黑曲霉葡萄糖氧化酶基因在毕赤酵母中的表达及产酶条件的优化

doi:10.13560/j.cnki.biotech.bull.1985.2020-1353

摘要/Abstract

摘要：

从黑曲霉中克隆葡萄糖氧化酶(glucose oxidase,E.C.1.1.3.4,GOD)基因,使其在毕赤酵母GS115中高效表达,为葡萄糖氧化酶的工业化生产提供理论依据。通过设计简并引物,扩增GOD,将该基因连接到质粒pPICZαA上并在毕赤酵母中表达,依据摇瓶水平优化最佳诱导产酶条件,在30 L发酵罐中进行发酵放大试验,共表达His4,采用DO-STAT的甲醇流加策略进行高密度发酵。结果表明,重组毕赤酵母GS115/pPICZαA-GOD在摇瓶水平经0.5%甲醇诱导96 h,发酵液上清GOD酶活达到32.25 U/mL。经优化得到最佳发酵条件为:在30℃、pH 6.0、250 r/min条件下,发酵液体积1%的甲醇诱导96 h,GOD酶活达到50.1 U/mL,相比初始发酵,酶活提高了55.3%。经30 L发酵罐放大试验,GOD酶活达到307.52 U/mL,提高了5.1倍。共表达His4使细胞湿重提高了72.5%,GOD酶活达到了461 U/mL,相比初始重组酵母提高了50.2%。SDS-PAGE分析发现GOD分子量约90 kD。重组GOD能在毕赤酵母中高效表达,且表达产物纯度高,杂蛋白少,有利于纯化。

关键词: 毕赤酵母, 葡萄糖氧化酶, 异源表达, 产酶优化

Abstract:

Glucose oxidase gene(GOD)was cloned from Aspergillus niger and highly expressed in Pichia pastoris GS115. The GOD gene was amplified by designing degenerate primers,and the gene was connected to the plasmid pPICZαA and expressed in P. pastoris. The optimized induction conditions for enzyme production were optimized according to the shaking flask level. The fermentation scale-up experiment was carried out in a 30 L bioreactor. The His4 was co-expressed and the methanol feed strategy of DO-STAT was used for high-density fermentation. The results showed that the GOD enzyme activity in the supernatant of the fermentation broth reached 32.25 U/mL after the recombinant P. pastoris GS115/pPICZ-GOD was induced by 0.5% methanol at the shake flask level for 96 h. The optimal fermentation conditions were obtained as follows:under the condition of 30℃,pH 6.0 and 250 r/min,the activity of GOD enzyme induced by 1.0% methanol for 96 h reached 50.1 U/mL,which was 55.3% higher than that of the initial fermentation. Through the scale-up experiment in a 30 L bioreactor,the GOD enzyme activity reached 307.52 U/mL,which increased by 5.1 times. Co-expression of the His4 increased the wet cell weight by 72.5% and the GOD enzyme activity reached 461 U/mL,which was an increase of 50.2% compared to the original recombinant yeast. SDS-PAGE analysis showed that the molecular weight of GOD was about 90 kD. Recombinant GOD can be highly expressed in P. pastoris with high purity and few miscellaneous proteins,which is beneficial to purification.

Key words: Pichia pastoris, glucose oxidase, heterologous expression, optimization of enzyme production

廖兆民, 蔡俊, 林建国, 杜馨, 王常高. 黑曲霉葡萄糖氧化酶基因在毕赤酵母中的表达及产酶条件的优化[J]. 生物技术通报, 2021, 37(6): 97-107.

LIAO Zhao-min, CAI Jun, LIN Jian-guo, DU Xin, WANG Chang-gao. Expression of Glucose Oxidase Gene from Aspergillus niger in Pichia pastoris and Optimization of Enzyme Production Conditions[J]. Biotechnology Bulletin, 2021, 37(6): 97-107.

图/表 13

图1 部分黑曲霉来源的葡萄糖氧化酶氨基酸序列比对

Fig.1 Alignment of glucose oxidase amino acid sequences from partial A. niger

图2 黑曲霉全基因组的琼脂糖凝胶电泳 M:DNA marker. 1:黑曲霉基因组DNA

Fig.2 Agarose gel electrophoresis of whole genome extraction from Aspergillus niger M: DNA marker. 1: A. niger genomic DNA

图3 简并引物Z1、Z2的PCR扩增产物的琼脂糖凝胶电泳 M:DNA marker. 1:简并引物Z1、Z2的PCR扩增产物

Fig. 3 Agarose gel electrophoresis of PCR products of degenerate primers Z1 and Z2 M: DNA marker. 1: PCR products of degenerate primer Z1 and Z2

图4 葡萄糖氧化酶的PCR扩增 M:DNA marker. 1:葡萄糖氧化酶的PCR扩增产物

Fig.4 PCR amplification of glucose oxidase M: DNA marker. 1: PCR amplification of glucose oxidase

图5 重组质粒pPICZαA-GOD构建及双酶切鉴定 A:重组表达载体的质粒图谱;B:双酶切鉴定结果;M:DNA marker.1:pPICZαA-GOD的双酶切产物

Fig.5 Construction of recombinant plasmid pPICZαA-GOD and identification by double restriction endonuclease digestion A: The plasmid map of the recombinant expression vector. B: The results of double restriction endonuclease digestion. M: DNA marker. 1: The product of double restriction endonuclease digestion of pPICZαA-GOD

图6 pPICZαA-GOD和pPICZαA线性化产物的琼脂糖凝胶电泳 M:DNA marker. 1:pPICZαA-GOD;2:pPICZαA-GOD线性化产物;3:pPICZαA;4:pPICZαA线性化产物

Fig.6 Agarose gel electrophoresis of the linearized products of pPICZαA-GOD and pPICZαA M: DNA marker. 1: pPICZαA-GOD; 2: pPICZαA-GOD linearization product;3: pPICZαA; 4: pPICZαA linearization product

图7 酵母转化子的高浓度zeocin抗性筛选

Fig.7 Screening of high concentration zeocin resistance of yeast transformants A:500 μg/mL. B:1 000 μg/mL. C:2 000 μg/mL

图8 高zeocin抗性酵母转化子表达葡萄糖氧化酶的活力曲线

Fig.8 Activity curve of glucose oxidase expressed by trans-formants with high zeocin resistance in yeast

图9 GS115/pPICZαA-GOD与GS115/pPICZαA-GOD/His4的摇瓶水平诱导结果

Fig.9 GS115/pPICZαA-GOD and GS115/pPICZαA-GOD/His4 shake flask level induction results

图10 重组酵母产GOD发酵条件的优化

Fig.10 Optimization of fermentation conditions for GOD production by recombinant yeast

图11 重组毕赤酵母GS115/pPICZαA-GOD的高密度发酵曲线

Fig.11 High-density fermentation curve of recombinant P. pastoris GS115/pPICZαA-GOD

图12 30 L发酵罐中重组毕赤酵母表达葡萄糖氧化酶蛋白的SDS-PAGE M:蛋白Maker;1-9:经甲醇诱导0、12、24、36、48、60、72、84和96 h葡萄糖氧化酶表达量

Fig.12 SDS-PAGE of glucose oxidase protein expressed in recombinant P. pastoris in 30 L bioreactor M: Protein Maker; 1-9: the expression of glucose oxidase was induced by methanol at 0, 12, 24, 36, 48, 60, 72, 84 and 96 h

图13 重组毕赤酵母GS115/pPICZαA-GOD/His4的高密度发酵曲线

Fig.13 High density fermentation curve of recombinant P. pastoris GS115/pPICZαA-GOD/His4

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