Gene cloning, heterologous expression and enzyme characterizations of halo-tolerant endoglucanase from Alicyclobacillus D-1

Abstract:

[Objective] An endoglucanase gene (gluE1) was cloned from a thermalacidophilus (Alicyclobacillus tengchongensis CGMCC1504) isolated from a hot spring, and the sequence and biochemical characterization of enzyme were analyzed. [Methods] The full-length gluE1 was obtained based on genome sequencing, analysis of amino acid sequence of GluE1. gluE1 was ligated into pEASY-E2 vector and expressed in Escherichia coli BL21 (DE3) cells. GluE1 was purified to electrophoretic homogeneity by Ni²⁺-NTA metal chelating affinity chromatography, and then the enzyme characterizations were determined. [Results] The 1020 bp full-length gluE1 (50.5% GC content) encodes a 339 residues polypeptide (GluE1: 40.45 kDa). GluE1 showed the highest identity of 97% with endoglucanase in public databases, and <60% identities with other endoglucanase. GluE1 efficiently hydrolyzed CMC-Na, soluble starch and barley-β-glucan, which showed apparent optimal at pH 6.5 and 55℃. GluE1 was stable and active (>60%) at pH 5.0-10.0, and had a high stability at 37℃; and it exhibited K_m, V_max and k_cat values of 8.58 mg/mL, 416.67 U/mg and 280.90 s^-1 respectively. GluE1 was strongly inhibited by Ag⁺, Hg²⁺ and SDS, partial promoted by β-Mercaptoethanol, Pb²⁺, Mg²⁺, Ca²⁺ and Na⁺, 30% NaCl still retains more than 64% of the activity. The residual enzyme activity kept 93% after pre-incubation of the enzyme in 30% NaCl. [Conclusion] Endoglucanase gene gluE1 from Alicyclobacillus was first reported, and GluE1 showed a good pH stability and strong halo-tolerant property. GluE1 might have greater potential applications.