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3-ketosteroid-[delta]1-dehydrogenase mutant and construction method thereof

A mutant and dehydrogenase technology, applied in the field of genetic engineering, can solve the problems of low protein expression and low enzymatic activity of -dehydrogenase, and achieve the effect of improving the potential of industrial application

Inactive Publication Date: 2017-04-19
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Heterologous expression of 3-sterone-Δ 1 -Prominent problems with dehydrogenases are low protein expression, 3-sterone-Δ 1 - Low dehydrogenase activity

Method used

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  • 3-ketosteroid-[delta]1-dehydrogenase mutant and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 contains 3-sterone-Δ 1 - Construction of recombinant vectors for dehydrogenase mutants

[0024] (1) Obtaining the S366A mutant: using the nucleotide sequence shown in SEQ ID NO.4 as a template, Fprimer (sequence shown in SEQ ID NO.5), Rprimer (sequence shown in SEQ ID NO.6) as primers, PCR is performed to obtain the recombinant gene shown in SEQ ID NO.3.

[0025] (2) Digest the recombinant gene and pMA5 with BamHI and HindIII, respectively, and ligate with T4 DNA ligase overnight at 16°C after purification. The ligation product was chemically transformed into JM109 competent cells. The transformation solution was applied to an LB plate containing kanamycin (50mg / L), the plasmid was extracted, and the recombinant plasmid constructed was verified by double enzyme digestion, which was named pMA5-S366A. The sequencing work was completed by Shanghai Sangong.

Embodiment 2

[0026] Example 2 produces 3-sterone-Δ 1 -Dehydrogenase Bacillus subtilis engineering bacteria construction

[0027] The recombinant plasmid pMA5-S366A obtained in Example 1 was chemically transformed into B. subtilis168 competent cells, the specific method is as follows:

[0028] (1) The solution required for the transformation experiment is as follows (g / L):

[0029] Sp-A: (NH 4 ) 2 SO 4 4,K 2 HPO 4 28. Sodium citrate 12Sp-B: MgSO 4 ·7H 2 O 0.4

[0030] 100×CAYE: Casaaminoacid20, yeast powder 100SpI medium: Sp-A49%, Sp-B49%, 50% glucose 2%, 100×CAYE2% SpII medium: SpI medium 98%, 50mmol / LCaCl21%, 250mmol / LMgCl21 %. 115°C damp heat sterilization.

[0031] (2) Inoculate a single colony of B.Subtilis168 into 2mL SpI medium (50mL centrifuge tube), and culture overnight at 37°C and 200r / min;

[0032] (3) Take 100 μL of the culture solution into 5 mL of SpI medium, and culture at 37°C and 200 r / min until the logarithmic phase (OD600 value is about 1), about 4 to 5 hou...

Embodiment 3

[0035] Example 3: Recombinant bacteria pMA5-S366A / B.subtilis1683-sterone-Δ 1 -Dehydrogenase high-level expression and enzyme activity determination.

[0036](1) Inoculate the recombinant strain pMA5-S366A / B.subtilis168 constructed in Example 2 and the original strain pMA5-ksdd / B.subtilis168 in 10 mL of LB medium containing kanamycin, respectively, culture at 37°C overnight with shaking, 8000rpm Centrifuge for 10 min, wash with 50 mM PBS buffer solution of pH 7.5 for 3 times, suspend in the buffer solution, and ultrasonically break to prepare crude enzyme solution for enzyme activity determination.

[0037] (2) KSDD enzyme activity assay method: 3ml reaction mixture is composed of 100μL crude enzyme solution, 50mM PBS (PH7.5), 40μM 2,6-dichlorophenol indophenol, 1.5mM phenazine methyl sulfate, 500μM AD (dissolved in 2% methanol), and detect the change of absorbance at 600nm. The enzyme activity unit is defined as: the amount of enzyme that can cause a change of 0.01 absorbanc...

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Abstract

The invention discloses a 3-ketosteroid-[delta]1-dehydrogenase mutant with enzyme activity improved and a construction method of the 3-ketosteroid-[delta]1-dehydrogenase mutant, and belongs to the field of genetic engineering. According to the mutant provided by the invention, on the basis of amino acid shown as SEQ ID NO.2, valine on the 366th site is mutated into serine. The mutant provided by the invention can achieve expression in bacillus subtilis, the enzyme activity of the mutant is improved to 2.15U / mL by conducting shake flask fermentation for 24h, mutation enzyme activity is improved by 23%, substrate affinity is improved by 67% in comparison with that of an original enzyme, a catalysis efficiency is increased by 43%, and meanwhile, specific enzyme activity is improved by 176%. The invention shows that the amino acid residue on the 366th site has a relatively high influence on the catalytic action of the enzyme (3-ketosteroid-[delta]1-dehydrogenase), a certain foundation is provided for researches on the catalysis mechanism of the enzyme, and the industrial application potential of the enzyme is improved.

Description

technical field [0001] The invention relates to a 3-sterone-Δ with improved enzyme activity 1 - Dehydrogenase mutants and construction methods thereof, belonging to the technical field of genetic engineering. Background technique [0002] Steroidal drugs can be obtained through total synthesis or degradation of natural steroidal compounds and transformation of their functional groups. Steroidal drugs have strong pharmacological effects such as anti-infection, just allergy, anti-virus and anti-shock. With the continuous development of the times, steroid drugs have become the second largest class of drugs after antibiotics. In 2000, the sales of steroid drugs in the global drug market have exceeded 20 billion US dollars, accounting for about 66% of the world's total pharmaceutical sales. %. [0003] Classification of steroid hormone drugs: adrenocortical hormones, including hydrocortisone, prednisone, etc. It can treat Addison's disease, anti-inflammation, anti-allergy, ant...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N1/21C12N15/75C12R1/125
CPCC12N15/75C12N9/0073C12N2800/101C12Y114/13054
Inventor 饶志明邵明龙沙宗焱张显杨套伟徐美娟
Owner JIANGNAN UNIV
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