苹果酸脱氢酶协同纤维素酶水解纤维素：理论分析与实验

doi:10.16085/j.issn.1000-6613.2023-0117

摘要/Abstract

摘要：

纤维素分子的刚性结构被认为是限制纤维素酶与底物接触的主要障碍，通过添加纤维素降解辅助蛋白破坏纤维素结构可以有效解决酶对底物的可及性问题。基于文献报道糖苷水解酶家族4（GH4）成员中α-葡萄糖苷酶对纤维素酶有协同作用，以及α-葡萄糖苷酶与苹果酸脱氢酶（MDH）的蛋白同源性和蛋白结构分析，本文提出MDH也具有协同作用的功能猜想，并开展了MDH协同纤维素酶水解滤纸纤维素的理论以及实验研究。通过分子对接模拟MDH与结晶纤维素的相互作用，理论分析发现MDH可能通过氢键和疏水的作用来降低纤维素的凝聚。实验考察了MDH添加量、纤维素酶量、水解时间等条件对MDH协同纤维素酶水解纤维素的影响。结果表明，在(50±0.50)mg滤纸体系中添加0.06FPU纤维素酶、150μg苹果酸脱氢酶，50℃水浴震荡24h，测定的还原糖产量是纤维素酶单独作用时的(3.47±0.28)倍，MDH对纤维素酶的增效活性为247%±28%。利用扫描电子显微镜（SEM）、X射线衍射（XRD）以及傅里叶变换红外光谱（FTIR）等手段对MDH作用前后的滤纸进行分析，所得结果表明MDH对纤维素的结晶区域具有破坏作用，协同促进了纤维素酶对纤维素的水解。作为一种有效的纤维素降解辅助蛋白，MDH为提高纤维素的酶法水解效率提供了更多选择。

关键词: 辅助蛋白, 苹果酸脱氢酶, 纤维素酶, 纤维素, 水解

Abstract:

The rigid structure of cellulose molecules is considered a serious impediment to the cellulose hydrolysis by cellulase. The disaggregation of cellulose by the addition of auxiliary proteins is an effective strategy to make the contact of cellulase with cellulose easier. Based on the documented α-glucosidase, a member of the glycoside hydrolase family 4, exhibiting synergism with the cellulase, and the analysis of protein homology as well as structure, a conjecture that the malate dehydrogenase (MDH) had a similar synergistic function was proposed. The theoretical and experimental studies on the hydrolysis of filter paper by the MDH synergism with cellulase were carried out. The interaction between MDH and crystalline cellulose was simulated by molecular docking. Theoretical analysis showed that the MDH could reduce the aggregation of cellulose by the effect of hydrogen bonds and hydrophobic interaction. The cellulose hydrolysis by the MDH synergism with cellulase, involving the addition of MDH, the amount of cellulase and the hydrolysis time were investigated. It was found that the yield of reducing sugar in the system of (50±0.50)mg filter paper, 150μg MDH and 0.06FPU cellulase was approximately 3.47-fold higher than the system of cellulase alone under the condition of hydrolyzing 24h in a water bath at 50℃. An enhanced activity of 247%±28% was acquired for cellulase by the synergic effect of MDH. The scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR) were used to analyze the filter papers before and after adding the MDH. Results showed that the MDH could destroy the crystalline regions of the cellulose, synergistically improving the efficiency of cellulose hydrolysis by cellulase. As an effective auxiliary protein, the MDH would provide an alternative for cellulose hydrolysis.

Key words: auxiliary protein, malate dehydrogenase, cellulase, cellulose, hydrolysis

中图分类号:

董友情, 唐爱星, 刘幽燕, 李青云. 苹果酸脱氢酶协同纤维素酶水解纤维素：理论分析与实验[J]. 化工进展, 2023, 42(12): 6641-6648.

DONG Youqing, TANG Aixing, LIU Youyan, LI Qingyun. The malate dehydrogenase synergistically hydrolyzes cellulose with cellulase: Theoretical analysis and experimental investigation[J]. Chemical Industry and Engineering Progress, 2023, 42(12): 6641-6648.

图/表 7

图1 MDH蛋白结构和功能的理论分析[(a)中蓝色为MDH，橙色为α-葡萄糖苷酶]

图2 添加MDH对纤维素酶的协同作用

图3 MDH与纤维素酶协同作用的影响因素

图4 MDH分别与EG、CBH、BG的协同作用

图5 不同处理后的滤纸SEM图

图6 不同处理后的滤纸XRD图

图7 不同处理后的滤纸FTIR图

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