产乙醇酸氧化酶的重组毕赤酵母的构建及催化合成乙醛酸的研究

乙醇酸氧化酶（Go）是植物光呼吸途径中的一种关键酶，可以催化乙醇酸生产乙醛酸。从新鲜菠菜叶中提取总RNA，利用RT-PCR技术获得编码GO基因的cDNA片断。通过基因重组将GO基因克隆到载体pA0815中，构建了胞内表达载体pA0815／GO，重组质粒经电转整合至甲醇营养酵母GS115染色体。在混合碳源为10g/L山梨醇和0．5g/L甲醇的培养条件下，细胞的GO酶活达到474IU／g（DCW）。利用该重组毕赤酵母作为催化剂生产乙醛酸，结果表明：在乙醇酸浓度为0．25mol／L，重组酵母湿菌体为10dL，黄素单核苷酸（FMN）浓度为0．01mmol／L，pH8．0，20℃，反应18h后乙醛酸的产率达到51．8％。

Construction of recombinant Pichia pastoris producing glycolate oxidase and its catalytic property in synthesis of glyoxylic acid

Glycolate oxidase （GO） is a key-enzyme in the photorespiration of plants, which can catalyze glycolic acid into glyoxylic acid. A cDNA coding for glycolate oxidase was isolated from fresh spinach leaves by RT-PCR. The GO gene was cloned into the pAO815 under the control of AOX1 promoter and integrated into the genome of methylotrophic yeast Pichia pastoris GSll5. The GO activity of cells could reach up to 474 IU/g （DCW） in batch culture on mixed substrates of 1% sorbitol and 0.05 % methanol. Recombinant Pichia pastoris cells were used as bio-catalyst in the bio-producfion process of glyoxylic acid. Results showed that yield of glyoxylic acid reached 51.8% under the conditions of 0.25 mol/L glycolic acid, 0.01 mmol/L flavin mononuleotide, pH 8.0, temperature 20℃ and reaction time 18 h.