持续性内切纤维素酶BvCel5的异源表达及酶学性质研究

doi:10.13560/j.cnki.biotech.bull.1985.2025-1159

摘要/Abstract

摘要：

目的将来源于贝莱斯芽胞杆菌（Bacillus velezensis）的持续性内切纤维素酶（processive endoglucanase）BvCel5构建至枯草芽胞杆菌（Bacillus subtilis）RIK 1285中进行分泌表达，并对其酶学性质进行研究，为该酶进一步的开发及利用奠定基础。方法利用酶切连接法将BvCel5构建至pBE-S载体，转入B. subtilis RIK 1285实现异源表达。利用镍离子亲和层析法纯化蛋白。以羧甲基纤维素钠（CMC）为底物，通过2,5-二硝基水杨酸比色法（DNS）和二喹啉甲酸法（BCA）测定BvCel5酶活力、酶学性质以及持续性降解能力。最后以CMC、Whatman滤纸和微晶纤维素PH-101为底物，利用薄层色谱法（TLC）及离子色谱（IC）对BvCel5产物进行分析。结果成功构建表达载体pBE-S-BvCel5及重组菌株，实现了BvCel5（分子量56.3 kD）的分泌表达及纯化，测得纯酶比酶活为（47.48±0.78） U/mg。酶学性质分析表明，该酶最适温度及pH分别为60 ℃和5.5，温度稳定区间为25-55 ℃，pH在3.5-12时其相对酶活保持90%以上。BvCel5产物滴定实验及持续性降解能力分析证实BvCel5兼具内切纤维素酶活性与持续性降解能力。该酶降解CMC的主要产物为纤维二糖（34.07%）和纤维三糖（47.15%），对Whatman滤纸及微晶纤维素PH-101也有降解活性。结论成功将BvCel5在枯草芽胞杆菌中表达，其酶学性质优良，具备工业化应用潜力。

关键词: 枯草芽胞杆菌, 持续性内切纤维素酶, 异源表达, 酶学性质, 产物分析

Abstract:

Objective To construct the BvCel5 gene encoding a processive endoglucanase from Bacillus velezensis into Bacillus subtilis RIK 1285 for expression, and to investigate its enzymatic properties. This lays the foundation for the enzyme’s further development and utilization. Method BvCel5 was constructed into the pBE-S vector via restriction enzyme digestion and ligation, then transferred into B. subtilis RIK 1285 for heterologous expression. nickel ion affinity chromatography was used to purify the protein. Using carboxymethyl cellulose sodium (CMC) as substrate, the BvCel5 activity, enzymatic properties, and sustained degradation capacity were determined via the 2,5-dinitrosalicylic acid (DNS) and 2,2-bicinchoninate assay (BCA) method. Finally, the BvCel5 product was analyzed using thin-layer chromatography (TLC) and ion chromatography (IC) with CMC, Whatman filter paper, and microcrystalline cellulose PH-101 as substrates. Result The expression vector pBE-S-BvCel5 and recombinant strain were successfully constructed, enabling the secretory expression and purification of BvCel5 (molecular weight 56.3 kD) . The purified enzyme exhibited an enzyme activity of (47.48 ± 0.78) U/mg. Enzymatic characterization revealed an optimal temperature of 60 ℃ and pH of 5.5. The enzyme exhibited thermal stability within 25-55 ℃ and maintained over 90% relative activity across a pH range of 3.5-12. Titration and persistent degradation analyses confirmed that BvCel5 possessed both endo-cellulase activity and persistent degradation capability. The primary degradation products of CMC by BvCel5 were cellobiose (34.07%) and cellotriose (47.15%). The enzyme also presented degradation activity towards Whatman filter paper and Avicel PH-101. Conclusion BvCel5 was successfully expressed in Bacillus subtilis, showing excellent enzymatic properties with potential for industrial application.

Key words: Bacillus velezensis, processive endocellulase, heterologous expression, enzymatic properties, product analysis

彭海燕, 刘佳鑫, 史禹涵, 李宪臻, 郭小宇, 袁悦. 持续性内切纤维素酶BvCel5的异源表达及酶学性质研究[J]. 生物技术通报, 2026, 42(7): 351-360.

PENG Hai-yan, LIU Jia-xin, SHI Yu-han, LI Xian-zhen, GUO Xiao-yu, YUAN Yue. Heterologous Expression and Enzymatic Properties of the Processive Endoglucanase BvCel5[J]. Biotechnology Bulletin, 2026, 42(7): 351-360.

图/表 6

图1 RIK 1285-pBE-S-BvCel5表达菌株的构建A：载体构建流程图；B：pBE-S-BvCel5的DH5α转化子的菌落PCR结果图（M：10 000 bp Marker；Lane 1-4：pBE-S-BvCel5的DH5α转化子的菌落PCR产物）；C：pBE-S-BvCel5质粒酶切产物鉴定结果图（M：10 000 bp Marker；Lane 1-2：pBE-S-BvCel5质粒酶切产物）；D：RIK 1285-pBE-S-BvCel5中质粒的提取及酶切产物鉴定结果图（M：5 000 bp Marker；Lane 1-3：RIK 1285-pBE-S-BvCel5的3个转化子）

Fig. 1 Construction of the RIK 1285-pBE-S-BvCel5 expression strainA: Vector construction flowchart. B: Graph of the colony PCR results of DH5α transformants of pBE-S-BvCel5 (M: 10 000 bp Marker; lane 1-4: DH5α transformants of pBE-S-BvCel5). C: graph of the identification results of enzyme digestion products of pBE-S-BvCel5 plasmid (M: 10 000 bp marker; lanes 1-2: pBE-S-BvCel5). D: Extraction of the RIK 1285-pBE-S-BvCel5 plasmid and identification results of enzyme-digestion product (M: 5 000 bp marker; lane 1-3: Three transformants of RIK 1285-pBE-S-BvCel5)

图2 BvCel5的表达及纯化A：RIK 1285-pBE-S-BvCel5的SDS-PAGE电泳结果图（M：预染Marker；Lane 1：RIK 1285-pBE-S-BvCel5发酵上清；Lane 2：RIK 1285-pBE-S发酵上清）；B：RIK 1285-pBE-S-BvCel5的Western Blot实验结果图（M：预染Marker；Lane 1：RIK 1285-pBE-S-BvCel5发酵上清；Lane 2：RIK 1285-pBE-S发酵上清）；C：RIK 1285-pBE-S-BvCel5纯蛋白的SDS-PAGE电泳结果图（M：Low Marker；Lane 1：粗酶液；Lane 2：流穿液；Lane 3-6：50 mmol/L咪唑缓冲液洗脱液）

Fig. 2 Expression and purification of BvCel5A: SDS-PAGE electrophoresis gel of RIK 1285-pBE-S-BvCel5 (M: pre-stained marker; lane 1: RIK 1285-pBE-S-BvCel5 fermentation supernatant; lane 2: RIK 1285-pBE-S fermentation supernatant). B: Western blot results for RIK 1285-pBE-S-BvCel5 (M: pre-stained marker; lane 1: RIK 1285-pBE-S-BvCel5 fermentation supernatant; lane 2: RIK 1285-pBE-S fermentation supernatant). C: SDS-PAGE electrophoresis gel image of pure protein RIK 1285-pBE-S-BvCel5 (M: Low molecular weight marker; lane 1: crude enzyme solution; lane 2: perfusion fluid; lane 3-6: 50 mmol/L imidazole buffer eluate)

图3 BvCel5酶学性质分析

Fig. 3 Enzymatic properties analysis of BvCel5

图4 BvCel5与CMC反应产物黏度变化曲线

Fig. 4 Viscosity change curve of the reaction product between BvCel5 and CMC

图5 BvCel5持续性降解能力分析黄色柱形：可溶性还原端；蓝色柱形：不溶性还原端；折线：可溶性还原端与不溶性还原端的比值；误差线：3次重复实验的平均值±标准误差

Fig. 5 Analysis of BvCel5 sustained degradation capacityYellow column: soluble reduction end. Blue column: insoluble reduction end. Broken line: Ratio of soluble reduction end to insoluble reduction end. Error bars: mean values ± standard error of three replicate experiments

表1 BvCel5与不同底物反应产物中各组分占比

Table 1 Proportions of components in reaction products between BvCel5 and different substrates

底物	G1		G2		G3
底物	含量（mg/L）	占比（%）	含量（mg/L）	占比（%）	含量（mg/L）	占比（%）
CMC	66.06±2.76	18.77	119.90±4.16	34.07	165.93±0.96	47.15
滤纸	1.84±0.13	1.92	21.93±2.33	22.87	72.10±2.04	75.21
PH-101	1.49±0.23	1.55	25.77±1.51	26.75	69.07±2.44	71.70

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