A kind of acid-resistant zearalenone detoxification enzyme and its coding gene and application

Abstract

The invention relates to the field of enzyme engineering, and in particular discloses acid-resistant zearalenone detoxifying enzyme and an encoding gene thereof, and application of the enzyme and the gene. The amino acid sequence of the zearalenone detoxifying enzyme is optimized, 8 amino acid residues (with 8 amino acid difference from zlhy-6 detoxifying enzyme) of the detoxifying enzyme are changed, the optimal reaction pH of recombinant expression zearalenone detoxifying enzyme is reduced from 8.5 to 6.5 and is very identical with the pH of pig and chicken intestinal canals, and the enzyme activity of the detoxifying enzyme still maintains 40 percent or more when the pH is reduced to 5.0, so the detoxifying efficiency of the zearalenone detoxifying enzyme serving as a feed additive to actual application is greatly improved. The zearalenone detoxifying enzyme gene is subjected to codon optimization, and the copy number of the gene in a recombinant yeast genome is increased to be 4, so the expression quantity of the zearalenone detoxifying enzyme is greatly increased.

Description

technical field [0001] The invention relates to the field of enzyme engineering, in particular to an acid-resistant zearalenone detoxification enzyme, its coding gene and its application. Background technique [0002] Zearalenone (Zearalenone, ZEN), also known as F-2 toxin, is a 2,4-dihydroxybenzoic acid lactone compound, a mycotoxin mainly produced by the secondary metabolism of Fusarium graminearum. ZEN has estrogenic activity and is toxic to the reproductive system. Excessive intake of ZEN can cause vulva and mammary gland enlargement and even vaginal and rectal prolapse; affect mammalian breast development, lead to delayed lactation, false pregnancy, and even infertility or miscarriage , fetal malformation and stillbirth. ZEN can induce tumors, not only stimulate the growth of human breast cancer MCF-7 cells by increasing the activity of cytochrome CYP1A1 enzyme, but also one of the causes of the increased incidence of esophageal cancer. ZEN also has obvious toxic effe...

AI Technical Summary

Problems solved by technology

However, the optimum pH of the enzyme is 8.5, and the degradation ability of the enzyme at pH 5.0 and 6.5 is only 15% and 25% of that at pH 8.5, respectively, which shows that the acidic condition seriously inhibits the detoxification enzyme of zearalenone activity; the detoxification enzyme is mainly mixed into pig and chicken feed as an additive to degrade zearalenone in pigs and chickens, and the pH value of the intestinal tract of pigs and chickens is between 5.5 and 6.5, so there is an urgent need for corn The acid-resistant transformation of erythralenone detoxification enzyme makes the optimal pH of its activity change from 8.5 to slightly acidic

Not only that, the above technical scheme also connects the zearalenone detoxification enzyme coding gene obtained by cloning to the plasmid pPIC9K and transforms Pichia pastoris to obtain a recombinant Pichia pastoris strain capable of expressing zearalenone detoxification enzyme, but zearalenone detoxification enzyme The activity (expression level) of mycocetone detoxification enzyme is very low, and 1mL of recombinant yeast fermentation supernatant can only degrade 18.8μg of ZEN in 2 hours

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