| Glucose oxidase（EC 1.1.3.4, GOD） is a flavoprotein. Its enzyme molecule is a dimer which contains two subunits. GOD catalyzes the oxidation of β-D-glucose to gluconic acid and hydrogen peroxide（H2O2） using molecular oxygen as an electron acceptor. GOD has gained considerable commercial importance in the food, pharmaceutical, beverage, and textile industries for its unique properties. But now the existing microbial production strain still cannot meet the requirements of safety production for food grade. Therefore, food-grade recombinant production strain is desperately needed.In previous studies, Pichia pastoris GS115-pPIC9K-GOD,which could produce GOD, was constructed. However, methanol co-feeding limited the application of GOD in food area. In order to construct food grade recombinant strains with high yield, GOD gene from Aspergillus niger was extracellularly expressed in Yarrowia lipolytica and fermentation conditions of recombinant strains were examined in this study. A GOD high-yielding mutant was achieved through Atmospheric and Room Temperature Plasma（ARTP） mutagenesis and its enzyme characteristics were studied. The main results are listed as following:（1） Expression of Aspergillus niger GOD gene in Yarrowia lipolytica and fermentation optimizationThe GOD gene from Aspergillus niger was obtained by PCR and cloned to the expression vector pINA1297 to construct recombinant plasmid p INA1297/GOD. They were transformed into Y. lipolytica Polh strains. Yarrowia lipolytica 1-28 with relatively high GOD yield was obtained. Its GOD enzyme activity reached 6.4 U·m L-1 when cultured in shaker flasks for 120 h, which is 2.8 times of the wild Aspergillus niger strain.In order to improve the yield of GOD, the medium composition was determined by single factor optimization experiment and orthogonal experiment. When cultured in shaker flasks with the medium consisted of 20 g·L-1 glycerol, 2.64 g·L-1 yeast extract, 2.64 g·L-1 NH4 Cl, 0.32 g·L-1 KH2PO4, 0.13 g·L-1 MgSO4, 3.34×10-4 g·L-1 Vitamin B1 at pH 6.0 and 28°C for 120 h, the enzyme activity of GOD got improved obviously and reached 11.0 U·mL-1, increased by 72% compared to the original medium. In the 3 L fermentation tank, the pH control and glycerol feeding strategies were developed based on the optimized culture medium to further improve the yield. When fermented in the 3 L tank at pH 5.0 and glycerol feeding at the concentration of 30 g·L-1, the enzyme activity of GOD reached 81.6 U·mL-1, which increased 6.4 times compared with the highest enzyme activity in shaker flasks.（2） Screening of GOD high-yield strains by ARTP mutagenesis and high-throughput screening technologiesARTP mutagenesis was conducted with Y. lipolytica 1-28 to further improve the GOD yield. The mutagenesis time was set at 30 s, 60 s and 90 s with the lethal rate over 90%. With the combined use of high-throughput screening technologies, a positive mutant named T2 was finally achieved. Its GOD enzyme activity reached 15.8 U·mL-1 in shaker flasks, increased by 44% compared to the original strain. When fermented in the 3 L tank at 28?C for 96 h, the GOD enzyme activity of T2 reached 134 U·mL-1 at pH 5.0 and glycerol feeding at the concentration of 30 g·L-1, which increased by 64.2% compared to the original strain. The colony morphology and microscopic morphology of T2 have no obvious change compared with Y. lipolytica 1-28. It turned out that its GOD enzyme activity remained stable after the eighth generation by analysis of genetic stability.（3）Purification and characterization of the recombinant GODThe recombinant GOD from T2 were purified by ammonium sulfate precipitation and FF HitrapTM Q column purification. After purification, the characterization of recombinant GOD was performed. The optimum temperature and pH were 30?C and 6.0. Metallic ions Mg2+, Mn2+ and Ca2+ had positive effects on the enzyme activity of GOD, while Hg2+, Ag+ and Cu2+ obviously inhibited it. Its kinetic parameter KmA is 36.45 mmol·L-1. |
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