异源表达和生物化学特性分析来自于白蚁台湾乳白蚁的 GHF9 内切葡聚糖酶在毕赤酵母中的表达。
Heterologous expression and biochemical characterization of a GHF9 endoglucanase from the termite Reticulitermes speratus in Pichia pastoris.
机构信息
Sichuan Normal University, College of Life Science, Chengdu, 610101, China.
National Key Laboratory of Biochemical Engineering, National Engineering Research Center for Biotechnology (Beijing), Key Laboratory of Biopharmaceutical Production & Formulation Engineering, PLA, Institute of Process Engineering, Chinese Academy of Sciences, No. 1 North 2nd Street, Beijing, 100190, China.
出版信息
BMC Biotechnol. 2018 Jun 1;18(1):35. doi: 10.1186/s12896-018-0432-3.
BACKGROUND
Cellulases are of great significance for full utilization of lignocellulosic biomass. Termites have an efficient ability to degrade cellulose. Heterologous production of the termite-origin cellulases is the first step to realize their industrial applications. The use of P. pastoris for the expression of recombinant proteins has become popular. The endoglucanase from Reticulitermes speratus (RsEG), belonging to glycoside hydrolase family 9 (GHF9), has not been produced in P. pastoris yet.
RESULTS
A mutant RsEG (G91A/Y97W/K429A) was successfully overexpressed in P. pastoris. RsEG, with optimum pH 5.0, was active over the pH range of 4.0 to 9.0, and exhibited superior pH stability over between pH 4.0 and pH 11.0. It displayed the highest activity and good stability at 40 °C, but lost activity quickly at 50 °C. The apparent kinetic parameters of RsEG against Carboxymethyl Cellulose (CMC) were determined, with K and V of 7.6 mg/ml and 5.4 μmol/min•mg respectively. Co, Mn and Fe enhanced the activity of RsEG by 32.0, 19.5 and 11.2% respectively, while Pb and Cu decreased its activity by 19.6 and 12.7% separately.
CONCLUSIONS
RsEG could be overexpressed in P. pastoris. It was stable between pH 4.0 and pH 11.0, and exhibited higher stability at temperatures ≤ 40 °C. This endoglucanase may have potential to be used in the field of laundry, textile and lignocellulose-based biofuels and chemicals.
背景
纤维素酶对于木质纤维素生物质的充分利用具有重要意义。白蚁具有高效降解纤维素的能力。异源生产白蚁来源的纤维素酶是实现其工业应用的第一步。利用毕赤酵母表达重组蛋白已经变得很流行。属于糖苷水解酶家族 9（GHF9）的锯白蚁内切葡聚糖酶（RsEG）尚未在毕赤酵母中生产。
结果
成功地在毕赤酵母中过表达了突变体 RsEG（G91A/Y97W/K429A）。RsEG 的最适 pH 为 5.0，在 pH 4.0 到 9.0 的范围内具有活性，并在 pH 4.0 到 pH 11.0 之间表现出优异的 pH 稳定性。它在 40°C 时表现出最高的活性和良好的稳定性，但在 50°C 时迅速失去活性。测定了 RsEG 对羧甲基纤维素（CMC）的表观动力学参数，K 和 V 分别为 7.6mg/ml 和 5.4μmol/min·mg。Co、Mn 和 Fe 分别提高了 RsEG 的活性 32.0%、19.5%和 11.2%，而 Pb 和 Cu 分别降低了其活性 19.6%和 12.7%。
结论
RsEG 可以在毕赤酵母中过表达。它在 pH 4.0 到 pH 11.0 之间稳定，在温度≤40°C 时表现出更高的稳定性。这种内切葡聚糖酶在洗衣、纺织和木质纤维素基生物燃料和化学品领域可能具有应用潜力。
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