Study On The Expression Of Glucose Oxidase And Its Application In The Hydrolysis Of Hemicellulose

Xylose is a monosaccharide traditionally produced from plant hemicellulose using enzymatic saccharification. Because it dose not lead to metabolic disorder such as diabetes and obesity, xylose can be used as a substitute for table sugar. The pretreatment of bagasse with hydrogen peroxide(H2O2) facilitates the fractionation, solubilization, hydrolysis and separation of cellulose, hemicellulose and lignin components. Glucose oxidase(GOD) catalyzes the oxidation of β-D-glucose and the reduction of oxygen to generate H2O2. Therefore, GOD could replace H2O2 in order to enhance hydrolysis of hemicellulose. In order to verify this conjecture, we designed a series of experiments and analyzed the obtained results.In order to improve the expression of GOD and lay a foundation for further studies, we built and acquired several single gene recombinant yeasts. First, Aspergillus niger CICC 40179 god gene was cloned into p MD19-T vector. Sequence and homology analysis by BLAST revealed it was a new god gene that has not been reported before. The structure comparison of different proteins through 3D structure modeling showed that the two variable residues were located at the external of the protein, which did not alter their stuctures.The new god gene was cloned into p2μRCMB and p GAPZαA for expression in Saccharomyces cerevisiae AS2.489 and Pichia pastoris X33 strain, respectivly.The molecular weight of the recombinant GOD from S. cerevisiae is approximately 100 k Da, while that from P. pastoris is approximately 70 k Da. GOD activity of the recombinant P. pastoris was significantly higher than that of the recombinant S. cerevisiae. Therefore, the recombinant P. pastoris was uesd to study enzymology characteristics, orthogonal optimization and enzymatic hydrolysis. The maximum activity of GOD was obtained at p H 7.5 and 35 °C, and the residual activity had little change at 25 °C to 40 °C after 2 h of incubation. Thus, the new GOD is probably a mesophilic enzyme with a relatively wide working temperature. The metal ions Mn2+ and Co2+ significantly improved the enzyme activity of GOD, whereas Zn2+ significantly inhibited it. The enzymatic activity of new GOD was robustly elevated(from 4.9 U/m L in shaken flasks to 16.31 U/m L in SL-05 fermenter) using orthogonal experiment.The catalytic characteristic of GOD was considered to apply to the production of xylose from hemicellulose. The p H value, temperature and concentration of GOD were optimized using the Box-Behnken design to improve the yield of xylose from sugarcane bagasse(SCB) hemicellulose. GOD(31.68 U/g) significantly increased the efficiency of xylose production from 67.21 % to 76.72 % when the reaction was conducted under the optimal conditions(p H 5.1, 44.9 °C, for 36 h). In addition, glucose was not detected in supernatants supplemented with GOD. This study prospectively demonstrated the application of GOD on the enzyme-catalysed production of xylose from SCB hemicellulose. This newly developed enzymatic hydrolysis system can contribute to industrial xylose production.