Patent Publication Number: US-10316303-B2

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

Description:
FIELD OF THE INVENTION 
     The present invention relates to a lactone hydrolase, and more particularly to a lactone hydrolase having increased efficiency for degrading α-zearalenol. 
     BACKGROUND OF THE INVENTION 
     Mycotoxin is the secondary metabolites secreted by mold or other fungi. The growth of mold and the production of the mycotoxin may occur in various processes including the food&#39;s maturity, transportation, processing and storage. Feeds contaminated by mycotoxin can cause animal poisoning and affect their immune systems, and even cause serious public health problems. Mycotoxin pollution has been one of the key factors limiting the development of feed and breeding industry. A quarter of crops are contaminated by mycotoxin in varying degrees in the USA and Canada every year, giving rise to a loss of 50 hundred million dollars in feed and breeding industry. The direct losses caused by mycotoxin in China reaches 100 hundred million RMB every year. Even worse situation occurs in south China due to the humid climate. 
     In order to get rid of the damage to breeding industry, various physical and chemical methods have been developed to absorb or degrade the mycotoxin in feed. Currently, the most popular methods are to remove the mycotoxin by absorption. However, the absorbent is often not selective, resulting in the loss of other nutrients. In addition, mycotoxin excreted from the body can cause secondary pollution. In contrast, the biological detoxification, using enzymes to specifically degrade mycotoxin in mild conditions, with no involvement of hazard chemicals and no loss of nutrients, is considered to be the best method. Developing high efficiency mycotoxin degrading enzymes is the most important way to solve the problems of mycotoxin pollution and to recover the great loss in feed and breeding industry. 
     Zearalenone (ZEN) is a kind of estrogen-like toxin produced by  Fusarium  specie, possessing the resorcylic acid lactone structure (as shown in  FIG. 1 ). ZEN is one of the three most seriously spread mycotoxin, identified firstly in moldy corn by Baldwin (Caldwell, R. W., Tuite, J., Stob, M., Baldwin, R., Zearalenone production by  Fusarium  species.  Applied Microbiology,  1970, 20 (1), 31-4). ZEN is mainly existed in corn, wheat, barley, and millet, etc, and can cause the estrogen disordering such as precocity and reproductive cycle disordering, bringing huge losses to crop farming and breeding industry. In addition, ZEN also has a strong carcinogenicity and can cause breast cancer and esophageal cancer. There are six common natural derivatives of ZEN, of which zearalenol (ZOL, as shown in  FIG. 1 ) is often coexisted with ZEN. ZOL has two isomers, α-ZOL and β-ZOL ( FIG. 1  shows a generic representation of ZOL comprising α-ZOL and β-ZOL), of which α-ZOL is the major form with a 30-fold higher toxicity than ZEN. 
     Naoko Takahashi-Ando (Takahashi-Ando, N., Kimura, M., Kakeya, H., Osada, H., Yamaguchi, I., A novel lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning.  The Biochemical Journal  2002, 365 (Pt 1), 1-6) identified a lactone hydrolase ZHD101 from  Gliocladium roseum , which becomes the most extensively studied ZEN degradation enzyme currently. ZHD101 hydrolyzes the lactone bond in the resorcylic acid lactone structure, opening the ring structure into a straight chain structure (as shown in  FIG. 2 ), which further spontaneously decarboxylized and isomerized to form the final product. The hydrolysis product cannot combine with estrogen receptors, thereby eliminating toxicity. The gene of ZHD101 has been successfully expressed in heterologous hosts. However, it was found that the α-ZOL was still remained in the solution after ZEN had been completely hydrolyzed, due to the lower activity of ZHD101 towards α-ZOL. Since α-ZOL has an even stronger toxicity than ZEN, a huge amount of ZHD101 is needed to remove α-ZOL and to achieve the complete detoxification. Therefore, it is of great value to improve the degradation activity of ZHD101 towards high toxic α-ZOL. 
     Therefore, in order to improve the efficiency of detoxification of zearalenone and the derivatives, the present invention intends to increase the activity of ZHD101 towards α-ZOL by genetic modification, while maintaining the activity towards ZEN. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to modify a current lactone hydrolase ZHD101 by means of structural analysis and site-directed mutagenesis to efficiently improve its activity towards α-ZOL, which is a natural derivative of ZEN and has high toxicity, improve its detoxification efficiency, and further increase its economic value of industrial application. 
     An another object of the present invention is to provide a method of degrading α-ZOL by using the modified ZHD101 mutant to degrade α-ZOL so as to increase the degradation efficiency towards α-ZOL. 
     According to an aspect of the present invention, there is provided a lactone hydrolase comprising a modified amino acid sequence of SEQ ID NO: 5, wherein the modification is a substitution of valine at position 167 with histidine. The amino acid sequence of SEQ ID NO: 5 adds a pET46 vector sequence of 14 amino acids in N terminal of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 2 is encoded by zhd101 gene isolated from  Gliocladium roseum . The lactone hydrolase has a full length amino acid sequence of SEQ ID NO: 8, and is used to increase the degradation efficiency towards α-zearalenol. 
     According to an another aspect of the present invention, there is provided a method of degrading α-zearalenol comprising a step of using a lactone hydrolase to degrade α-zearalenol, wherein the lactone hydrolase comprises a modified amino acid sequence of SEQ ID NO: 5, and the modification is a substitution of valine at position 167 with histidine. The amino acid sequence of SEQ ID NO: 5 adds a pET46 vector sequence of 14 amino acids in N terminal of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 2 is encoded by zhd101 gene isolated from  Gliocladium roseum . The lactone hydrolase has a full length amino acid sequence of SEQ ID NO: 8. 
     The above objects and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed description and accompanying drawings, in which: 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows the structures of ZEN and ZOL; 
         FIG. 2  shows the hydrolyzation reaction of ZHD101 towards ZEN; 
         FIG. 3  shows the nucleotide sequence of ZHD101; 
         FIG. 4  shows the amino acid sequence of ZHD101; 
         FIG. 5  shows the amino acid sequence of ZHD101 expressed by pET46 vector; 
         FIG. 6  shows the sequences of the mutagenic primers; 
         FIG. 7  shows the amino acid sequence of the mutant ZHD101; and 
         FIG. 8  shows the activity analysis of the wild type and the mutant lactone hydrolase ZHD101. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     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. 
     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 α-ZOL. Following are the details of engineering the lactone hydrolase and description of the improved lactone hydrolase. 
     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 ). 
     In order to improve the activity of ZHD101 towards α-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. 
     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. 
     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 α-ZOL were determined to verify the difference therebetween. The assay of lactone hydrolase was shown as following. 
     The assay solution (210 μl) contains 5 μl substrate (5 mg/ml ZEN or 5 mg/ml α-ZOL) and 5 μl 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 μl 1 N HCl and 300 μl methanol were added to stop the reaction. 20 μl 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. 
       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 α-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 α-ZOL and maintained the activity towards ZEN. 
     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 α-ZOL. According to the present invention, the mutant V167H can increase the activity towards α-ZOL, and the activity of the mutant V167H towards α-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 α-ZOL by using the mutant V167H to degrade α-ZOL so as to increase the degradation efficiency towards α-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. 
     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.