Mutant of 7 beta-hydroxyl steroid dehydrogenase, application of mutant and synthesis method

A technology of mutant and hydroxyl, applied in the field of 7β-hydroxysteroid dehydrogenase mutant and its application and synthesis, which can solve the problem of unpublished sequence information and the like

Active Publication Date: 2016-01-27
苏州天绿生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Hirano and Masuda described a NADP+-dependent 7β-HSDH from Collinsella aerogenes ATCC25986 (ApplEnvironMicrobiol, 1982, 43(5):1057-1063), but no sequence information was published

Method used

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  • Mutant of 7 beta-hydroxyl steroid dehydrogenase, application of mutant and synthesis method
  • Mutant of 7 beta-hydroxyl steroid dehydrogenase, application of mutant and synthesis method
  • Mutant of 7 beta-hydroxyl steroid dehydrogenase, application of mutant and synthesis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Escherichia coli BL21(DE3) containing recombinant DNA pET21a(+)-RUHSDH, pET21a(+)-RU-8C2 and pET21a(+)-RU-4F9 were inoculated into 250 mL triangles containing 50 mL LB medium (100 μg / mL ampicillin) After culturing overnight at 37°C and 300 rpm in shake flasks, transfer 10% of the inoculum into a 2L shake flask containing 400 mL of LB medium (100 μg / mL ampicillin), and continue to grow at 37°C and 300 rpm Incubate for 2 hours until OD600 reaches 1.0. Add IPTG at a final concentration of 0.2 mM, induce expression at 25°C for 4 hours, and collect cells by centrifugation. The cells were suspended in 20 mL of 100 mM phosphate buffer (pH 8.0), ultrasonically disrupted, and centrifuged to obtain a recombinant crude enzyme solution. The enzyme activities (forward / reverse) of RUHSDH wild-type enzyme, mutant RU-8C2 and mutant RU-4F9 were 5.3 / 10.6 units / mL, 8.5 / 25.6 units / mL, and 16.7 / 48.5 units / mL, respectively.

Embodiment 2

[0047] Inoculate a single colony on the Escherichia coli BL21(DE3) plate containing recombinant DNA pET21a(+)-RUHSDH into two 1L shake flasks filled with 250mL of LB medium (100μg / mL ampicillin), at 37°C and 300 rpm Incubate with shaking for 15 hours. Combine the seed liquids in the two shake flasks into 500mL and inoculate them into 5L of initial medium containing: 30g / L glycerol, 25g / L potassium dihydrogen phosphate, 10g / L ammonium sulfate , 10g / L magnesium sulfate heptahydrate, 0.4g / L iron sulfate heptahydrate. Recombinant Escherichia coli was cultured with aeration and stirring in a 10L fermenter at a temperature of 30°C and a pH of 7.0. Stirring and aeration were adjusted to control dissolved oxygen at 25%. After the glycerol in the starting medium was exhausted, the induction medium (lactose: 50 g / L, glycerol: 200 g / L) was added to induce the production of the enzyme. The flow rate is 60-250mL / hour, and the maximum flow rate is gradually reached within 3 hours. Temper...

Embodiment 3

[0049] Escherichia coli BL21(DE3) containing recombinant DNA pET21a(+)-RU-8C2 was fermented using the same method as in Example 2, the total induction time was 9 hours, and the wet cell weight reached 110g / L. Cells were collected by centrifugation and stored at -20°C. 50 g of wet cells were weighed, suspended in 450 mL of 100 mM phosphate buffer (pH 8.0), ultrasonically disrupted, and centrifuged to obtain a RU-8C2 mutant recombinant enzyme solution. The enzyme activity (forward / reverse) of this recombinant enzyme solution is 9.8 / 25.2 units / mL, and the enzyme solution is stored at -20°C for UDCA synthesis.

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Abstract

The invention provides a mutant of 7 beta-hydroxyl steroid dehydrogenase, application of the mutant and a synthesis method. The mutant of the 7 beta-hydroxyl steroid dehydrogenase is characterized in that amino acid sequences of the mutant are Seq ID NO:4, and coded nucleotide sequences are Seq ID NO:3; or amino acid sequences of the mutant are Seq ID NO:6, and coded nucleotide sequences are Seq ID NO:5. The mutant, the application and the synthesis method have the advantages that cholic acid compounds, particularly ursodeoxycholic acid, can be catalytically synthesized by the efficient 7 beta-hydroxyl steroid dehydrogenase, mutant enzymes of the 7 beta-hydroxyl steroid dehydrogenase and coenzyme regeneration systems, accordingly, the substrate concentration can reach 100 g/L, the conversion rate is 99.2-99.5%, and the weight yield can reach 94-96%; and the enzymes can be inexpensively and easily obtained by the aid of a fermentation process, accordingly, the production cost and the product quality are superior to the production cost and the product quality of chemical synthesis methods, and the mutant and the synthesis method are applicable to industrial production.

Description

Technical field: [0001] The present invention relates to novel mutants of 7β-steroid dehydrogenase (7β-hydroxsteroiddehydrogenase, 7β-HSDH) from bacteria of the genus Ruminococcus, in particular of the genus Ruminococcus gnavus, mutants encoding these enzymes sequences, methods for producing such enzymes, and their use in the enzymatic synthesis of cholic acid compounds, in particular in the synthesis of ursodeoxycholic acid (UDCA); furthermore, the invention also includes novel methods for the synthesis of UDCA using mutants method, and the post-extraction method of UDCA. Background technique: [0002] Ursodeoxycholic acid (I, UDCA) is the main active ingredient contained in the precious traditional Chinese medicine bear bile, and it and its corresponding diastereoisomer chenodeoxycholic acid (II, CDCA) are clinically used to treat various Gallstone disease, various acute and chronic liver diseases, has good effect. The yield of extracting UDCA from bear bile of artificia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/04C12P33/06
CPCC12N9/0006C12P33/06C12Y101/01201
Inventor 刘志斌刘经辉吴庆斌周敦秀
Owner 苏州天绿生物制药有限公司
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