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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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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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