Lactone hydrolase and method of degrading alpha-zearalenol using the same

Abstract

A lactone hydrolase having improved activity towards -zearalenol is disclosed. The lactone hydrolase has a modified amino acid sequence of SEQ ID NO: 5, wherein the modification is a substitution of valine at position 167 with histidine. A method of degrading -zearalenol using such lactone hydrolase is also disclosed.

Claims

1. A method of degrading -zearalenol comprising steps of: (a) cloning zhd101 gene into pET46 vector to form pET46-zhd101 clone which is capable of expressing the protein of SEQ ID NO: 5; (b) substituting valine at position 167 in SEQ ID NO: 5 with histidine by site-directed mutagenesis and expressing the protein of SEQ ID NO: 8; and (c) incubating -zearalenol with the protein of SEQ ID NO: 8 to degrade -zearalenol.

2. The method according to claim 1 wherein the zhd101 gene is isolated from Gliocladium roseum.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the structures of ZEN and ZOL;

(2) FIG. 2 shows the hydrolyzation reaction of ZHD101 towards ZEN;

(3) FIG. 3 shows the nucleotide sequence of ZHD101;

(4) FIG. 4 shows the amino acid sequence of ZHD101;

(5) FIG. 5 shows the amino acid sequence of ZHD101 expressed by pET46 vector;

(6) FIG. 6 shows the sequences of the mutagenic primers;

(7) FIG. 7 shows the amino acid sequence of the mutant ZHD101; and

(8) FIG. 8 shows the activity analysis of the wild type and the mutant lactone hydrolase ZHD101.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

(9) The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for purpose of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.

(10) In order to improve the industrial application of ZHD101, the present invention cloned the zhd101 gene from Gliocladium roseum, which encodes a lactone hydrolase. By studying the molecular structure, a novel residue interacting with substrates in the catalytic site is mutated to improve the activity towards -ZOL. Following are the details of engineering the lactone hydrolase and description of the improved lactone hydrolase.

(11) First, the zhd101 gene from Gliocladium roseum was selected as the target gene, which consists of 795 base pairs (SEQ ID NO: 1, as shown in FIG. 3) and encodes 264 amino acids (SEQ ID NO: 2, as shown in FIG. 4). The gene was amplified by polymerase chain reaction (PCR) with the forward primer 5-GACGACGACAAGATGCGTACTCGTAGCACTATATCTA-3 (SEQ ID NO: 3) and the reverse primer 5-GAGGAGAAGCCCGGTTAAAGG TGTTTCTGAGTAGTCTCA-3 (SEQ ID NO: 4). The PCR amplification product was inserted into pET46 vector by using the pET46EK/LIC kit. With this plasmid construction strategy, the expressed ZHD101 protein has a vector sequence of 14 amino acids in the N terminal of the protein, so the expressed ZHD101 protein includes 278 amino acids (SEQ ID NO: 5, as shown in FIG. 5).

(12) In order to improve the activity of ZHD101 towards -ZOL, the site-directed mutagenesis was used to modify ZHD101. pET46-zhd101 was used as the PCR template and the primers was shown in FIG. 6, in which the forward primer was numbered as SEQ ID NO: 6 and the reverse primer was numbered as SEQ ID NO: 7. In this mutation design, the 153rd residue in SEQ ID NO: 2 was mutated from valine to histidine. Since the protein expressed by pET46 vector has a vector sequence of 14 amino acids in the N terminal of the protein, the mutant protein has a modified amino acid sequence of SEQ ID NO: 5, wherein the modification is a substitution of valine at position 167 with histidine. The mutant protein is represented as V167H, and the amino acid sequence thereof was numbered as SEQ ID NO: 8, as shown in FIG. 7. Afterwards a restriction enzyme DpnI was added to remove the template plasmid at 37 C. The purified PCR product was transformed into Escherichia coli competent cells, and the clones was first screened by antibiotic and further checked by DNA sequencing.

(13) The wild type and mutant clones were cultured in 5 ml LB culture as the primary culture seed, and then transferred to 200 ml secondary culture seed, and finally transferred into the 6 l culture, respectively. 1 mM IPTG was added to induce protein expression when OD of culture reached 0.6-0.8. After inducing for 3 hours, cells were harvested by centrifuge for 10 min at 6000 rpm. The cell pellets were resuspended in lysis buffer and disrupted by sonicator. The lysis solution was centrifuged for 30 min at 16000 rpm and the supernatant was collected for further purification. The Ni affinity chromatography and the DEAE anion exchange chromatography were used consecutively for purification with fast protein liquid chromatography (FPLC). Finally, the purified wild type and mutant proteins were obtained with the purity higher than 95%, and were stored at the concentration of 5 mg/ml at 80 C. in the solution of 25 mM Tris, 150 mM NaCl, pH 7.5.

(14) The activities of the wild type and the mutant lactone hydrolase ZHD101, which are proteins of SEQ ID NO: 5 and SEQ ID NO: 8, respectively, towards ZEN and -ZOL were determined to verify the difference therebetween. The assay of lactone hydrolase was shown as following.

(15) The assay solution (210 l) contains 5 l substrate (5 mg/ml ZEN or 5 mg/ml -ZOL) and 5 l enzyme (0.25 mg/ml ZHD101, wild type or mutant) in the solution of 25 mM Tris, 150 mM NaCl, pH 7.5. After incubated for 10 min at 30 C., 50 l 1 N HCl and 300 l methanol were added to stop the reaction. 20 l assay solution was tested by HPLC. The sample was eluted by 60% acetonitrile at the rate of 0.6 ml/min and the absorbance was detected at 254 nm. The amount of residual substrate was calculated according to the peak area. All data were determined three times and the average values were adopted.

(16) FIG. 8 shows the activity analysis of the wild type and the mutant lactone hydrolase ZHD101. After Val167 was mutated to His, the activity towards -ZOL of the mutant protein (V167H) was 3.7 times of that of the wild type (WT). Meantime, the activity towards ZEN was maintained in the mutant protein. These results showed that the mutant V167H improved the activity towards -ZOL and maintained the activity towards ZEN.

(17) From the above, in order to increase the detoxification efficiency, the key residue in the catalytic site of ZHD101 was mutated to increase the activity towards -ZOL. According to the present invention, the mutant V167H can increase the activity towards -ZOL, and the activity of the mutant V167H towards -ZOL was increased by 3.7 times, while the activity of the mutant V167H towards ZEN remains the same when compared with the wild type. In other words, the present invention also provides a method of degrading -ZOL by using the mutant V167H to degrade -ZOL so as to increase the degradation efficiency towards -ZOL. Therefore, the mutagenesis chosen by structural analysis of the present invention can obviously increase the detoxification efficiency of the lactone hydrolase ZHD101 and further enhance its application value in feed industry.

(18) While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclosed embodiment. On the contrary, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.