| Glucose oxidase (GOD) is named asβ-D-glucose:oxygen 1-oxidoreductase (EC 1.1.3.4) according to systematic nomenclature. This enzyme has the ability of antioxidation, removal of oxygen and glucose, as well as production of gluconic acid due to catalyzing the oxidation of P-D-glucose to gluconic acid and hydrogen peroxide with utilization of oxygen. Thus, glucose oxidase is widely used in the fields of food engineering, medicine and biotechnology. Strains of Aspergillus niger or Penicillium are mostly used to produce glucose oxidase by industrial-scale fermentation. However, several problems like low enzyme yield, contamination of hybridprotein and complicated procedures of purification emerge in this process. It is suggested that constructing recombinant strains of glucose oxidase with genetic engineering techniques would be an efficient approach to achieve high expression of the enzyme and facilitate the process of purification.In this study, the gene of glucose oxidase was cloned from Aspergillus niger Z-25 maintained by our lab, and then integrated into chromosome genomes of Pichia pastoris, consequently accomplishing high expression of glucose oxidase. Meanwhile, expression conditions of recombinant yeat strain in shake flask and high-density fermentation, purification of recombinant glucose oxidase as well as its biochemical characterizations were investigated. The main results are as following.1. The whole open reading frame of glucose oxidase with 1818 bp (GenBank Accession No. FJ979866) was amplified by PCR method using genomic DNA of A. niger Z-25 as the template. The GOD nucleotide sequence from A. niger Z-25 displayed 93% identity to those from A. niger NRRL-3 and A. niger ATCC 9029 (GenBank Accession numbers X16061 and J05242), while comparison of the deduced amino acid sequence of A. niger Z-25 with its counterparts showed identity degree of 97% with 17 amino acids replaced. The deduced amino acid sequence of GOD from A. niger Z-25 consisted of a signal peptide of 22 amino acids residues and a mature peptide of 583 amino acids residues. It contained three cysteine residues at conserved position (Cys164,Cys206,Cys521) and severn potential N-glycosylation sites conforming to the consensus sequence Asn-X-Thr/Ser (Asn89,Asn161,Asn168,Asn258,Asn355,Asn388,Asn473).2. The gene of glucose oxidase from A. niger Z-25 was ligated to the secretory expression vector pPICZaA, which resulted in recombinant expression vector pPICZaA-GOD. The recombinant plasmid pPICZaA-GOD was linearized with Pme I and then transformed into P. pastoris SMD1168. After isolation with gradient concentrations of zeocin and identification by PCR method, six transformants were selected for induction of glucose ocidase. Eventually, the recombinant yeast strain with the highest activity of glucose oxidase was screened and named as SMD1168/GOD.3. SMD1168/GOD strain was incubated in YPG medium until the cells were in logarithmic phase growth. The inoculum was resuspended with BMMY medium (pH 5.0). Then the induction was performed in 30℃with addition of 2.0% methanol every 24 h at 240 rpm. The GOD activity of roughly 40 U/mL was obtained after 168 hrs, which was 20-fold higher than that from A. niger Z-25. High-density fermentation of SMD1168/GOD strain was carried out with 29.5℃, pH 5.5 and 25% dissolved oxygen. After glycerol batch phase (20.5 h) and glycerol fed-batch phase (7.5 h), the introduction was started by addition of methanol. The maximal activity of recombinant GOD reached up to 522.5 U/mL after 142 hrs, which was 13-fold higher than that attained in shake flask.4. The recombinant GOD was purified by 60-90% ammonium sulfate precipitate.153.46 U/mg of rGOD was pooled from the culture supernatant after 3.34-fold purification with a final yield of 95.6%. SDS-PAGE analysis of the purified enzyme showed that the subunit molecular weight of rGOD was 94 kDa and the carbohydrate content was confirmed to be 31.9%. The optimum temperature and pH of the purified enzyme were 40℃and 6.0, respectively. Over 90% of maximal activity was retained below 40℃. And the recombinant enzyme displayed a favorable stability in the pH range from 4.0 to 8.0. The rGOD activity was considerably activated by Ag+ while strongly inhibited by Cu2+ and Pb2+, and weakly inhibited by Fe3+. The Lineweaver-Burk plot revealed that rGOD exhibited a Km value of 16.95 mM and a Kcat value of 484.26 s-... |
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