Prolyl hydroxylase and application thereof

A technology for proline hydroxylase and proline hydroxylase activity, which is applied in the fields of genetic engineering and enzyme engineering, and can solve problems such as poor selectivity

Active Publication Date: 2017-06-13
ASYMCHEM LAB TIANJIN +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The main purpose of the present invention is to provide a kind of proline hydroxylase and application thereof, to solve the problem of poor selectivity of proline hydroxylase catalysis in the prior art

Method used

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  • Prolyl hydroxylase and application thereof
  • Prolyl hydroxylase and application thereof
  • Prolyl hydroxylase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Recombinant expression of proline hydroxylase

[0083] Codon optimization of the DNA coding sequence SEQ ID NO:1 from Kordia jejudonensis annotated as a hypothetical protein (codon improvement based on E. coli codon preference and degeneracy, designed by Suzhou Jinweizhi Biotechnology Co., Ltd. ), the optimized DNA sequence SEQ ID NO: 3 is obtained, and the encoded proline hydroxylase polypeptide sequence is SEQ ID NO: 4. The coding sequence of SEQ ID NO: 3 was ligated into the pET22b(+) expression vector (purchased from Novagen, product number 69744), then transformed into E. coli BL21 (DE3), and coated on LB containing 50 μg / ml ampicillin Incubate overnight at 37°C in a petri dish. Pick the single clone on the above petri dish and activate it in the test tube, then inoculate it in 500ml LB liquid medium containing 50μg / ml ampicillin, and shake culture at 37℃ to OD 600 = 0.6, add IPTG to a final concentration of 1 mM, and induce expression at 25°C. After 16 h...

Embodiment 2

[0084] Example 2: Preparation of variants of proline hydroxylase

[0085] Take the pET22b(+) expression vector containing SEQ ID NO: 3 as a template, and use primers with mutation sites to obtain a complete linear fragment by full plasmid PCR. After the above PCR product is digested with DPnI to remove the mother template, transform Into Escherichia coli BL21 (DE3), spread on an LB petri dish containing 50μg / ml ampicillin, and cultivate overnight at 37°C. Obtain a single clone containing the amino acid sequence of the proline hydroxylase variant, and then determine the mutation site through trial induction and gene sequencing. Finally, the mutant with a single mutation site is obtained, and then the mutation at the single mutation site is used as the mutant parent, and then the whole plasmid PCR is performed again with primers with mutations at other sites to determine the mutation site again.

[0086] After the mutant strain was activated, it was inoculated into 500ml LB liquid m...

Embodiment 3

[0087] Example 3: Activity screening of proline hydroxylase variants

[0088] The activity screening of proline hydroxylase variants with one amino acid residue difference compared with SEQ ID NO: 2 uses the following 10 mL reaction solution for screening, and the 10 mL reaction solution contains: L-pipecolic acid 30g / L , 5~10wt (1wt means 1g proline hydroxylase variant recombinant wet cell is required to transform 1g main raw material.) Recombinant crude enzyme, 37.3g / L α-ketoglutarate, 6.1g / L L-ascorbic acid, 5mM ferrous ammonium sulfate, reaction pH6.5, reaction temperature 10℃, reaction time 40h. At the end of the reaction, 200μL of the reaction system was taken, 200μL of acetonitrile was added, and 3000μL of purified water was added after mixing. The supernatant was collected by centrifugation at 10000rpm for 5min for HPLC to detect the conversion rate. The activity screening results are shown in Table 1 (Table 1 is based on all conversion rates Activity screening results o...

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Abstract

The invention provides prolyl hydroxylase and application thereof. The prolyl hydroxylase comprises (a) a protein with an amino acid sequence as shown by SEQ ID NO: 2; (b) a protein with the amino acid sequence as shown by the SEQ ID NO: 2 being mutated through one or more amino acids, and prolyl hydroxylase activity; or (c) a protein with mutation of one or more amino acids in the (b), prolyl hydroxylase activity, and at least 78 percent of homology with the amino acid sequence of the protein in the (b). Due to the protein with the amino acid sequence as shown by the SEQ ID NO: 2 and a mutant obtained through improving the protein by utilizing a gene engineering means, the prolyl hydroxylase provided by the invention has higher catalysis specifity compared with existing prolyl hydroxylase, or the catalytic activity is remarkably improved.

Description

Technical field [0001] The invention relates to genetic engineering and enzyme engineering, in particular to a proline hydroxylase and its application. Background technique [0002] Derivatives of proline are important structural units for drug synthesis, especially hydroxyproline is a rare amino acid in nature, and is the starting material for many important drug synthesis. According to the difference of proline hydroxylation position and spatial structure, there are 8 isomers of hydroxyproline, among which 3-hydroxy-L-proline and 4-hydroxy-L-proline are used as antibiotics and enzyme inhibitors , Anti-tumor, anti-hypertensive and new gastric drugs and other important raw materials, is the current focus of biosynthetic research. In addition, hydroxyproline can also be a source of natural products or chemically synthesized, for example, it can be a hydrolysate of plant materials and collagen, or it can be based on allyl bromide and diethylacetamidomalonic acid, D -Glutamic acid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N15/53C12P13/24
CPCC12N9/0071C12P13/24C12Y114/11002
Inventor 洪浩詹姆斯·盖吉卢江平张娜于文燕刘芳李艳君黄鑫高娟张克俭马玉磊魏俊璐
Owner ASYMCHEM LAB TIANJIN
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