Carnosine hydrolase, gene, mutant and application thereof

A technology of hydrolase and carnosine, applied in the field of bioengineering, can solve the problems of low catalytic activity, no published L-carnosine yield data, and high cost of synthesis process

Pending Publication Date: 2019-03-15
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2005, Yamaoka et al. improved on the basis of this method, using L-histidine hydrochloride and β-alanyl chloride hydrochloride as raw materials, and obtained L-carnosine through two-step reactions with a yield of 68%, but Nitrogen protection is required during the reaction, and the reaction time is as long as 40h, and the final product will partially racemize (JP2005306782A)
In 2007, Hildbrand et al. used ethyl cyanoacetate and L-histidine as substrates to synthesize L-carnosine without corresponding protection and deprotection, and the yield could reach 70%, but a high pressure of 45 bar was required during the reaction. Under pass hydrogen for 1h, the reaction conditions are relatively harsh (US7164028B2)
Nevertheless, the amount of substrate L-histidine added in the reaction need

Method used

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  • Carnosine hydrolase, gene, mutant and application thereof
  • Carnosine hydrolase, gene, mutant and application thereof
  • Carnosine hydrolase, gene, mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Screening of carnosine hydrolase-producing bacteria

[0063] More than 500 strains preserved in the laboratory were cultured separately, the culture medium was centrifuged, cells were collected, and carnosine hydrolysis activity was screened. The preserved strains were inoculated into a test tube containing 4 mL of culture medium, and shaken at 30°C and 180rpm for 48h. After the culture was over, the cells were collected by centrifugation at 4°C and 8000rpm, and resuspended in Tris-HCl (50mM , pH8.0) buffer solution, at 30 ° C, 1000rpm shaking reaction for 24h, using high performance liquid chromatography to analyze the hydrolysis conversion rate of carnosine. The chromatographic column used is a chiral crown ether column (CR(+), φ40mm×150mm). The specific analysis conditions are: use perchloric acid solution with pH 1.0 as the mobile phase, the mobile phase flow rate is 0.3mL / min, and the detection wavelength is 220nm. temperature 25°C. The results are sho...

Embodiment 2

[0068] Embodiment 2. Cloning of carnosine hydrolase SmPepD gene

[0069] The strain Serratia marcescens ECU1010 was inoculated into LB medium for cultivation, and the total genomic DNA with high purity and large fragments was extracted by cetyltrimethylammonium bromide (CTAB) method. Add appropriate amount of Serratia marcescens cell into liquid nitrogen to freeze, grind into powder, add appropriate amount of 2×CTAB extraction buffer (100mmol / L Tris-HCl, pH 8.0, containing 20mmol / L EDTA, 1.4moI / L NaCl, 20g / L CTAB, 40mmol / L mercaptoethanol), at 65°C for 10 minutes, shaking intermittently. Then add an equal volume of chloroform / isoamyl alcohol, gently invert the centrifuge tube to mix, centrifuge at 12,000 rpm for 10 min at room temperature, transfer the supernatant to another centrifuge tube, add an equal volume of chloroform / isoamyl alcohol, and centrifuge upside down The tube was mixed well and centrifuged at 12000 rpm for 10 minutes at room temperature. Transfer the upper ...

Embodiment 3

[0074] Embodiment 3. Preparation of recombinant carnosine hydrolase SmPepD

[0075] Forward primer 5'-CCG GAATTC GTGTCTGAATTGTCTCAGCTTT-3', reverse primer 5'-CCG CTCGAG TTACGCGCGCTCAGGGATCGCTTT-3', the nucleotide sequence of the carnosine hydrolase SmPepD obtained in Example 2 is amplified by polymerase chain reaction technology, and the obtained amplified DNA fragment containing the SmPepD sequence is used with the restriction endonuclease EcoRI and XhoI double digestion, and then ligated with the plasmid pET28a that was also cut with restriction endonucleases EcoRI and XhoI to obtain the recombinant plasmid pET28a-SmPepD.

[0076] The obtained recombinant plasmid pET28a-SmPepD was transformed into Escherichia coli E.coli BL21, and the recombinant Escherichia coli was inoculated to the LB medium containing 50 μg / ml kanamycin (peptone 10g / L, yeast extract 5g / L, (NaCl 10g / L, pH 7.0), shake culture overnight at 37°C, transfer 1% (v / v) inoculum into a 2L Erlenmeyer flask conta...

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Abstract

The invention belongs to the technical field of bioengineering, and relates to carnosine hydrolase and a mutant thereof, a recombinant expression vector containing the enzyme and a mutant gene, and arecombinant expression transformant, a preparation method of the recombinant enzyme, a method for immobilizing the recombinant enzyme, and a method for preparing levo form L-carnosine by using the recombinant enzyme through reverse hydrolysis reaction. Compared with the prior art, the carnosine hydrolase disclosed by the invention is high in activity and high in thermal stability, and the enzyme is used for catalyzing beta-alanine and L-histidine to prepare L-carnosine through direct condensation, so that the steps of protection and deprotection in a conventional chemical method of synthesizing peptides are avoided; the process is simple, and the conditions are mild and environmentally friendly. Therefore, the carnosine hydrolase has an extremely good application prospect in industrial production of the L-carnosine.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a carnosine hydrolase and its mutant, a nucleic acid encoding the carnosine hydrolase, a recombinant expression vector containing nucleic acid and a recombinant expression transformant, a method for preparing the recombinant carnosine hydrolase, and Method for L-carnosine synthesis. Background technique [0002] L-carnosine [L-carnosine, N-β-Alanyl-L-histidine], also known as β-alanyl-L-histidine, molecular formula: C 9 h 14 N 4 o 3 , the molecular weight is 226.23, and the CAS number is 305-84-0. It is a dipeptide obtained by condensation of two amino acids, β-alanine and L-histidine. It is a crystalline solid and is a kind of Natural antioxidant. [0003] Lipids in the human body will undergo auto-oxidation reactions. This chain reaction is a free radical reaction. The free radicals generated by the reaction further catalyze the attack on unsaturated fat...

Claims

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Application Information

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IPC IPC(8): C12N9/48C12N15/57C12P17/10
Inventor 潘江殷东亚许建和钱小龙
Owner EAST CHINA UNIV OF SCI & TECH
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