Abstract
BackgroundCellulases 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.ResultsAn 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 Km, Vmax, Kcat 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.ConclusionsAn 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.
Highlights
Cellulases played an important role in the production of bioenergy and bio-products
One percent (v/v) of the cultural broth was transferred into 50 ml mineral salt medium (MSM) containing 0.05% (w/v) carboxymethylcellulose sodium (CMC) and cultured in a shaker at 37 °C with 200 rpm for 80 h
Enzyme purification with two‐phase system Aqueous two-phase systems were prepared at room temperature by mixing certain amounts of 50% (w/w) of PEG solution, 40% (w/w) of phosphate buffer, 2 ml cell culture supernatant sonicated in 50 mM of citrated buffer, and NaCl in 15 ml centrifuge tubes with conical cap. pH was pre-adjusted to (6.0–8.0) in phosphate buffer
Summary
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 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 Km, Vmax, Kcat 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
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