Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Proline Hydroxylase and Its Application

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: 2021-07-06
ASYMCHEM LAB TIANJIN +4
View PDF19 Cites 0 Cited by
  • 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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Proline Hydroxylase and Its Application
  • Proline Hydroxylase and Its Application
  • Proline Hydroxylase and Its Application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1: Recombinant expression of proline hydroxylase

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

Embodiment 2

[0084] Example 2: Preparation of proline hydroxylase variants

[0085]Using the pET22b(+) expression vector containing the sequence of SEQ ID NO: 3 as a template, a complete linear fragment was obtained by whole plasmid PCR with primers with a mutation site, and the above PCR product was digested with DPnI to remove the parent template, and transformed into Escherichia coli BL21 (DE3), spread on LB dishes containing 50 μg / ml ampicillin, and cultured at 37°C overnight. A single clone containing the amino acid sequence of the proline hydroxylase variant was obtained, and the mutation site was determined by trial induction and gene sequencing. The mutant with a single mutation site is finally obtained, and then the mutation of the single mutation site is used as the mutation mother, 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 in...

Embodiment 3

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

[0088] Compared with SEQ ID NO: 2, the activity screening of proline hydroxylase variants with one amino acid residue difference was screened by using the following 10 mL reaction solution, and the 10 mL reaction solution contained: L-piperic acid 30g / L , 5 ~ 10wt (1wt refers to the transformation of 1g main raw material requires 1g proline hydroxylase variant recombinant wet cells.) recombinant crude enzyme, 37.3g / Lα-ketoglutaric acid, 6.1g / L L-ascorbic acid, 5mM ferrous ammonium sulfate, the reaction pH is 6.5, the reaction temperature is 10°C, and the reaction time is 40h. At the end of the reaction, take 200 μL of the reaction system, add 200 μL of acetonitrile, add 3000 μL of purified water after mixing, and centrifuge at 10000 rpm for 5 min to collect the supernatant for HPLC to detect the conversion rate. The activity screening results are shown in Table 1 (Table 1 is based on all conversion rates). ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a proline hydroxylase and application thereof. Proline hydroxylase includes (a) a protein having the amino acid sequence shown in SEQ ID NO:2; (b) the amino acid sequence shown in SEQ ID NO:2 is mutated by one or more amino acids and has proline A protein with hydroxylase activity; or (c) retaining one or more amino acid mutations in (b), has proline hydroxylase activity, and has at least 78% identity with the amino acid sequence of the protein in (b) source of protein. The protein with the amino acid sequence shown in SEQ ID NO: 2 and the mutants obtained by genetic engineering means have higher catalytic specificity or significantly improved catalytic activity than existing proline hydroxylases .

Description

technical field [0001] The present 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 the synthesis of many important drugs. Hydroxyproline has 8 isomers according to the hydroxylation position and spatial structure of proline, among which 3-hydroxy-L-proline and 4-hydroxy-L-proline are used as antibiotics and enzyme inhibitors It is an important raw material for various drugs such as anti-tumor, anti-hypertension and new gastric drugs, and is currently a hot spot in biosynthesis research. In addition, hydroxyproline can also be a source of natural products or chemically synthesized, such as hydrolysates of plant materials and collagen, or as allyl bromide and diethylacetamidomalonic acid, D - Glutamic acid a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(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
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com