Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

Abstract

Background

Cellulases played an important role in the production of bioenergy and bio-products. Cellulases from bacteria with some special characteristics drew great attention due to its fast growth speed, wide adaption to harsh environment, and production of multi-function cellulases.

Results

An endoglucanase gene egls from Bacillus velezensis A4 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme Egls was partially purified using aqueous two-phase system. The highest recovery rate of the enzyme was 90.39% at PEG 4000 (25% w/w), phosphate buffer 8.08% (w/w) (pH 6.0), and NaCl (5% w/w). The enzyme molecular weight was 55 KD estimated by zymogram. The optimal pH and temperature of recombinant enzyme Egls were pH 6.0 and 55 °C, respectively. The enzyme was stable at pH range of 5.0–7.0 at 55 °C for 60 min. The enzyme exhibited K_m, V_max, K_cat values as 63.38 mg/ml, 55.6 mg/min, and 3.93 × 10³/S, respectively. The addition of 10 mM of Mg²⁺, Mn²⁺, or 5% (w/w) of Triton-X 100 in the reaction system enhanced the enzyme activity significantly. The enzyme showed both endoglucanase and exoglucanase activity.

Conclusions

An endoglucanase gene egls from B. velezensis A4 was cloned and expressed in E. coli BL21 (DE3). The recombinant enzyme Egls was purified by aqueous two-phase system and characterized. The enzyme can be applied for the efficient pretreatment of lignocellulosic biomass for bioenergy and bio-products production.

Keywords

Purification and characterizations / Endoglucanase / Bacillus velezensis / Aqueous two-phase system

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Funding

Natural Sciences and Engineering Research Council of Canada(RGPIN-2017-05366)

BioFuelNet Canada(Project No. 67)

China Scholarship Council(201608330437)