Abstract
A novel endoglucanase gene (1425 bp), designated thrcel5A, was cloned from Thermoactinospora rubra YIM 77501Tand determined to be a member of glycoside hydrolase family 5. The putative amino acid sequence displayed 76% conservation with reported endoglucanases (GenBank: SCG57304.1) from Micromonospora siamensis. Thrcel5A was expressed in Escherichia coli BL 21 (DE3) and purified using Ni2+-affinity chromatography. The resulting purified protein displayed high hydrolytic activity against the sodium salt of carboxymethyl cellulose and β-(1, 3; 1, 4)-glucans from barley and beechwood xylan, with specific activities of 85.7 ± 1.5, 120.3 ± 2.6, and 22.9 ± 1.1 U/mg, respectively. The optimal pH and temperature for the recombinant enzyme were determined to be 8.5 and 60 °C, respectively. Additionally, ThrCel5A was thermotolerant as it retained more than 60% of its original activity after an incubation at 60 °C for 2 h. Moreover, ThrCel5A can hydrolyze β-(1, 3; 1, 4)-glucan into prebiotics, such as cellobiose, cellotriose, and cellotetrose. Its endoglucanase activity was significantly affected by link sequences and CBM2. Due to being an alkali-tolerant, thermostable, and multifunctional cellulolytic enzyme, ThrCel5A is an attractive candidate for use in production of prebiotics.
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