N端截短对嗜酸普鲁兰芽孢杆菌普鲁兰酶酶学特性及功能的影响

Effect of N-terminal truncation of Bacillus acidopullulyticus pullulanase on enzyme properties and functions

摘要:

采用N端截短方式对嗜酸普鲁兰芽孢杆菌Bacillus acidopullulyticus普鲁兰酶进行分子精简，构建不同形式的N端截短突变体，考察截短突变对表达水平、酶学特性及实用性能的影响。研究结果表明：缺失X45后，目标蛋白几乎全部以不溶形式的包涵体形式存在；缺失CBM41可引起可溶性表达量的大幅提升。缺失CBM41 (M1)、X25 (M3)、亦或两结构域同时缺失 (M5) 的变体最适温度 (60 ℃) 和最适pH (5.0) 与野生型相同。突变体M1和M5的Km值分别提高至野生型的2.4倍和3.1倍，kcat/Km测定结果显示M5催化效率下降明显。突变体M1、M3和M5对分子量较大底物的水解效率略有下降，但对小分子底物的水解效率影响不明显。野生型及突变体M1、M3和M5与糖化酶复配使用时，糖化值 (DE) 分别为93.6%、94.7%、94.5%和93.1%。上述结果表明，切除CBM41和/或X25结构域的普鲁兰酶变体不影响其在淀粉糖化过程中的应用，且由于分子量减小更易于异源表达，截短突变体可能更适合于工业化生产。

Abstract:

We constructed different N-terminal truncated variants based on Bacillus acidopullulyticus pullulanase 3D structure (PDB code 2WAN), and studied the effects of truncated mutation on soluble expression, enzymatic properties, and application in saccharification. Upon expression, the variants of X45 domain deletion existed as inclusion bodies, whereas deletion of CBM41 domain had an effective effect on soluble expression level. The variants that lack of CBM41 (M1), lack of X25 (M3), and lack both of CBM41 and X25 (M5) had the same optimal pH (5.0) and optimal temperature (60 ℃) with the wild-type pullulanase (WT). The Km of M1 and M5 were 1.42 mg/mL and 1.85 mg/ml, respectively, 2.4- and 3.1-fold higher than that of the WT. kcat/Km value of M5 was 40% lower than that of the WT. Substrate specificity results show that the enzymes exhibited greater activity with the low-molecular-weight dextrin than with high-molecular-weight soluble starch. When pullulanases were added to the saccharification reaction system, the dextrose equivalent of the WT, M1, M3, and M5 were 93.6%, 94.7%, 94.5%, and93.1%, respectively. These results indicate that the deletion of CBM41 domain and/or X25 domain did not affect the practical application in starch saccharification process. Furthermore, low-molecular-weight variants facilitate the heterologous expression. Truncated variants may be more suitable for industrial production than the WT.