Mutant of D-carbamyl hydrolysis enzyme and application thereof

A technology of carbamoyl hydrolase and mutant, which is applied in the application field of the mutant of D-carbamyl hydrolase and the production of D-p-hydroxyphenylglycine, and can solve the problems of not improving thermal stability and the like

Active Publication Date: 2009-09-30
洛阳华荣生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, although the protein soluble expression of these three mutants was greatly improved, their thermal stability was still not improved

Method used

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  • Mutant of D-carbamyl hydrolysis enzyme and application thereof
  • Mutant of D-carbamyl hydrolysis enzyme and application thereof
  • Mutant of D-carbamyl hydrolysis enzyme and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 , Cloning of DCase-M3 gene

[0033] 1.1, PCR amplification

[0034] Design a pair of primers:

[0035] 5'-GCTTTCAGAGTTCCGCGATCA-3'; and

[0036] 5'-TATGACACGTCAGATGATACTTGC-3'.

[0037] PCR amplification was performed using pET28b-Dcase-M3 (Jiang, S., et al., Biochem J, 2007.402(3): p.429-37) as a template.

[0038] PCR system: 17.75 μl redistilled water, 2.5 μl PCR buffer, 1 μl MgCl 2 (25 mM), 1.5 μl dNTP (2.5 mM), 0.5 μl of the above two primers, 15 ng of the above template, and 2 units of Taq enzyme (Shanghai Sangon Company).

[0039] PCR conditions: 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.

[0040] 1.2. Construction of plasmid containing DCase-M3 gene

[0041] After PCR amplification, first use 1% agarose gel to carry out nucleotide electrophoresis with a voltage of 100 volts. After cutting the gel...

Embodiment 2

[0046] Example 2 , Random mutation of DCase-M3 gene

[0047] Primer pair:

[0048] 5'-GCTTTCAGAGTTCCGCGATCA-3'; and

[0049] 5'-TATGACACGTCAGATGATACTTGC-3'.

[0050] Error-prone PCR reaction system (50μl): 20ng of template pET28b-Dcase-M3 (Jiang, S., et al., Biochem J, 2007.402(3): p.429-37), a pair of primers of 30pmol each, 7mM MgCl 2 , 50mM KCl, 10mM Tris-HCl (pH8.3), 0.2mM dGTP, 0.2mM dATP, 1mM dCTP, 1mM dTTP, 0.05mM MnCl 2 , and 5 units of Taq enzyme (Shanghai Sangon Company).

[0051] PCR reaction conditions: After 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 a full extension at 72°C for 10 min.

[0052] After PCR amplification, first use 1% agarose gel for nucleotide electrophoresis with a voltage of 100 volts. After cutting the gel, use the gel recovery kit (Shanghai Huashun Bioengineering Co., Ltd.) to recover the target DNA fragment of about 900 b...

Embodiment 3

[0057] Example 3 , screening of mutant libraries

[0058] 3.1. Transformation of mutants

[0059] The mutant constructed in Example 2 was transformed into E. coli DH10B (Invitrogen) by electric shock method. Transformed cells were smeared on LB plates (1% peptone, 0.5% yeast extract, 1% sodium chloride, 2% agar) containing ampicillin antibiotics, and cultured at 37°C.

[0060] 3.2 Mutant screening and identification

[0061] After the bacteria grew for 12 hours, a single colony was picked and added to a 96-well cell culture plate with 300 μl / well, 1 single colony / well, cultured on a shaker at 37° C. and 220 rpm. After 2 hours, the cells were induced with 1 mM IPTG, and cultured for 18 hours.

[0062] Take 100 μl / well of the cultured bacterial liquid into a 96-well microtiter plate, place the whole in a -70°C environment, and transfer it to a 37°C environment for recovery after 1.5 hours. The recovery time is about 30 minutes.

[0063] Mix the resuscitated bacterial solut...

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Abstract

The invention discloses a mutant of D-carbamyl hydrolysis enzyme and application thereof in producing D-p-hydroxyphenylglycine. The method comprises the following steps that: a mutant is obtained by the directed evolution technology and mutates into nonpolar amino acid in 12-glutamine of the D-carbamyl hydrolysis enzyme; another mutant mutates into methionine in 263-threonine of the D-carbamyl hydrolysis enzyme; a third mutant mutates into serine in 263-threonine of the D-carbamyl hydrolysis enzyme; a fourth mutant mutates into the threonine in 12-glutamine of the D-carbamyl hydrolysis enzyme; and the 263-threonine mutates into the serine. The mutant enzyme shows higher thermal stability and enzyme activity in the production of the D-p-hydroxyphenylglycine, and indicates that using the directed evolution technology to reconstruct industrial enzyme is a quite effective method.

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] D-p-hydroxyphenylglycine (DHPG) is a key chiral intermediate in the synthesis of β-semisynthetic lactam antibiotics such as amoxicillin and amoxicillin cephalosporins. The current global market demand is about 22,000 tons, equivalent to about 1.7 billion yuan. Organic synthesis of racemic p-hydroxyphenylglycine followed by chiral resolution was the main method for producing DHPG. After the 1980s, D-hydantoinase (Dhase) / D-carbamoylase (D-carbamoylase, Dcase) two-step asymmetric hydrolysis mixed-rotation p-hydroxyphenylhydantoin production method for DHPG is gradually adopted by large foreign companies, and its principle is as follows: [0003] [0004] The technical principle of the enzymatic method is: use D-hydantoinase (also known as D-hydantoin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/78C12N15/55C12N15/63C12N1/21C12P13/04C12R1/19
Inventor 杨晟余宏李键张大龙姜卫红杨蕴刘
Owner 洛阳华荣生物技术有限公司
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