RAHMAN M.M.

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Gene Cloning of Hyper-thermo-stable Cellulaseand its Characterizationjournal orpublication titleReport of National Food Research Institutevolume 67page range 42-42year 2003-03-01URL http://doi.org/10.24514/00002666doi: 10.24514/00002666 42 Gene Cloning of Hyper . thermo . stable Cellulase and its Characterization M.M. Rahman UNK-Kirin Fellow from Bangladesh Enzyme Laboratory, National Food Research Institute A cellulase gene (TM1751, Sw1ssprot Q9X273, GenBank AAD36816) of Thermotoga maritima was cloned and ex-pressed in E.coli. The recombinant protem was punfied by a combination of Ni-NTA and Q-Sepharose FF column chro-matography. SDS-PAGE analysis of Q-Seph f arose active ractions showed a homogeneous protem band with expected mo-lecular mass of 38 kDa. The specific activity of the enzyme mcreased about 16-fold while the recovery was io % after punfication. The optimal temperature of the enzyme was found to be 90℃ while that of pH was 6.6. The enzyme was ac-t1ve over a wide pH range (4-9.5) and stable up to 85℃ . Kinetic studies showed that the PNP-b-D-cellotetr~oside and PNP-b-D-cellopentaos1de had similar Km values of 0.25 and 0.24 mM respectively. However, maximum catalytic effic1en .cy (kcat/Km) was observed with PNP-/3-D-cellotetraos1de. Further analysis of kcat and Km revealed that the addition of even one more glucopyranose umt to the non reducing end of PNP-b-D-cellotetraoside had litle or no effect on binding or catalysis, suggesting that the carbohydrate binding cleft of the enzyme may be composed of four subs1tes for glucopyra-nose umts. Hydrolysis of PNP-oligosacchandes (DP up to 5) yielded a range of products with cellobiose and cellotnose being the maJor products. CMC hydrolysis also predormnantly produced cellobiose and cellotriose. The enzyme hydro!-yzed mixed linked /3 -1,3/1,4 soluble substrates such as barley glucan and lichenan more strongly than CMC. Sttong trans-glycosylation act:tvity was also found to be talcing place with smaller soluble cellooligosacchar1des. 

Gene Cloning of Hyper-thermo-stable Cellulase and its Characterization

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Gene Cloning of Hyper-thermo-stable Cellulase and its Characterization

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