Abstract
A β-glucosidase cDNA belonging to glycoside hydrolase family 1 was cloned from RNA isolated from lactose grown cellulolytic fungus, Penicillium funiculosum NCL1 by reverse-transcriptase polymerase chain reaction. The gene (bgl6) contained an open reading frame of 1470bp that encoded 490 amino acids. bgl6 gene was overexpressed in Escherichia coli BL21-CodonPlus and the recombinant enzyme (rBgl6) was purified. The rBgl6 was a monomer with the molecular weight of 52kDa. The enzyme was optimally active at pH 6.0, 50°C and stable in the wide range of pH and temperature. rBgl6 showed higher activity with pNPG, pNPGal cellobiose and lactose, while it showed substantial activity on pNPX. When the substrate was pNPG, the enzyme showed glucose tolerance at a concentration of 500mM. rBgl6 exhibited enhanced activity with metal cations Ca2+, Mn2+, Mg2+ and Zn2+, while it showed stability with DTT and EDTA. The enzyme showed Km values of 0.125 and 0.25mM, whereas Vmax of 256 and 166μmol/mg with pNPG and cellobiose respectively. rBgl6 possessed transglycosylation activity and it converted glucose into gentiobiose, a rare oligosaccharide with greater prebiotic value. The yield of gentiobiose was 0.57mg/ml for 5mg/ml of glucose. These results indicated that rBgl6 could be a valuable candidate for the enzymatic synthesis of gentiobiose.
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