Heterologous Expression and Enzymatic Properties of the Processive Endoglucanase BvCel5

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

Abstract

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

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.

Figures/Tables 6

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)

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)

Fig. 3 Enzymatic properties analysis of BvCel5

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

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

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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