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Steroid C14 alpha hydroxylase, expression vector, engineering bacterium and application of steroid C14 alpha hydroxylase

An expression vector, hydroxylase technology, applied in application, genetic engineering, oxidoreductase and other directions, can solve the problem of poor P450 enzyme selectivity and achieve excellent performance

Pending Publication Date: 2022-03-18
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to obtain a steroid 14-position α hydroxylase with high regioselectivity, which can realize efficient and specific hydroxylation of various steroid compounds C14α , so as to solve the problem of poor selectivity of P450 enzymes for α-hydroxylation at the C14 position of steroids

Method used

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  • Steroid C14 alpha hydroxylase, expression vector, engineering bacterium and application of steroid C14 alpha hydroxylase
  • Steroid C14 alpha hydroxylase, expression vector, engineering bacterium and application of steroid C14 alpha hydroxylase
  • Steroid C14 alpha hydroxylase, expression vector, engineering bacterium and application of steroid C14 alpha hydroxylase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, cultivation and fermentation detection of Curvularia lunae

[0071] 1. Cultivation of Curvularia lunae

[0072] Inoculate Curvularia lunata on PDA slant medium, culture in a 30°C incubator at constant temperature for 3 to 5 days, take appropriate amount of Curvularia lunata CGMCC 3.4381 and CGMCC 3.3589 mycelia and inoculate them into two 100mL fermentation tanks. Medium (soy bean cake powder 5g / L, glucose 20g / L, yeast extract 5g / L, NaCl 5g / L, K 2 HPO 4 5g / L, PH6.1 Erlenmeyer flask, and respectively marked No. 1 and No. 2, 28 ° C, 180r / min shaker after 24 hours, weigh the substrate progesterone (PG), with L cosolvent N-N dimethyl After dissolving the substrate with DMF, it was added to No. 1 and No. 2 Erlenmeyer flasks respectively, with a final concentration of 250 mg / L.

[0073] 2. The fermentation broth is processed for samples, and UPLC is used to detect the components of the fermentation broth.

[0074] Add 2 times the volume of ethyl acetate to ...

Embodiment 2

[0075] Example 2, Cloning and Expression of Curvularia lunae Steroidal C14α Hydroxylase P450 (ClCYP450-14) The cloning and expression of the gene is divided into the following three steps

[0076] 1) Extraction of total DNA of Curvularia lunata

[0077] The strains were cultured in PDB for 2 days at a temperature of 30°C. The bacteria were collected by centrifugation, washed 2-3 times with sterile water, and an appropriate amount of mycelium was placed in a 2mL EP tube containing magnetic beads, and the genome was extracted with a high-speed oscillating instrument. The oscillating program: 8m / s, 15s for a cycle 6 cycles. The thalline after the above shaking was centrifuged at high speed (12000rpm, 10min), and the supernatant was taken as a PCR template.

[0078] 2) Cloning of the complete DNA sequence of the gene ClCYP450-14

[0079] Using the reported 14-position hydroxylation gene sequence of steroids (CYP450lun) as a reference, design 3 pairs of ClCYP450-14 gene amplific...

Embodiment 3

[0088] Example 3, Construction of Saccharomyces cerevisiae recombinant engineering bacteria Sc-CLCYP14-WT and Sc-CYP450lun

[0089] The construction of engineering bacteria Sc-CLCYP450-WT and Sc-CYP450lun is divided into the following 3 steps

[0090] 1) Preparation of Saccharomyces cerevisiae YPH499 Competent State

[0091] Saccharomyces cerevisiae (Saccharomyces cerevisiae) YPH499 was cultured overnight at 30°C in YPD liquid medium. YPD liquid medium formula: yeast extrat, inoculate 1mL of the above bacterial solution into 10mL of fresh YPD medium, culture at 30°C, 220rpm for 3-4h, collect under sterile conditions, centrifuge at 1500rpm for 5min, discard the supernatant and use 10mL Resuspend the solution 1 solution in the Frozen-EA Yeast Transformation IITM Kit, centrifuge at 1500rpm for 5min, discard the supernatant, resuspend with 1mL solution 2 (OD about 0.6-1.0) and dispense into 1.5ml EP tubes, 30μl per tube, Freeze at -80°C for later use.

[0092] 2) Transformation...

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Abstract

The invention discloses a steroid C14 alpha hydroxylase, an expression vector, an engineering bacterium and an application of the steroid C14 alpha hydroxylase, and relates to the fields of steroid drug conversion, bioengineering technology and enzyme engineering, and an active bacterium with the steroid C14 alpha hydroxylase is Curvularia lunata CGMCC 3.3589; the invention relates to an amino acid sequence of steroid C14 alpha hydroxylase and an expression vector of the steroid C14 alpha hydroxylase. The invention relates to an engineering bacterium of a C14 alpha hydroxylation steroid method. The engineering bacterium is applied to catalysis of steroid compounds. The invention provides the steroid C14 alpha hydroxylase with wide substrate and stronger regioselectivity and stereospecificity, a C14 alpha hydroxylase mutant library is obtained through a recombinant saccharomycetes mutant steroid transformation experiment, the transformation specificity of the mutants I111A and V124A on the substrate progesterone is obviously higher than that of a gene wild type strain, and the mutant C14 alpha hydroxylase mutant library can be used for preparing the steroid C14 alpha hydroxylase. The method has great application potential and economic value.

Description

technical field [0001] The present invention relates to the fields of steroid drug transformation, bioengineering technology and enzyme engineering, in particular to a Curvularia lunata strain with high-efficiency C14α hydroxylase activity, steroid C14α hydroxylase, expression vector and engineering Bacteria and their applications. Background technique [0002] Steroids, also known as sterols, are a class of compounds with cyclopentane polyhydrophenanthrene as the core. Because of its good anti-inflammatory, anti-infection, anti-allergic and other pharmacological activities, it is called "just-needed drug" and "life-saving drug" for many critical diseases such as organ transplantation and severe infection. At present, there are about 400 steroid drugs approved for marketing in the world. As essential drugs for the basic health system, steroid drugs are often reserved as strategic materials. [0003] The introduction of functional groups at specific sites on the steroid ske...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/80C12N15/81C12N1/15C12N1/19C12P33/06C12R1/645C12R1/865
CPCC12N9/0071C12N15/80C12N15/81C12P33/06
Inventor 瞿旭东郑梦梦林芝
Owner SHANGHAI JIAO TONG UNIV
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