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Recombinant carnosine hydrolase mutant and application thereof

A hydrolase and mutant technology, applied in the field of bioengineering, can solve the problems of unsuitability for industrial production, low catalyst activity, and few varieties of carnosine hydrolase, etc.

Active Publication Date: 2021-01-26
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the existing reports, there are few types of carnosine hydrolase, relatively low catalyst activity, long reaction time, and low product concentration, which are not suitable for industrial production requirements

Method used

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  • Recombinant carnosine hydrolase mutant and application thereof
  • Recombinant carnosine hydrolase mutant and application thereof
  • Recombinant carnosine hydrolase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1. Molecular transformation of recombinant carnosine hydrolase

[0060] A random mutant library of carnosine hydrolase SmPepD was established by error-prone PCR technology, and high-throughput screening of the mutant library was performed using OPA derivatization to screen for mutants with increased activity in a low pH environment.

[0061] Design primers at both ends:

[0062] Forward primer 5'-CCG GAATTC GTGTCTGAATTGTCTCAGCTTT-3',

[0063] reverse primer 5

[0064] '-CT CGAGTG CGGCCGCAAGCTTACCGAGGCTCGAGATGAA-3'

[0065] The recombinant plasmid pET28a-SmPepD was used as a template for amplification. Polymerase chain reaction PCR system (50 μL): rTaq 0.25 μL, 10×Buffer 5 μL, dNTP Mix 4 μL, template plasmid about 100 ng, Primer F 2 μL, Primer R 2 μL, MnCl 2 (10mM) 0.5μL, diH 2 O to make up to 50 μL. The PCR program was: 98°C for 3min, 98°C for 30s, 55°C for 30s, 72°C for 10min, 30 cycles, 72°C for 10min. The PCR products were stored at 4°C for futur...

Embodiment 3

[0082] Example 3 Different pH to recombinant SmPepD M13 Effect on Synthetic Activity

[0083] 20 μL of SmPepD M13 The crude enzyme solution (freeze-dried enzyme powder concentration 5mg / mL) was added to 180μL of buffer solutions with different pHs containing 20mmol / LL-carnosine, and the reaction was shaken at 37°C for 5-20min to investigate the carnosine hydrolase SmPepD M13 Activity in different pH buffers. The buffer systems used were phosphate buffer (pH 5.5-7.5) and Tris-HCl (pH 7.5-9.0). The results are shown in Table 2. In the Tris-HCl buffer solution with a pH of 7.5, the relative activity of the enzyme is the highest, which is defined as 100%. Compared with the parent enzyme SmPepD, the mutant SmPepD M13 The optimum pH shifted from 8.0 to 7.5, and the activity at pH 6.0 was significantly improved.

[0084] Table 2. Carnosine hydrolase SmPepD M13 Activity in different pH buffers

[0085]

Embodiment 4

[0086] Example 4 Different pH to recombinant SmPepD M13 Effect of concentration on catalyzed synthesis of carnosine

[0087] (12) Replace the 134th proline with alanine in the amino acid sequence shown in SEQ ID No.2 in the sequence listing, replace the 309th alanine with isoleucine, and replace the 360th leucine is asparagine;

[0088] (13) Replace the 97th alanine with isoleucine in the amino acid sequence shown in SEQ ID No.2 in the sequence listing, replace the 275th alanine with tyrosine; replace the 301st valine is tryptophan;

[0089] (14) The 134th proline of the amino acid sequence shown in SEQ ID No.2 in the sequence listing is replaced with alanine, the 183rd alanine is replaced with valine, and the 281st arginine is replaced with half Cystine; asparagine at position 479 is replaced by alanine;

[0090] (15) Replace the 217th alanine with glutamic acid in the amino acid sequence shown in SEQ ID No.2 in the sequence listing, replace the 250th glycine with asparti...

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Abstract

The invention relates to a recombinant carnosine hydrolase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene of the enzyme mutant, a preparation method of the recombinase, and a method for preparing L-carnosine through reverse hydrolysis of the recombinase. Compared with the prior art, the recombinant carnosine hydrolase mutant provided by the invention has good thermal stability and low optimal pH, relatively cheap L-histidine hydrochloride can be directly used as a raw material for catalytic preparation of L-carnosine, higher product concentration can be reached, and the recombinant carnosine hydrolase mutant has good industrial application prospects in production of the L-carnosine.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to a recombinant carnosine hydrolase mutant, a recombinant expression vector and a recombinant expression transformant containing the gene of the recombinant carnosine hydrolase mutant, and the application of the recombinant carnosine hydrolase mutant to L- Methods of Carnosine Synthesis. Background technique [0002] L-carnosine, also known as β-alanyl-L-histidine, is a dipeptide obtained by condensation of two amino acids, β-alanine and L-histidine. It is a white crystalline solid and is a biological A natural antioxidant found in the body. As a natural dipeptide, L-carnosine exists in large quantities in animal muscle, brain tissue, and eye lens tissue, and the concentration can be as high as 20mM. In organisms, carnosine has pH buffering, anti-oxidation, anti-free radical and anti-aging effects. Clinically, it is used to reduce visual fatigue and treat catar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/48C12N15/57C12N15/70C12N1/21C12P13/02C12R1/19
CPCC12N9/485C12N15/70C12P13/02Y02P20/55
Inventor 潘江许建和殷东亚钱小龙
Owner EAST CHINA UNIV OF SCI & TECH
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