不同材种木质素影响底物吸附纤维素酶的水解机制

doi:10.11707/j.1001-7488.LYKX20220547

摘要/Abstract

摘要：

目的: 以杨木、桉木和相思木木质素为原料，探究纤维素酶在不同材种木质素和模拟底物上的吸附行为，为木质纤维原料预处理技术选择和酶水解转化成本降低提供参考。方法: 采用二维核磁方法分析提取木质素的组成结构，应用旋涂法制备木质素和木质素/纤维素薄膜，并对酶吸附前后的表面形貌进行表征，利用耗散型石英晶体微天平（QCM-D）技术原位、实时探究不同材种木质素吸附纤维素酶影响底物酶水解的规律。结果: 杨木、桉木和相思木分离木质素纯度高，均为GS型木质素（杨木、桉木和相思木木质素S/G比分别为1.09、1.73和1.22），木质素结构单元之间的主要连接方式为β—O—4结构。原子力显微镜（AFM）观察显示，底物水解过程中纤维素酶可均匀分布在底物和木质素表面且结合紧密，酶水解后桉木木质素薄膜的粗糙度高于杨木和相思木。经QCM-D分析木质素吸附纤维素酶的能力大小为杨木<相思木<桉木。结论: 原料酶水解过程可分为吸附、快速水解、缓慢水解和水解平衡4个阶段。同结构单元类型木质素对纤维素酶表现出的吸附能力不同，较高S/G比的木质素与纤维素酶之间的非生产吸附越多，导致酶与纤维素之间吸附位点减少，达到最大转化率所需时间更长，水解程度较低。

关键词: 木质素, 酶水解, 结构单元, 酶吸附, 石英晶体微天平

Abstract:

Objective: In order to explore the effect of lignin on enzyme adsorption in substrate enzyme hydrolysis, using the proto lignin as material, isolated from different species of poplar, eucalyptus and acacia wood, and the adsorption behavior of cellulase on lignin films was studied by quartz crystal microbalance with dissipation (QCM-D) technology. Method: The structures of isolated lignins were characterized by 2D NMR. The lignin/cellulose biosensor was prepared by spin coating method and characterized by the surface morphology before and after enzyme adsorption, and the QCM-D technology was used to explore the influence of lignin with different structural unit on cellulase adsorption in situ and in real time. Result: Lignins from poplar, eucalyptus and acacia were GS lignin, and the main connection mode between structural units was β—O—4, and their S/G ratios were 1.09, 1.73 and 1.22, respectively. After the injection of the cellulase, the cellulase was evenly distributed on the surface of the lignin film, and they were bound tightly. The roughness of the lignin film of eucalyptus was higher than that of poplar and acacia at the end of injection. By QCM-D analysis, the poplar lignin film had minimal capacity to adsorb cellulase while eucalyptus lignin largest capacity. Conclusion: Different structural unit types in lignin structure also show different adsorption capacity to cellulase. The enzymatic hydrolysis process of composite film can be divided into four stages: adsorption, rapid hydrolysis, slow hydrolysis and hydrolysis equilibrium. Combined with the kinetic parameters of enzymatic hydrolysis, eucalyptus lignin with higher S/G ratio takes longer time to reach the maximum enzymatic hydrolysis conversion and lower hydrolysis degree.

Key words: lignin, enzymatic hydrolysis, structural unit, enzymatic adsorption, QCM-D

中图分类号:

S785

蒋康杰,吴文娟,黄丽菁,李家全,黎孔燕. 不同材种木质素影响底物吸附纤维素酶的水解机制[J]. 林业科学, 2024, 60(7): 140-148.

Kangjie Jiang,Wenjuan Wu,Lijing Huang,Jiaquan Li,Kongyan Li. Effect Mechanism of Lignin Isolated from Diverse Types Woods on Cellulase Adsorption[J]. Scientia Silvae Sinicae, 2024, 60(7): 140-148.

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参考文献 0

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标签Sign	δC/δH/ppm	归属Assignments
C_α	84.8/4.63	树脂醇结构C_α—H_α C_α—H_α in resinol substructures
C_β	53.9/3.06	树脂醇结构C_β—H_β C_β—H_β in resinol substructures
C_γ	71.9/4.19&71.7/3.83	树脂醇结构C_γ—H_γ C_γ—H_γ in resinol substructures
A_γ	60.3/3.72	β-O-4结构中C_γ—H_γ C_γ—H_γ in β-O-4 substructures
A_α(G)	71.6/4.74	β—O—4连接G单元C_α—H_α C_α—H_α in β—O—4 substructures linked to a G unit
A_α(S)	72.2/4.86	β—O—4连接S单元C_α—H_α C_α—H_α in β—O—4 substructures linked to a S unit
A_β(G)	84.3/4.29	β—O—4连接S单元C_β—H_β C_β—H_β in β—O—4 substructures linked to a S unit
A_β(S)	86.4/4.11	β—O—4连接G单元C_β—H_β C_β—H_β in β—O—4 substructures linked to a G unit
B_α	87.5/5.44	苯基香豆满结构C_α—H_α C_α—H_α in phenylcoumaran substructures
B_γ	62.6/3.67	苯基香豆满结构C_γ—H_γ C_γ—H_γ in phenylcoumaran substructures
I_γ	62.0/4.07	肉桂醇末端基C_γ—H_γ C_γ—H_γ in cinnamyl alcohol end-groups
I_α	129.1/6.46	肉桂醇末端基C_α—H_α C_α—H_α in cinnamyl alcohol end-groups
S_2,6	104.5/6.70	紫丁香基单元C_2,6—H_2,6 C_2,6—H_2,6 in syringyl units
S'_2,6	106.9/7.31	氧化(C_αOOH)紫丁香基单元C_2,6—H_2,6 C_2,6—H_2,6 in oxidized (C_αOOH) syringyl units
G₂	111.6/6.99	愈创木基单元C₂—H₂ C₂—H₂ in guaiacyl units
G₅	115.1/6.71	愈创木基单元C₅—H₅ C₅—H₅ in guaiacyl units
G₆	119.6/6.77	愈创木基单元C₆—H₆ C₆—H₆ in guaiacyl units
PAC_2,6	131.9/7.51	对香豆酸结构C_2,6—H_2,6 C_2,6—H_2,6 in p-coumarate
J_β	127.0/6.77	肉桂醛末端基C_β—H_β C_β—H_β in cinnamyl aldehyde end-groups
J_α	154.3/7.36	肉桂醛末端基C_α—H_α C_α—H_α in cinnamyl aldehyde end-groups

结构 Structure	杨木 Poplar（%）	桉木 Eucalyptus（%）	相思木 Acacia（%）
β—O—4(A)	61.38	57.35	54.61
β—5(B)	5.24	3.83	4.99
β—β(C)	13.48	14.28	13.68
愈创木基 Guaicyl (G)	47.90	36.53	44.96
紫丁香基 Syringyl (S)	52.10	63.47	55.04
S/G	1.09	1.73	1.22

原料 Raw material	杨木Poplar	桉木Eucalyptus	相思木Acacia
葡聚糖含量 Glucan content（%）	38.07	37.80	35.27
木质素含量 Lignin content（%）	25.13	25.25	25.69
葡聚糖转化率 Glucan conversion（%）	67.85	63.38	52.71

木质素薄膜 Lignin film	粗糙度（吸附前） Roughness (before adsorption) /nm	粗糙度（吸附后） Roughness (after adsorption) /nm
杨木Poplar	3.37	1.89
桉木Eucalyptus	3.14	2.35
相思木Acacia	3.22	1.56

木质素/纤维素复合薄膜 MWL/MCC composite films	B/Hz	V₅₀/min	1/C/min^?1	R²
杨木Poplar	208.85	20.26	0.25	0.995
桉木Eucalyptus	152.51	22.19	0.29	0.987
相思木Acacia	180.47	20.97	0.37	0.994