Carnosine hydrolase, gene, mutant and application thereof

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...

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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 needs to be more than 16 times that of β-alaninamide, and the price of another substrate β-alaninamide is 40 times that of β-alanine. The price of L-carnosine is basically the same, so the synthesis process is expensive and difficult to realize industrial application

In 2011, Japanese Takeshita et al. found that the aminopeptidase RhDmpA3 from RhodotoruLa minuta IFO0879 can catalyze the condensation of β-alanine ester and L-histidine to generate L-carnosine (US2011 / 0081678 A1), but the report did not Published yield data for enzyme-catalyzed synthesis of L-carnosine

Nevertheless, in the prior art, there are few carnosine hydrolase species publicly reported, low catalytic activity, long reaction time, and low product concentration, which cannot meet the requirements of industrial production.

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