阅读说明：本技术 玉米赤霉烯酮水解酶突变体zhdm1及其编码基因和应用 (Zearalenone hydrolase mutant ZHDM1 and coding gene and application thereof ) 是由 姚斌 罗会颖 于心蕊 涂涛 柏映国 黄火清 苏小运 王亚茹 王苑 张�杰 于 2019-09-02 设计创作，主要内容包括：本发明属于农业生物技术领域,具体涉及玉米赤霉烯酮水解酶突变体ZHDM1及其编码基因和应用。玉米赤霉烯酮水解酶突变体ZHDM1,通过将野生型玉米赤霉烯酮水解酶ZH607的第136-142位氨基酸突变得到。本发明的玉米赤霉烯酮水解酶突变体ZHDM1的酶活力是野生型的2.9倍,因此,本发明高酶活力突变体能够满足能源、食品和饲料等领域中对于玉米赤霉烯酮降解活性的要求,具有广阔的应用前景。(the invention belongs to the technical field of agricultural biology, and particularly relates to a zearalenone hydrolase mutant ZHDM1, and a coding gene and application thereof. Zearalenone hydrolase mutant ZHDM1 is obtained by mutating 136-142 th amino acids of wild zearalenone hydrolase ZH 607. The enzyme activity of the zearalenone hydrolase mutant ZHDM1 is 2.9 times of that of a wild type, so that the high-enzyme-activity mutant can meet the requirement on the zearalenone degradation activity in the fields of energy, food, feed and the like, and has a wide application prospect.)

1. The zearalenone hydrolase mutant ZHDM1 is characterized in that the zearalenone hydrolase mutant ZHDM1 is obtained by mutating 136-142 th amino acids of a wild zearalenone hydrolase ZH607 from ASVTGME to DILLHIH, wherein the amino acid sequence of the wild zearalenone hydrolase ZH607 is shown as SEQ ID No. 1.

2. A zearalenone hydrolase mutant ZHDM1 gene encoding the zearalenone hydrolase mutant ZHDM1 according to claim 1.

3. The zearalenone hydrolase mutant ZHDM1 gene according to claim 2, characterized in that its nucleotide sequence is represented by SEQ ID No. 3.

4. A recombinant expression vector comprising the zearalenone hydrolase mutant ZHDM1 gene of claim 2.

5. A recombinant strain comprising the zearalenone hydrolase mutant ZHDM1 gene of claim 2.

6. A method of preparing zearalenone hydrolase mutant ZHDM1 according to claim 1, characterized in that the method comprises the steps of:

(1) Transforming a host cell by using a recombinant expression vector containing a zearalenone hydrolase mutant ZHDM1 encoding gene to obtain a recombinant strain;

(2) Culturing the recombinant strain, and inducing and expressing zearalenone hydrolase mutant ZHDM 1;

(3) isolating and purifying the expressed zearalenone hydrolase mutant ZHDM 1.

7. The use of zearalenone hydrolase mutant ZHDM1 according to claim 1.

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to a zearalenone hydrolase mutant ZHDM1, and a coding gene and application thereof.

Background

zearalenone (ZEN), also known as F-2 toxin, is a non-steroidal, estrogenic mycotoxin produced by a variety of fusarium species. The estrogen-responsive toxicity of ZEN is manifested not only by impairment of female reproductive ability, but also by impairment of male reproductive system. In addition, high dose ZEN has hepatotoxicity, intestinal toxicity, immunotoxicity and combined toxicity of mycotoxins thereof, which seriously endanger the health of animals.

The elimination of ZEN pollution by microbial degradation is a process of utilizing the action of enzyme generated by microorganisms in the metabolic process and ZEN to destroy the toxic groups of ZEN molecules so as to degrade or convert the ZEN molecules into non-toxic products. The degradation ZEN by the biodegradation method has strong specificity, the ZEN molecule is converted efficiently, no environmental pollution is caused, and the original nutritive value of the feed can be reserved. However, the zearalenone hydrolase activities found at present are all low, which hinders large-scale industrial production and application of the zearalenone hydrolase.

Disclosure of Invention

the invention aims to provide a zearalenone hydrolase mutant ZHDM 1.

It is still another object of the present invention to provide a gene encoding the above mutant.

It is still another object of the present invention to provide a recombinant expression vector containing the above-mentioned coding gene.

It is still another object of the present invention to provide a recombinant strain containing the above-mentioned encoding gene.

Still another object of the present invention is to provide a method for preparing the zearalenone hydrolase mutant ZHDM 1.

The invention further aims to provide application of the zearalenone hydrolase mutant ZHDM 1.

According to a specific embodiment of the invention, the zearalenone hydrolase mutant ZHDM1 is obtained by mutating the 136-142 amino acids of the wild-type zearalenone hydrolase ZH607 from ASVTGME to dillhh, wherein the amino acid sequence of the wild-type zearalenone hydrolase ZH607 is shown in SEQ ID No. 1:

MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAASGFRVTTFDMPGMSRSRDAPPETYRDVTGHKLAGYVDTLLEELKIPIASVWGCSSGATTVLALCAAFPERVRNAMPHELPTVNPASVTGMEEKDPAVISQEMAAVSRSISGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPVDWTVGGGTPTAMFFDNIVIAVKEGLNIGLLPGGHFPYVSHPEAFAEYVVETCRKYI

the amino acid sequence of the zearalenone hydrolase mutant ZHDM1 is shown as SEQ ID NO. 2:

MSTTRATKTVTTKDGIKWHVEQEGNGPDIVLVPDGLGECQMFDKPMSIIAASGFRVTTFDMPGMSRSRDAPPETYRDVTGHKLAGYVDTLLEELKIPIASVWGCSSGATTVLALCAAFPERVRNAMPHELPTVNPDILLHIHEKDPAVISQEMAAVSRSISGGTEAWDALGPEVHARLHDNYVRWARGYPVTIPPSAPTKSEVLHKRPVDWTVGGGTPTAMFFDNIVIAVKEGLNIGLLPGGHFPYVSHPEAFAEYVVETCRKYI

The nucleotide sequence of the zearalenone hydrolase mutant ZHDM1 encoding gene is shown as SEQ ID NO. 3:

ATGTCCACCACCAGAGCCACCAAGACTGTTACCACCAAGGATGGAATTAAGTGGCATGTTGAGCAAGAAGGAAACGGTCCAGACATTGTGTTGGTTCCAGATGGTTTGGGTGAGTGTCAAATGTTTGATAAGCCCATGTCCATTATTGCAGCCTCCGGTTTTAGAGTCACTACCTTTGATATGCCAGGTATGAGTAGATCCAGAGATGCTCCTCCAGAAACTTACAGAGATGTTACCGGTCATAAGCTGGCTGGTTACGTTGACACTTTGCTTGAGGAATTGAAGATTCCAATTGCTTCTGTTTGGGGTTGTTCCTCTGGTGCTACTACTGTTCTGGCCTTGTGTGCTGCTTTTCCAGAAAGAGTTAGAAATGCCATGCCACACGAGTTGCCAACTGTTAACCCAGATATTTTACTTCATATTCATGAGAAGGACCCTGCTGTTATTTCTCAAGAAATGGCTGCTGTTTCAAGATCAATTAGTGGTGGTACTGAAGCCTGGGATGCTTTAGGACCAGAAGTTCATGCTAGACTTCATGATAATTACGTTAGATGGGCTAGAGGTTACCCAGTGACTATTCCACCATCTGCCCCAACCAAGTCTGAGGTTTTGCATAAGAGACCAGTTGATTGGACAGTTGGTGGTGGTACCCCAACTGCTATGTTTTTTGATAATATTGTCATCGCTGTCAAGGAAGGTTTGAACATTGGTTTGTTGCCAGGTGGTCATTTCCCATACGTTTCTCATCCTGAAGCTTTTGCTGAATACGTTGTTGAGACTTGTAGAAAGTACATTTAA

The above sequence contains a terminator of 798bp in total.

The invention also provides a recombinant expression vector containing the coding gene, preferably pPICZ (alpha) A-zh 607-AH.

The invention also provides a recombinant strain containing the coding gene, preferably pichia pastoris X33(pPICZ (alpha) A-zh 607-AH).

The preparation method of zearalenone hydrolase mutant ZHDM1 according to the embodiment of the invention comprises the following steps:

(1) Transforming a host cell by using a recombinant expression vector containing a zearalenone hydrolase mutant ZHDM1 encoding gene to obtain a recombinant strain;

(2) Culturing the recombinant strain, and inducing and expressing zearalenone hydrolase mutant ZHDM 1;

(3) Isolating and purifying the expressed zearalenone hydrolase mutant ZHDM 1.

the invention also provides application of the zearalenone hydrolase mutant ZHDM1, in particular to the fields of energy, food and feed.

The invention has the beneficial effects that:

the enzyme activity of the zearalenone hydrolase mutant ZHDM1 is improved to 14419U/mg from 4940U/mg of a wild type, and is 2.9 times of the wild type, so that the mutant with high enzyme activity can meet the requirement on the zearalenone degradation activity in the fields of energy, food, feed and the like, and has wide application prospect.

Drawings

FIG. 1 shows SDS-PAGE electrophoresis detection results of gibberellin ketone hydrolase wild type and mutant expressed in Pichia pastoris X33.

Detailed Description

1. Bacterial strain and carrier: the expression host Pichia pastoris X33 of the invention expresses plasmid vector pPICZ (alpha) A.

2. Enzymes and other biochemical reagents: the endonuclease was purchased from TaKaRa, the ligase was purchased from Invitrogen, and the others were made reagents in China.

3. Coli medium LLB (1% peptone, 0.5% yeast extract, 0.5% NaCl, pH Nature). Pichia pastoris medium YPD (Yeast Extract 1%, Trytone 2%, Glucose 2% pH natural); BMGY (YeastExtract 1%, Trytone 2%, YNB 10%, biotin 0.1%, pH natural); BMMY (Yeast Extract 1%, Trytone 2%, methanol 0.5%, YNB 10%, biotin 0.1%, pH Natural).

Description of the drawings: the molecular biological experiments, which are not specifically described in the following examples, were performed according to the methods listed in molecular cloning, a laboratory manual (third edition) J. SammBruker, or according to the kit and product instructions.