High-level expression and characterization of a thermophilic β-mannanase from Aspergillus niger in Pichia pastoris
High-level expression and characterization of a thermophilic β-mannanase from Aspergillus niger in Pichia pastoris
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DOI：
10.1007/s10529-015-1848-7
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发表时间：
2015-05
影响因子：
2.7
通讯作者：
Shi Yu;Zhezhe Li;Yaping Wang;Wanping Chen;Ling Fu;Wei Tang;Cheng Chen;Yunyun Liu;Xue Zhang;Lixin Ma
中科院分区：
文献类型：
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作者：
Shi Yu;Zhezhe Li;Yaping Wang;Wanping Chen;Ling Fu;Wei Tang;Cheng Chen;Yunyun Liu;Xue Zhang;Lixin Ma
目的 工业应用迫切需要一种新型、高水平表达、耐热的甘露聚糖内切1,4-β-甘露糖苷酶。结果根据毕赤酵母密码子使用偏好，对来自黑曲霉CBS 513.88的甘露聚糖内切-1,4-β-甘露糖苷酶基因（MAN）进行优化，并通过重叠PCR合成，产生MAN-P。它以P表示。来自组成型表达载体 pHBM-905 M.MAN-P 的巴斯德酵母 GS115 在摇瓶中诱导 192 小时后达到 594 mg/l。在 5 l 发酵罐中生产时，MAN-P 的产量达到约 3.5 mg/ml，酶活性为 1612 U/ml。该酶在 80°C 时表现出最大活性 3049 U/ml，并在 80°C 2 小时内保留 60% 的酶活性。最适pH为4.5，该酶在pH 1.5-11范围内稳定。结论 MAN-P的热稳定性高于其他已知的真菌甘露聚糖酶，其表达和嗜热特性使MAN-P可用于工业应用。
ObjectivesA novel, high-level expression, thermostable mannan endo-1,4-beta-mannosidase is urgently needed for industrial applications.ResultsThe mannan endo-1,4-β-mannosidase gene (MAN) fromAspergillus nigerCBS 513.88 was optimized based on the codon usage bias inPichia pastorisand synthesized by overlapping PCR to produceMAN-P. It was expressed inP. pastorisGS115 from a constitutive expression vector pHBM-905 M. MAN-P reached 594 mg/l in shake-flasks after 192 h induction. On production in a 5 l fermenter, the yield of MAN-P reached ~3.5 mg/ml and the enzyme activity was 1612 U/ml. The enzyme exhibited a maximum activity of 3049 U/ml at 80 °C and retained 60 % enzyme activity at 80 °C for 2 h. The pH optimum was 4.5 and the enzyme was stable over the pH range 1.5–11.ConclusionThe thermostability of MAN-P is higher than other known fungal mannanases and the expression and thermophilic properties make MAN-P useful for industrial applications.