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Novel proline 4-hydroxylase and application thereof

A proline, hydroxylase technology, applied in the biological field, can solve problems such as poor expression and catalytic performance

Active Publication Date: 2020-02-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it refers to the wild-type proline 4-hydroxylase of cystic bacteria, which mainly exists in the form of inclusion bodies with no activity or low activity when recombinantly expressed in prokaryotes, such as Escherichia coli, and the mutant type after codon optimization The proline 4-hydroxylase derived from cystic fungus is still poor in expression and catalytic performance

Method used

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  • Novel proline 4-hydroxylase and application thereof
  • Novel proline 4-hydroxylase and application thereof
  • Novel proline 4-hydroxylase and application thereof

Examples

Experimental program
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Embodiment

[0113] 1. Synthesis and cloning expression of proline 4-hydroxylase

[0114] First, the inventor synthesized the gene shown in the sequence SEQ ID NO: 1 through the whole gene (the amino acid sequence encoded by it is shown in SEQ ID NO: 2), the 5' end adopts the Nde I endonuclease site, and the 3' end The Hind III endonuclease site was used to connect to the high-copy plasmid pET21a, and the obtained recombinant plasmid was named DaWT, and then the recombinant plasmid DaWT was introduced into Escherichia coli BL21a (DE3) competent cells by chemical transformation, and the recombinant engineering strain DaWT was obtained , according to the following procedure, proline 4-hydroxylase was induced to express.

[0115] Take 5 μl of glycerol bacteria solution and inoculate 5ml of LB medium (10g / l peptone, 10g / l NaCl, 5g / l yeast extract) containing antibiotics (100μg / ml ampicillin), and culture overnight at 37°C and 220rpm; then inoculate with 1% The amount of inoculum was inoculate...

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Abstract

The invention discloses a method for reforming proline 4-hydroxylase. According to the method, a section between specific sites of proline 4-hydroxylase to be reformed is subjected to whole replacement, and / or the specific sites are subjected to amino acid mutation, so that the activity and the heat stability of the obtained proline 4-hydroxylase can be significantly improved, and the proline 4-hydroxylase which is excellent in stability and activity can be obtained. The proline 4-hydroxylase disclosed by the invention can be applied to production of trans-4-hydroxyl-L-proline or downstream products by using the trans-4-hydroxyl-L-proline as a precursor.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to novel proline 4-hydroxylase, its coding gene and host cells comprising the enzyme or its coding gene and their role in the production of trans-4-hydroxyl-L-proline application. Background technique [0002] Trans-4-hydroxy-L-proline (referred to as hydroxyproline), is an amino acid with unique physiological activity, easily soluble in water, widely used in medicine, chemical industry, animal feed and beauty industry, etc. Market prospects. Due to the high price, hydroxyproline is currently mainly used to synthesize side chains of carbapenem antibiotics (meropenem, etc.). Carbapenems are the third-generation antibiotics and a type of atypical β-lactam antibiotics with the broadest antibacterial spectrum. They have the characteristics of broad antibacterial spectrum and strong antibacterial activity. The overall global market has exceeded 3 bill...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N1/21C12P13/24
CPCC12N9/0071C12P13/24C12Y114/11002
Inventor 孙际宾赵晶刘超周文娟郑平马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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