Abstract
A gene (TM1751, Swissprot Q9X273, GenBank AAD36816) of Thermotoga maritima encoding endo-β-1, 4-glucanase of family 5 was cloned and expressed in Escherichia coli. The recombinant protein was purified by combination of Ni-NTA and Q-Sepharose FF column chromatography. SDS-PAGE analysis of purified fractions showed a homogeneous protein band with an expected molecular mass of 38 kDa. The specific activity of the enzyme increased about 16-fold while the recovery was 21% after purification. The optimal temperature of the enzyme was found to be 90°C while its optimal pH was 6.6. The enzyme was active over a wide pH range (4-9.5) and stable up to 85°C. Kinetic studies showed that the PNP-β-D-cellotetaoside and PNP-β-D-cellopentaoside had similar Km values of 0.25 and 0.24 mM respectively. However, maximum catalytic efficiency (kcat/ Km) was observed with PNP-β-D-cellopentaoside. Further analysis of the enzyme hydrolyzate by HPLC suggests that the carbohydrate binding cleft of the enzyme may be composed of four sub-sites for glucopyranose units. At the initial stage, the hydrolysis of PNP-oligosaccharides (DP up to 5) yielded a range of products with cellobiose, cellotriose and cellotetraose being the major prod-ucts. CMC hydrolysis also predominantly produced cellobiose and cellotriose as end products. The enzyme hydrolyzed mixed linked β-1, 3/1, 4 soluble substrates such as barley glucan and lichenan more strongly than CMC. Transglycosylation activity was also found to be taking place with smaller soluble cellooligosaccharides.
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