嗜热毁丝菌裂解性多糖单加氧酶TtLPMO9I的酶学性质及其功能研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0877

摘要/Abstract

摘要：

【目的】为挖掘新型裂解性多糖单加氧酶（LPMO）酶资源，探究LPMO在辅助降解纤维素过程中起到的重要作用。【方法】从Thermothelomyces thermophilus基因组中克隆表达了一个新型LPMO酶TtLPMO9I，系统地分析了其序列及结构的进化特征；采用DNS法表征了TtLPMO9I的酶学性质；在反应体系中添加不同浓度的抗坏血酸探究外部电子供体对TtLPMO9I活性的影响；以玉米秸秆和微晶纤维素为底物，通过检测还原糖的生成量计算获得TtLPMO9I与纤维素酶的协同作用效果。【结果】TtLPMO9I在60℃，pH 5.0时表现出最佳酶活力。在60℃孵育12 h后，仍能剩余54%的活性。经pH 6.0-8.0处理12 h后，酶活无损失。添加外部电子供体抗坏血酸使TtLPMO9I的活性提高至184%。在玉米秸秆和微晶纤维素降解过程中，TtLPMO9I与纤维素酶表现出良好的协同作用效果。将50-200 μg的TtLPMO9I添加至降解体系中，还原糖产量分别提高了34%-142%和6%-46%。【结论】TtLPMO9I不仅具有良好的温度稳定性和pH稳定性，在木质纤维素的降解过程中也具有突出的作用效果，为工业生产应用提供了潜在的优质酶资源。

关键词: 裂解性多糖单加氧酶（LPMO）, 异源表达, 酶学性质, 协同降解

Abstract:

【Objective】 To explore a novel lytic polysaccharide monooxygenase（LPMO）enzyme and to elucidate its crucial role in cellulose degradation.【Method】 A new LPMO enzyme TtLPMO9I was cloned from Thermothelomyces thermophilus genome, and its sequence and structure were systematically analyzed to determine its evolutionary characteristics. The enzymatic properties of TtLPMO9I were characterized using the DNS method; the impact of different concentrations of ascorbic acid on the activity of TtLPMO9I was investigated by adding external electron donors to the reaction system. By detecting the production of reducing sugars, the synergistic effect between TtLPMO9I and cellulase was calculated when pretreated corn straw and Avicel were used as substrates. 【Result】 TtLPMO9I presented the highest activity at 60℃ and pH 5.0. After 12 h of incubation at 60℃, approximately 54% of its activity remained. TtLPMO9I still maintained initial enzyme activity at pH 6.0-8.0 for up to 12 h. Adding an external electron donor, ascorbic acid, to the reaction system, the activity of TtLPMO9I increased to 184%. In addition, TtLPMO9I has shown promising synergistic effects with cellulase in the degradation of pretreated corn straw and Avicel. Adding 50-200 μg of TtLPMO9I into the degradation system increased reducing sugar yields by 34%-142% and 6%-46% respectively. 【Conclusion】 TtLPMO9I not only has excellent thermal and pH stability, but also has outstanding performance in degrading lignocellulose, providing potential high-quality enzyme resources for industrial production.

Key words: lytic polysaccharide monooxygenase（LPMO）, heterologous expression, enzymatic properties, co-degradation

郑菲, 杨俊钊, 牛羽丰, 李蕊麟, 赵国柱. 嗜热毁丝菌裂解性多糖单加氧酶TtLPMO9I的酶学性质及其功能研究[J]. 生物技术通报, 2024, 40(2): 289-299.

ZHENG Fei, YANG Jun-zhao, NIU Yu-feng, LI Rui-lin, ZHAO Guo-zhu. Characterization and Functional Analysis of Lytic Polysaccharide Monooxygenase TtLPMO9I from Thermothelomyces thermophilus[J]. Biotechnology Bulletin, 2024, 40(2): 289-299.

图/表 7

表1 嗜热毁丝菌AA9家族LPMO蛋白性质预测

Table 1 Protein property prediction of the LPMO AA9 family from T. thermophilus

序号No.	蛋白 Protein	氨基酸数目 Number of amino acids	分子量 Molecular weight/kD	参考文献 Reference
1	MYCTH_112089	232	26.1	[19]
2	MtLPMO9B	323	32.4	[20]
3	MtLPMO9D	255	27.0	[19,21]
4	MtLPMO9A	225	24.4	[22]
5	AEO59955.1	235	24.4	未报道
6	AEO54509.1	303	31.5	未报道
7	MtLPMO9	342	34.9	[23]
8	TtLPMO9I	306	31.4	本文研究材料
9	MtLPMO9J	246	26.0	[24-25]
10	AEO59836.1	227	24.1	未报道
11	AEO59482.1	198	21.5	未报道
12	AEO58921.1	340	35.8	未报道
13	AEO58412.1	338	35.2	未报道
14	AEO61257.1	241	25.9	未报道
15	AEO56498.1^a	151	16.2	未报道
16	AEO59823.1	254	28.4	未报道
17	AEO55776.1	444	46.6	未报道
18	AEO61305.1	230	25.0	未报道
19	AEO55082.1	226	24.5	未报道
20	MtLPMO9C	237	24.9	[20]
21	AEO56547.1	245	26.5	未报道
22	AEO55652.1	242	25.5	未报道

图1 嗜热毁丝菌AA9家族LPMO蛋白系统发育树分析

Fig. 1 Phylogenetic tree of LPMO proteins in the AA9 family of T. thermophilus

图2 TtLPMO9I生物信息学分析 A：TtLPMO9I与已解析出晶体结构LPMO的多序列比对；B：TtLPMO9I三维结构模拟图

Fig. 2 Bioinformatics analysis of TtLPMO9I A: Multiple sequence alignment of TtLPMO9I with LPMO in crystal structure. B: 3D structural simulation diagram of TtLPMO9I

图3 重组蛋白TtLPMO9I的验证 A：TtLPMO9I的SDS-PAGE图；B：TtLPMO9I MALDI-TOF质谱分析结果图。 Marker：蛋白分子质量标准；1：TtLPMO9I纯化蛋白

Fig. 3 Validation of the recombinant protein TtLPMO9I A: SDS-PAGE analysis of TtLPMO9I. B: MALDI-TOF mass spectrometry analysis results diagram for TtLPMO9I. Marker: Protein molecular weight standard. 1: Purified protein of TtLPMO9I

图4 抗坏血酸对TtLPMO9I酶活性的影响 * P<0.05; ** P<0.01, *** P<0.001; **** P<0.000 1；ns: 差异不显著，下同

Fig. 4 Effect of ascorbic acid concentration on the enzyme activity of TtLPMO9I ns: Not significance, the same below

图5 TtLPMO9I的酶学性质 A：温度对TtLPMO9I酶活力的影响；B：温度对TtLPMO9I酶活力稳定性的影响；C：pH对TtLPMO9I酶活力的影响；D：pH对TtLPMO9I酶活力稳定性的影响

Fig. 5 Enzymatic properties of TtLPMO9I A: Effect of temperature on the cellulase activity of TtLPMO9I. B: Effect of temperature on the stability of TtLPMO9I. C: Effect of pH on the cellulase activity of TtLPMO9I. D: Effect of pH on the viability stability of TtLPMO9I

图6 TtLPMO9I与纤维素酶（Novozymes 188）降解玉米秸秆、微晶纤维素的协同反应 A：以玉米秸秆为底物反应12 h；B：以玉米秸秆为底物反应24 h；C：以微晶纤维素为底物反应12 h；D：以微晶纤维素为底物反应24 h。上图中蓝色均代表分别添加50、100、200 μg的TtLPMO9I所产生的还原糖的量；绿色均代表添加100 μL纤维素酶所产生的还原糖的量；橙色均代表50 μg TtLPMO9I+100 μL纤维素酶、100 μg TtLPMO9I+100 μL纤维素酶、200 μg TtLPMO9I+100 μL纤维素酶所产生的还原糖的量

Fig. 6 Synergistic reaction between TtLPMO9I and cellulase（Novozymes 188）for the degradation of pretreated corn straw and Avicel A: Pretreated corn straw was used as substrate for 12 h. B: Pretreated corn straw was used as substrate for 24 h. C: Reaction with Avicel as substrate for 12 h. D: Reaction with Avicel as substrate for 24 h. The blue block in the figure above indicates the amount of reducing sugar produced by adding 50, 100, and 200 μg TtLPMO9I, respectively. The green block indicates the amount of reducing sugar produced by adding 100 μL cellulase. The orange block indicates the amount of reducing sugar produced by 50 μg TtLPMO9I+100 μL cellulase, 100 μg TtLPMO9I+100 μL cellulase, and 200 μg TtLPMO9I+100 μL cellulase

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