β-甘露聚糖酶Man5A和木聚糖酶Tlxyn11B的融合表达
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国家自然科学基金 (No. 31601976) 资助。


Fusion expression of β-mannanase Man5A and xylanase Tlxyn11B in Pichia pastoris
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National Natural Science Foundation of China (No. 31601976).

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    摘要:

    甘露聚糖酶和木聚糖酶是主要的半纤维素降解酶,在食品、饲料、纺织、造纸等工业应用广泛且通常搭配使用。文中将蓝状菌Talaromyces leycettanus JCM12802来源的性质优良的甘露聚糖酶编码基因man5A的CBM (Carbohydrate-binding module) 编码区去除,留下连接区和催化区,并将木聚糖酶基因Tlxyn11B成熟区编码序列与man5A的连接区进行融合,形成Tlxyn11B-linker-man5A融合基因,并在毕赤酵母中成功表达,获得了融合蛋白Tlxyn11B-Man5A。Tlxyn11B、不含CBM区的Man5A和Tlxyn11B-Man5A的理论分子量分别为21.6 kDa、41.0 kDa、62.6 kDa。对纯化后的融合蛋白进行了性质分析,融合蛋白同时具有高的木聚糖酶和甘露聚糖酶活性。融合后的木聚糖酶的最适温度为70 ℃,较单独表达时提高了5 ℃。甘露聚糖酶的最适温度为90 ℃,与融合前一致。融合后的木聚糖酶热稳定性明显提高,60 ℃处理1 h剩余48%的酶活力,单独表达的木聚糖酶60 ℃处理20 min仅剩余20%的酶活力。融合后的木聚糖酶和甘露聚糖酶的最适pH分别为4.0和5.0,较单独表达时分别提高了0.5和1.0个单位,融合后酶的作用pH范围有所拓宽。融合前后的蛋白均具有较好的pH稳定性。融合后木聚糖酶和甘露聚糖酶的比活分别为1 784.3 U/mg和1 639.6 U/mg,较单独表达时比活 (8 300 U/mg和1 979 U/mg) 降低,与融合酶分子量增大相关。融合后的木聚糖酶和甘露聚糖酶的Km值分别为1.2 mg/mL和1.7 mg/mL, Vmax分别为2 000.0 μmol/(min·mg)和2 831.6 μmol/(min·mg)。综合其性质特点,融合木聚糖酶和甘露聚糖酶在饲料、食品等工业生产中有较大应用潜力,并为酶的性能改良提供了新的思路。

    Abstract:

    Mannanase and xylanase, the main hemicellulolytic enzymes, are widely used in food, feed, textile and papermaking industries, and usually they are used in combination. Mannanase Man5A from Talaromyces leycettanus JCM12802 consist of the carbohydrate binding module (CBM), linker region and catalytic domain. The CBM coding region of Man5A was removed and fused to C-terminal of the xylanase gene Tlxyn11B. The fusion gene Tlxyn11B-linker-man5A was successfully expressed in Pichia pastoris and the fusion protein Tlxyn11B-Man5A was purified and characterized. The theoretical molecular weights of Tlxyn11B, Man5A without CBM region, and Tlxyn11B-Man5A are 21.6 kDa, 41.0 kDa, and 62.6 kDa, respectively. The fusion protein had high xylanase and mannanase activities. The optimal temperature of the fused xylanase is 70 °C, which is 5 °C higher than Tlxyn11B-w (xylanase before fusion). The fused mannanase exhibited maximal activity at 90 °C, which is similar to Man5A-w (mannanase before fusion). More than 48% of xylanase activity of Tlxyn11B-Man5A was residual after the condition of 60 °C with 1 h, which is significantly higher than Tlxyn11B-w (only 20% of activity was left at 60 °C for 20 minutes). The optimal pHs of Tlxyn11B-Man5A for xylanase and mannanase activity are 4.0 and 5.0, respectively, which are 0.5 and 1.0 units higher than those of Tlxyn11B-w and Man5A-w. The pH range of fused enzymes got wider and the pH stability is improved. The specific activities of xylanase and mannanase of Tlxyn11B-Man5A are 1 784.3 U/mg and 1 639.6 U/mg, respectively, which is lower than those of Tlxyn11B and Man5A (8 300.0 U/mg and 1 979.0 U/mg). It may be due to of the high molecular weight of fusion enzyme. The Km and Vmax of the fused xylanase and mannanase are 1.2 mg/mL and 1.7 mg/mL, 2 000.0 μmol/(min·mg) and 2 831.6 μmol/(min·mg), respectively. Tlxyn11B and Man5A were successfully fusion expressed in P. pastoris, and the good properties of fusion of xylanase and mannanase make it has great application potential in animal feed, food and other industrial production, and it provided new ideas for the improvement of enzyme performance.

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孔海洋,蒋肖,王苑,黄火清,罗会颖. β-甘露聚糖酶Man5A和木聚糖酶Tlxyn11B的融合表达[J]. 生物工程学报, 2020, 36(9): 1849-1858

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  • 收稿日期:2020-02-04
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  • 在线发布日期: 2020-09-25
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