黑曲霉葡萄糖氧化酶在毕赤酵母中的表达及其高效发酵研究

葡萄糖氧化酶是一类能催化β-D-葡萄糖生成葡萄糖酸-δ-内酯的酶类,在食品、药品以及生物传感器等领域具有广泛的应用前景。从黑曲霉中克隆出葡萄糖氧化酶编码序列,在毕赤酵母中实现了组成型和诱导型表达并分析了其酶学性质和发酵条件。结果表明:黑曲霉葡萄糖氧化酶的编码基因长度为1 818 bp,编码605个氨基酸,具有21个氨基酸组成的信号肽。毕赤酵母组成型表达葡萄糖氧化酶的发酵上清液中葡萄糖氧化酶的酶活可达1.2 U/m L,利用镍离子亲和层析纯化后得到了分子质量约为100 kDa的葡萄糖氧化酶。利用糖苷内切酶H(Endo H)对葡萄糖氧化酶去糖基化后,其分子质量约为70 kDa,与推测的分子质量相近。对黑曲霉葡萄糖氧化酶的酶学性质研究表明,该酶的最适反应pH 6.0,最适反应温度35℃,比活力60.9 U/mg。为了进一步提高葡萄糖氧化酶在毕赤酵母中的表达量,构建了诱导型表达的毕赤酵母菌株并通过G418抗生素筛选获得了高效表达菌株。对该菌株进行高密度发酵(湿菌体200 g/L),甲醇添加量为2.5%,发酵至第7天时葡萄糖氧化酶发酵量可达133 U/m L,蛋白表达量约1.9 g/L。

Study on the expression of Aspergillus niger glucose oxidase in pichia pastoris and its high-efficiency fermentation

Glucose oxidase is a group of enzymes that can catalyze β-D-glucose to produce glucono-δ-lactone,and has broad application prospects in the production of food,medicine,and biosensors. In this study,the coding sequence of glucose oxidase was cloned from Aspergillus niger,and recombined to the constitutive expression vectors pGAPZαA. The recombined plasmid was linearized by restriction endonuclease BspH1 and transformed to Pichia patoris GS115 strain using a MicroPulser electroporator. The Pichia pastoris transformants were cultivated in liquid PDA medium,and the enzymatic properties of recombinant glucose oxidase were analyzed. The results showed that the coding gene of Aspergillus niger glucose oxidase was 1 818 bp,encoding 605 amino acids with a signal peptide composed of 21 amino acids. The enzyme activity of glucose oxidase in the fermentation supernatant of Pichia pastoris constitutively expressing glucose oxidase can reach 1. 2 U/mL. The purified glucose oxidase,which is about 100 kDa,was obtained by nickel ion affinity chromatography. After removing glycosylation of glucose oxidase by endoglycosidase H( Endo H),its molecular weight was about 70 kDa,which was similar to its estimated molecular weight. The study of its enzymatic properties showed that the optimal reaction pH value of the enzyme was 6. 0,the optimal reaction temperature was 35 ℃,and the specific activity was 60. 9 U/mg. To further increase the expression level of glucose oxidase in Pichia pastoris,the coding sequence of glucose oxidase from Aspergillus niger was recombined to the inducible expression vectors pPIC9 K. The recombined plasmid was linearized by restriction endonuclease Pme1 and transformed to Pichia patoris GS115 strain. The inducible expression of Pichia pastoris strain was cultivated and screened through G418 antibiotic,and a highly expressing strain that can grow on the YPD agar plate containing 4 mg/mL G418 antibiotic was obtained. The high-density fermentation of this strain( 200 g wet cells/L) showed that the optimal methanol addition amount was 2. 5%,and the fermentation capacity of glucose oxidase could reach 133 U/mL by the 7 th day of fermentation,and the protein concentration in fermentation broth was about 1. 9 g/L. This study provides experimental basis for the scale-up production of Aspergillus niger glucose oxidase.