC CGATCGGGAC AAGCATGCTCTTCAGCCACC TCGCCGTGCG GCACATGGGC ACCGAGGACG CGCGATGA。
the sequence of SEQ ID NO.4 is based on SEQ ID NO.3, the sequence of 151-153 is replaced by TGC.
SEQ ID NO.5 sequence is:
ATGGACTGGG CAGAGGAGTA CGACGTACTG GTGGCGGGCT CCGGCGCCGG CGGCATGGCCGGGACCTACA CCGCGGCCCG CGAGGGGCTC AGCGTGTGCC TGGTCGAGGC CGGGGACAAGTTCGGCGGGA CGACCGCCTA CTCCGGCGGC GGTGGGGCCT GGTTCCCCGC GAACCCGGTGCTGCTGCGGG CGGGCACCGA CGACACGATC GAGGACGCTC TCGAGTACTA CCGAGCGGTCGTCGGCGACC GCACCCCCGC GGACCTGCAG GAGACCTACG TCCGCGGCGG CGCCGGCCTGGTCGCCTACC TCGAGGAGGA CGACCACTTC TCCTTCGAGT CCTACCCGTG GCCGGACTAC
TTCGGCGACG CCCCCAAGGC CCGTCGCGAC GGCCAGCGGC ACATCATCCC GACGCCGCTGCCGGTGCCCT CCGCACCCGA GCTGCGCGAG GTGGTCCGCG GGCCGCTCGA CAACGACCGGCTCGGCACGC CGCAGCCCGA CGACCTGTTC ATCGGCGGAC GGGCGCTCGT CGCCCGCTTCCTGACCGCGC TCGCGACCTA CCCCCACGCC ACGCTCGTGC GCGAGACCGC ACTGGCCGAGCTCGTCGTCG AGGACGGCGT CGTGGTCGGC GCGATCGTCG AGACCGACGG CGTCCGCCGCGCGATCCGGG CCCGCCGCGG CGTCCTCCTG AACGCGGGCG GCTTCGAGGC CAATGACGAGCTCCGCCAGA AGTACGGCGT CCCCGGCGTC GCGCGCGACA CGATGGGCCC GCCGACCAACGTCGGCGCCG CGCACCAGGC CGCGATCGCG GTCGGCGCCG ACACCGACCT GATGGGCGAGGCCTGGTGGT CCCCCGGGCT GACCCACCCC GACGGACGAT CGGCGTTCGC GCTCTGGTTCACCGGCGGCA TCTTCGTCGA CGGCGCCGGC CGGCGCTTCG TCAACGAGTC GGCGCCGTACGACCGGCTCG GCCGCGCCGT CATCGACCAC CTCACCGAGG GCGGCGTCAC CCCGCGGTACTGGATGGTCT ACGACCACAA GGAGGGCTCG ATCCCCCCGG TGCGCGCCAC CAACGTCTCGATGGTCGACG AGGAGCAGTA CGTCGCCGCG GGCCTGTGGC ACACCGCCGA CACGCTGCCCGAGCTGGCCG CGCTGATCGG CGTCCCCGCC GACGCGCTGG TCGCCACGGT CGCGCGCTTCAACGAGCTCG TCGCCGACGG GTACGACGCG GACTTCGGCC GCGGCGGCGA GGCCTACGACCGGTTCTTCT CCGGCGGCGA GCCGCCGCTG GTGAGCATCG ACGAGGGGCC GTTCCACGCGGCCGCCTTCG GCATCTCCGA CCTCGGCACC AAGGGCGGGC TGCGCACCGA CACGTCCGCGCGCGTGCTGA CCGCGGACGG CACGCCGATC GGGGGCCTCT ACGCAGCCGG CAATACGATGGCGGCGCCGA GCGGCACCAC CTACCCGGGC GGTGGCAACC CGATCGGGAC AAGCATGCTCTTCAGCCACC TCGCCGTGCG GCACATGGGC ACCGAGGACG CGCGATGA。
the sequence of SEQ ID NO.6 is based on SEQ ID NO.5, the sequence of 691-693 is replaced by AAT.
The invention provides engineering bacteria, which contain the genes. The present application may comprise the means of integrating the gene into the chromosome of the host microorganism or the means of placing the gene in a recombinant plasmid and the recombinant plasmid in the host microorganism. The host microorganism of the engineering bacteria is preferably Escherichia coli.
The invention provides an application of engineering bacteria in transforming steroid hormone into dehydrogenation product.
The steroid hormone is 11 beta, 17 alpha-dihydroxyl-6 alpha-methyl pregna-1, 4-diene-3, 20-dione.
Mixing bacterial liquid containing engineering bacteria with substrate steroid hormone, electron acceptor and organic solvent, catalytic reacting, and treating to obtain dehydrogenation product, i.e. 6-methyl dehydrogenation product, i.e. dehydrogenation product of substrate, usually C 1,2 And (3) a site dehydrogenation product.
The electron acceptor is phenazine methosulfate or 2, 6-dichlorophenol indophenol, and the organic solvent is isopropanol.
The preparation method of the bacterial liquid containing the engineering bacteria comprises the steps of culturing the engineering bacteria under the induction of IPTG (isopropyl-beta-D-thiogalactoside) for a period of time, collecting bacterial cells, and adding a buffer solution to obtain the bacterial liquid containing the engineering bacteria. The concentration of IPTG is preferably 0.5mM, the culture temperature is room temperature, the rotating speed is 180rpm, and the culture time is 8-12h. Before adding the buffer solution, the thalli can be washed by using 0.9 percent sodium chloride solution; the buffer is preferably Tris-HCl pH 7.0.
When the engineering bacteria are adopted for dehydrogenation, the reaction formula for converting the substrate 6-methyl dehydrogenation into the product is as follows:
Figure BDA0004039785830000051
the dehydrogenase has the advantages of high dehydrogenation activity, strong stereoselectivity and regioselectivity and no generation of other conformational impurities. The constructed engineering bacteria can efficiently secrete the dehydrogenase, effectively improve the conversion efficiency of a substrate, and particularly has low cost and obvious industrialization potential for the substrate 6-A format (11 beta, 17 alpha-dihydroxyl-6 alpha-methyl pregna-1, 4-diene-3, 20-dione); the dehydrogenase mutant has definite physicochemical properties, omits complicated steps of enzyme purification by a whole-cell catalysis method, reduces the cost and is simple and convenient to operate.
A related research is carried out on biological dehydrogenation of a methylprednisolone intermediate, namely 6-formazan (11 beta, 17 alpha-dihydroxyl-6 alpha-methylprednisolone-1, 4-diene-3, 20-dione), so as to construct a mutant engineering strain for efficiently reacting the 6-formazan dehydrogenation.
Detailed Description
Comparative example 1
Constructing unmutated steroid C1, 2-dehydrogenase engineering bacteria E.coli BL21 (DE 3)/pET 28a-PsKstD, and the operation steps are as follows:
the nucleotide SEQ ID NO. 7 (NCBI: NZ_BJMC 01000016.1) is taken as a template, and the amino acid sequence encoded and expressed by the nucleotide SEQ ID NO. 7 is the nucleotide SEQ ID NO. 8. Codon optimization is carried out on the expression vector pET-28a (+), the gene sequence is synthesized through complete genes, enzyme cutting sites BamHI and XhoI are designed at two ends, and subcloning is carried out on the corresponding sites of the vector pET28a (+), so that the recombinant plasmid pET28a-PsKstD is obtained.
The constructed recombinant plasmid pET28a-PsKstD is transformed into competent cells E.coli BL21 (DE 3) prepared by a calcium chloride method, the competent cells are coated on LB culture medium (containing 10g/L peptone, 5g/L yeast powder and 10g/L NaCl) containing 50mg/L kanamycin for overnight culture, positive clones are selected, and the positive clones are sent to a sequencing company for verification to obtain recombinant escherichia coli E.coli BL21 (DE 3)/pET 28a-PsKstD expressing wild-type steroid C1 and 2 dehydrogenase.
The sequence of SEQ ID NO. 7 is:
ATGGACTGGG CAGAGGAGTA CGACGTACTG GTGGCGGGCT CCGGCGCCGG CGGCATGGCCGGGACCTACA CCGCGGCCCG CGAGGGGCTC AGCGTGTGCC TGGTCGAGGC CGGGGACAAGTTCGGCGGGA CGACCGCCTA CTCCGGCGGC GGTGGGGCCT GGTTCCCCGC GAACCCGGTGCTGCTGCGGG CGGGCACCGA CGACACGATC GAGGACGCTC TCGAGTACTA CCGAGCGGTC
GTCGGCGACC GCACCCCCGC GGACCTGCAG GAGACCTACG TCCGCGGCGG CGCCGGCCTGGTCGCCTACC TCGAGGAGGA CGACCACTTC TCCTTCGAGT CCTACCCGTG GCCGGACTACTTCGGCGACG CCCCCAAGGC CCGTCGCGAC GGCCAGCGGC ACATCATCCC GACGCCGCTGCCGGTGCCCT CCGCACCCGA GCTGCGCGAG GTGGTCCGCG GGCCGCTCGA CAACGACCGGCTCGGCACGC CGCAGCCCGA CGACCTGTTC ATCGGCGGAC GGGCGCTCGT CGCCCGCTTCCTGACCGCGC TCGCGACCTA CCCCCACGCC ACGCTCGTGC GCGAGACCGC ACTGGCCGAGCTCGTCGTCG AGGACGGCGT CGTGGTCGGC GCGATCGTCG AGACCGACGG CGTCCGCCGCGCGATCCGGG CCCGCCGCGG CGTCCTCCTG GCCGCGGGCG GCTTCGAGGC CAATGACGAGCTCCGCCAGA AGTACGGCGT CCCCGGCGTC GCGCGCGACA CGATGGGCCC GCCGACCAACGTCGGCGCCG CGCACCAGGC CGCGATCGCG GTCGGCGCCG ACACCGACCT GATGGGCGAGGCCTGGTGGT CCCCCGGGCT GACCCACCCC GACGGACGAT CGGCGTTCGC GCTCTGGTTCACCGGCGGCA TCTTCGTCGA CGGCGCCGGC CGGCGCTTCG TCAACGAGTC GGCGCCGTACGACCGGCTCG GCCGCGCCGT CATCGACCAC CTCACCGAGG GCGGCGTCAC CCCGCGGTACTGGATGGTCT ACGACCACAA GGAGGGCTCG ATCCCCCCGG TGCGCGCCAC CAACGTCTCGATGGTCGACG AGGAGCAGTA CGTCGCCGCG GGCCTGTGGC ACACCGCCGA CACGCTGCCCGAGCTGGCCG CGCTGATCGG CGTCCCCGCC GACGCGCTGG TCGCCACGGT CGCGCGCTTCAACGAGCTCG TCGCCGACGG GTACGACGCG GACTTCGGCC GCGGCGGCGA GGCCTACGACCGGTTCTTCT CCGGCGGCGA GCCGCCGCTG GTGAGCATCG ACGAGGGGCC GTTCCACGCGGCCGCCTTCG GCATCTCCGA CCTCGGCACC AAGGGCGGGC TGCGCACCGA CACGTCCGCGCGCGTGCTGA CCGCGGACGG CACGCCGATC GGGGGCCTCT ACGCAGCCGG CAATACGATGGCGGCGCCGA GCGGCACCAC CTACCCGGGC GGTGGCAACC CGATCGGGAC AAGCATGCTCTTCAGCCACC TCGCCGTGCG GCACATGGGC ACCGAGGACG CGCGATGA。
the sequence of SEQ ID NO. 8 is:
MDWAEEYDVL VAGSGAGGMA GTYTAAREGL SVCLVEAGDK FGGTTAYSGG GGAWFPANPVLLRAGTDDTI EDALEYYRAV VGDRTPADLQ ETYVRGGAGL VAYLEEDDHF SFESYPWPDYFGDAPKARRD GQRHIIPTPL PVPSAPELRE VVRGPLDNDR LGTPQPDDLF IGGRALVARFLTALATYPHA TLVRETALAE LVVEDGVVVG AIVETDGVRR AIRARRGVLL AAGGFEANDELRQKYGVPGV ARDTMGPPTN VGAAHQAAIA VGADTDLMGE AWWSPGLTHP DGRSAFALWFTGGIFVDGAG RRFVNESAPY DRLGRAVIDH LTEGGVTPRY WMVYDHKEGS IPPVRATNVSMVDEEQYVAA GLWHTADTLP ELAALIGVPA DALVATVARF NELVADGYDA DFGRGGEAYDRFFSGGEPPL VSIDEGPFHA AAFGISDLGT KGGLRTDTSA RVLTADGTPI GGLYAAGNTMAAPSGTTYPG GGNPIGTSML FSHLAVRHMG TEDAR。
comparative example 2
Site-directed saturation mutagenesis is carried out on sites 12, 14, 17, 38, 45, 196, 255, 263, 449, 488 and 495 on the nucleotide SEQ ID NO. 7 of the comparative example 1 as a reference, so that the obtained amino acid sequence is other 19 amino acids except the original amino acid.
The whole cell catalyst was obtained in the same manner as in comparative example 1 from all the mutated genes of comparative example 2, and the 6-formazan was subjected to catalytic conversion in the same manner as in example 4, with a conversion of 0.
Examples 1 to 2
The 51 st and 231 st sites on the nucleotide SEQ ID NO. 7 of the comparative example 1 are mutated based on the nucleotide SEQ ID NO. 7 to obtain SEQ ID NO. 3-6 respectively, and the sequence of the mutated dehydrogenase is shown as SEQ ID NO. 1-2 respectively.
The site-directed mutagenesis primer was designed as shown in Table 1, and the recombinant plasmid pET28a-PsKstD of comparative example 1 was subjected to site-directed mutagenesis by inverse PCR to obtain the dehydrogenase gene shown in examples 1-2. The inverse PCR product was treated with DpnI enzyme (purchased from Takara) and purified, and then transformed into competent cells E.coli BL21 (DE 3), and positive clones were screened for correct sequencing to give mutant strains E.coli BL21 (DE 3)/pET 28a-PsKstD-G51C and E.coli BL21 (DE 3)/pET 28a-PsKstD-A231N.
TABLE 1 primer sequences
Primer name Primer sequences Sequence number
G51C-F TACTCCGGCGGCTGCGGGGCCTGGTTC SEQ ID NO:9
G51C-R GAACCAGGCCCCGCAGCCGCCGGAGTA SEQ ID NO:10
A231N-F GGCGTCCTCCTGAACGCGGGCGGCTTC SEQ ID NO:11
A231N-R GAAGCCGCCCGCGTTCAGGAGGACGCC SEQ ID NO:12
Example 3
Preparation of whole cell catalytic liquid
(1) Inducing and culturing E.coli BL21 (DE 3)/pET 28a-PsKstD-G51C and E.coli BL21 (DE 3)/pET 28a-PsKstD-A231N of the engineering bacteria E.coli obtained in the example 2 with IPTG with the final concentration of 0.5mM at 25 ℃, collecting the bacteria after fermentation is finished for 8 hours, and weighing the wet weight of the bacteria;
the wet weight thalli are washed 3 times by 0.9% NaCl solution, and the thalli are resuspended by borate buffer solution containing 0.2M boric acid and 0.05M borax with pH value of 7.0, thus obtaining E.coli BL21 (DE 3)/pET 28a-PsKstD-G51C and E.coli BL21 (DE 3)/pET 28a-PsKstD-A231N whole cell catalytic solution.
Example 4
The E.coli BL21 (DE 3)/pET 28a-PsKstD-G51C and E.coli BL21 (DE 3)/pET 28a-PsKstD-A231N whole-cell catalytic solution obtained in example 3 are used for catalyzing and reacting 6-A format (11 beta, 17 alpha-dihydroxyl-6 alpha-methyl pregna-1, 4-diene-3, 20-dione), and the operation steps are as follows:
to 1G/L of E.coli BL21 (DE 3)/pET 28a-PsKstD-G51C and E.coli BL21 (DE 3)/pET 28a-PsKstD-A231N whole cell catalyst were added 0.1G/L of 6-methyl form, 0.02% (W/V) PMS (phenazine methosulfate) and 1% (W/V) isopropanol, respectively, and the reaction was stopped after shaking reaction for 12 hours at 30℃and 220rpm and 35℃respectively, the reaction solution was extracted with ethyl acetate several times, the organic phase was combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The substrate conversion was detected as 99% and 98%, respectively, as 1.98-fold and 1.96-fold of the reaction conversion of the wild-type enzyme (comparative example 1), and the ee and de values of the products were both >98%.
Example 5
The E.coli BL21 (DE 3)/pET 28a-PsKstD-G51C and E.coli BL21 (DE 3)/pET 28a-PsKstD-A231N whole-cell catalytic solution obtained in example 3 are used for catalyzing and reacting 6-A format (11 beta, 17 alpha-dihydroxyl-6 alpha-methyl pregna-1, 4-diene-3, 20-dione), and the operation steps are as follows:
to 1G/L of E.coli BL21 (DE 3)/pET 28a-PsKstD-G51C and E.coli BL21 (DE 3)/pET 28a-PsKstD-A231N whole cell catalyst solution were added 0.05G/L of 6-A format, 0.05% (W/V) DCPIP (2, 6-dichlorophenol) and 1% (W/V) isopropanol, respectively, and after shaking reaction at 30℃at 220rpm and 35℃at 220rpm for 12 hours, the reaction was stopped, ethyl acetate was extracted several times, the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The conversion rate of the detection substrate is 98% and 95%, 2.45 times and 2.375 times of the conversion rate of the wild enzyme reaction, and the ee and de values of the products are both more than 98%.
The results indicate that steroid C with wild type 1,2 Compared with the site dehydrogenase, the steroid C of the invention 1,2 The mutant protein of the site dehydrogenase can obviously improve the yield, ee and de value of the 6-formazan (11 beta, 17 alpha-dihydroxyl-6 alpha-methyl pregna-1, 4-diene-3, 20-dione) dehydrogenate.
Those of ordinary skill in the art will appreciate that: the discussion of any of the embodiments above is merely exemplary and is not intended to imply that the scope of the present application is limited to such examples; combinations of features of the above embodiments or in different embodiments are also possible within the spirit of the application, steps may be implemented in any order, and there are many other variations of the different aspects of one or more embodiments described above which are not provided in detail for the sake of brevity.
One or more embodiments herein are intended to embrace all such alternatives, modifications and variations that fall within the broad scope of the present application. Any omissions, modifications, equivalents, improvements, and the like, which are within the spirit and principles of the one or more embodiments in the present application, are therefore intended to be included within the scope of the present application.

Claims (10)

1. A dehydrogenase, characterized in that the amino acid sequence of the dehydrogenase is SEQ ID NO.1 or SEQ ID NO.2.
2. A gene encoding the dehydrogenase according to claim 1.
3. An engineered bacterium comprising the gene of claim 2.
4. The engineered bacterium of claim 3, wherein the host microorganism of the engineered bacterium is escherichia coli.
5. Use of the engineered bacterium of claim 3 or 4 for converting steroid hormone to a dehydrogenation product.
6. The use according to claim 5, wherein the steroid hormone is 11 β,17 α -dihydroxy-6 α -methylpregna-1, 4-diene-3, 20-dione.
7. The use according to claim 5 or 6, wherein the dehydrogenation product is obtained by mixing the bacterial liquid containing the engineering bacteria with the substrate steroid hormone, the electron acceptor and the organic solvent, and performing a catalytic reaction.
8. The use according to claim 7, wherein the electron acceptor is phenazine methosulfate or 2, 6-dichlorophenol indophenol.
9. The use according to claim 7, wherein the organic solvent is isopropanol.
10. The method of claim 7, wherein the engineering bacteria-containing bacterial liquid is prepared by culturing engineering bacteria under the induction of IPTG for a period of time, collecting bacterial cells, and adding buffer solution to obtain engineering bacteria-containing bacterial liquid.
CN202310014700.5A 2023-01-05 2023-01-05 Dehydrogenase, gene, engineering bacteria and application Pending CN116240183A (en)

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