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D-carboxamide hydrolase mutant and its uses

A technology of carbamoyl hydrolase and hydrolase, applied in the production of D-p-hydroxyphenylglycine, the field of mutants of D-carbamyl hydrolase

Inactive Publication Date: 2007-07-11
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although natural enzyme molecules have evolved for millions of years under natural conditions, because the environment in which natural enzyme molecules exist is different from the environment in which they are actually used, enzyme molecules still have great potential for evolution

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 , Cloning of DCase gene

[0019] 1.1, PCR amplification

[0020] Design a pair of primers:

[0021] 5'-CGCCATATGACACGTCAGATGATA-3'; and

[0022] 5'-CCCAAGCTTTCAGAGTTCCGCGAT-3'

[0023] Using plasmid pXZ-total (Xu Zhen et al., Acta Bioengineering Sinica, 2002, 18(2): 149-54) DNA as a template, PCR amplification was performed. The PCR conditions were as follows: denaturation at 94°C for 10 min, denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 50 sec, a total of 30 cycles, and finally full extension at 72°C for 10 min.

[0024] 1.2. Construction of plasmid containing DCase gene

[0025] After PCR amplification, first use 1% agarose gel to carry out nucleotide electrophoresis with a voltage of 100 volts. After cutting the gel, use a gel recovery kit (Shanghai Huashun Bioengineering Co., Ltd.) to recover a DNA fragment of about 900 bp. Then, according to the method described in the 2005-2006 product catalog manual of Takara...

Embodiment 2

[0030] Example 2 , Random mutation of DCase gene

[0031] 2.1. Error-prone PCR

[0032] 100μl error-prone PCR reaction system: 20ng of DNA template pXZ-total, 30pmol each of a pair of primers 5'-CGCCATATGACACGTCAGATGATA-3' and 5'-CCCAAGCTTGAGTTCCGCGAT-3', 7mMMgCl 2 , 50mM KCl, 10mM Tris-HCl (pH8.3), 0.01% gelatin, 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 0.15mM MnCl 2 , and 5 units of Taq enzyme (Shanghai Sangon Company).

[0033] The PCR reaction conditions were as follows: denaturation at 94°C for 10 min, denaturation at 94°C for 30 sec, annealing at 58°C for 30 sec, extension at 72°C for 50 sec, a total of 30 cycles, and finally full extension at 72°C for 10 min.

[0034] 2.2. DNA shuffling

[0035] PCR products were purified using a gel recovery kit (Shanghai Huashun Bioengineering Co., Ltd.). According to the method described in the literature (StemmerWPC.Nature, 1994, 370(6488): 389.), the obtained DNA fragments were subjected to DNA shuffling, including: degrad...

Embodiment 3

[0037] Example 3 , screening of mutant libraries

[0038] 3.1. Transformation of mutants

[0039] Transform the mutant constructed in step 2.2 into the DH5α strain by electric shock. Transformed cells were coated with Amp-containing r Antibiotics, IPTG inducers and chromogenic substrate X-gal were cultured on LB plates (peptone 1%, yeast extract 0.5%, sodium chloride 1%, agar 2%) at 37°C.

[0040] 3.2 Mutant screening and identification

[0041] After the strains grew for 16 hours, pick the dark blue clones on each plate, extract the plasmids with a small amount of plasmid extraction kit (Shanghai Huasun Bioengineering Co., Ltd.), and use Nde I and HindIII for double enzyme digestion identification and sequencing Determination.

[0042] 3.3. Mutant gene amplification

[0043] Design a pair of primers:

[0044] 5'-CGCCATATGACACGTCAGATGATA-3'; and

[0045] 5'-CCCAAGCTTTCAGAGTTCCGCGAT-3'

[0046] Use the mutant plasmid obtained in step 3.2 as a template for PCR amplific...

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Abstract

The invention discloses four mutants of D-carbamoyl hydrolase and application to manufacture D-p-hydroxybenzene glycine in the biological engineering domain, which is characterized by the following: mutating the amino acid at 18th position of mutation enzyme 1 from alanine into threonine and tyrosine at 30th position of mutation enzyme 2 to asparagine; switching the lysine at 30th of mutation enzyme 2 into glutacid; overlapping three mutation positions; obtaining the mutation enzyme 4.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a mutant of D-carbamyl hydrolase and its application in producing D-p-hydroxyphenylglycine. Background technique [0002] β-lactam antibiotics have the advantages of strong antibacterial activity, broad-spectrum and low toxicity, and are one of the first-choice drugs for clinical control of bacterial infections. D-p-Hydroxyphenylglycine (DHPG) is an important intermediate in the synthesis of such drugs, and is used to prepare the side chains of amoxicillin and amoxicillin cephalosporins. The preparation of D-p-hydroxyphenylglycine has two industrial methods: chemical synthesis and enzymatic method. Among them, the chemical synthesis method has high cost and large pollution; while the enzymatic method has the characteristics of low cost and no pollution, so it has been paid more and more attention. The technical principle of the enzymatic method is: use D-hydantoinase (al...

Claims

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Application Information

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IPC IPC(8): C12N9/14C12N9/78C12N15/55C12P13/04C12N15/09C12N1/21
Inventor 姜卫红姜世民杨蕴刘杨晟
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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