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Method for producing trans-4-hydroxy-L-proline

A proline and hydroxyl technology, applied in the field of producing trans-4-hydroxy-L-proline, can solve the problem of poor expression and catalytic performance, and can not better produce trans-4-hydroxy-L- Problems such as poor stability of proline and proline 4-hydroxylase

Active Publication Date: 2018-02-09
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Refers to the wild-type proline 4-hydroxylase of cystic bacteria in prokaryotes, such as Escherichia coli, mainly in the form of inclusion bodies with no activity or low activity, and the mutant proline after codon optimization Acid 4-hydroxylase is still poor in expression and catalytic performance
At present, only 33-35° C. (CN97117929.8) can be used to produce trans-4-hydroxyl-L-proline in Escherichia coli using proline 4-hydroxylase from the cyst fungus RH1, and usually When producing other products in Escherichia coli, the optimum growth temperature of Escherichia coli is generally used at 37°C. The revelation means that the proline 4-hydroxylase of Cystomyces RH1 cannot produce trans-4 better at 37°C -Hydroxy-L-proline
After our thermal stability test, it was found that the proline 4-hydroxylase of the cyst fungus RH1 had poor stability at 37°C, which was the main reason why it could not be produced at 37°C

Method used

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  • Method for producing trans-4-hydroxy-L-proline
  • Method for producing trans-4-hydroxy-L-proline
  • Method for producing trans-4-hydroxy-L-proline

Examples

Experimental program
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Effect test

Embodiment 1

[0117] Embodiment 1. Whole gene synthesis and clone expression of proline 4-hydroxylase

[0118] First, the inventor synthesized the gene whose sequence is shown in SEQ ID NO: 2 through whole gene synthesis. Subsequently, the gene synthesized by the whole gene (sequence shown in SEQ ID NO: 2) was cloned into pET21a plasmid (purchased from Novagen) through NdeI and HindIII restriction sites, and the obtained recombinant plasmid was named pSWXP1, which expressed The protein has 6 his tags at the C-terminus, and then the recombinant plasmid is introduced into the Escherichia coli Rossetta strain (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) to obtain the Escherichia coli Rossetta (pSWXP1) strain. Escherichia coli Rossetta (pSWXP1) is used to induce the expression of the protein shown in SEQ ID NO: 1, using LB medium, inoculated with 1%, adding 50ug / mL ampicillin and 34μg / mL chloramphenicol, and culturing at 220rpm at 37°C for 2-3h , OD grows to 0.6-0.8, add 0.5mM I...

Embodiment 2

[0119] Embodiment 2. The crude enzyme activity assay of proline 4-hydroxylase

[0120]The crude enzyme solution prepared in Example 1, and the crude enzyme solution of Escherichia coli Rossetta (pET21a) empty plasmid control strain were prepared by the same method. The BCA protein quantitative analysis kit (purchased from Bio-Rad, product number: 23227) was used to quantify the total protein of the crude enzyme solution. Enzyme activity assay system: 240mM MES (pH6.5), 6mM FeSO 4 , 24mM α-ketoglutarate, 8mM L-ascorbic acid, 12mM L-proline and an appropriate amount of crude enzyme, react at 35°C for 10min, stop the enzyme activity, and measure the content of trans-4-hydroxy-L-proline. The detection method of trans-4-hydroxy-L-proline refers to the national standard GB / T 9695.23-2008. One unit of enzyme activity U is defined as the amount of 1 nmol of trans-4-hydroxy-L-proline catalyzed per minute. The amount of enzyme required. The specific enzyme activity of the crude enzym...

Embodiment 3

[0123] Example 3. Proline 4-hydroxylase purification and stability assay

[0124] The performance of the enzyme of the present invention was compared with the proline 4-hydroxylase of Dactylocystis sp. RH1. The literature (Liu Hedong, Yuan Chunwei, Zhang Zhenyu. Codon-optimized expression of proline 4-hydroxylase in Escherichia coli and its effect on trans-4-hydroxyproline biosynthesis[J]. Bioprocessing, 2014, 12( 6): 44-51.) The reported codon optimization refers to the Cystomyces RH1 proline 4-hydroxylase (named as P4H-2) gene, which is synthesized by the whole gene through NdeI and HindIII enzyme cleavage sites The gene (sequence is shown in the literature: Liu Hedong. Construction and fermentation optimization of high-yielding trans-4-hydroxyproline recombinant Escherichia coli [D]. Wuxi: Jiangnan University, 2013.) was cloned into pET21a plasmid (purchased from Novagen) , the obtained recombinant plasmid was named as pSWXP2, and the expressed protein had 6 his tags at th...

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Abstract

The invention provides a method for producing trans-4-hydroxy-L-proline. Specifically, the invention provides an application of polypeptide in producing the trans-4-hydroxy-L-proline or a downstream product taking the trans-4-hydroxy-L-proline as a precursor. The invention also provides the method for producing the trans-4-hydroxy-L-proline, wherein the method comprises a step of cultivating and expressing a strain of polypeptide, so that the trans-4-hydroxy-L-proline is obtained. The invention also provides a trans-4-hydroxy-L-proline producing strain and a construction method of the trans-4-hydroxy-L-proline producing strain. With the application of the method provided by the invention, the efficient production of the trans-4-hydroxy-L-proline with low cost is achieved.

Description

technical field [0001] The present invention relates to the field of biotechnology. Specifically, the present invention relates to the use of a polypeptide in the production of trans-4-hydroxyl-L-proline or the production of downstream products that use trans-4-hydroxyl-L-proline as a precursor, and A method for producing trans-4-hydroxy-L-proline. 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 the fields of medicine, chemical industry, animal feed and beauty industry, etc. The market prospect is broad. Due to the high price, hydroxyproline is currently mainly used to synthesize the side chain 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 stron...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N1/21C12P13/24C12R1/19
CPCC12N9/0071C12P13/24C12Y114/11002
Inventor 孙际宾刘娇郑平王兴初周文娟马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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