SILPAKORN UNIVERSITY SCIENCE AND TECHNOLOGY JOURNAL, Vol 10, No 2 (2016)

Cloning and Expression of the Cellulase Gene from the King Oyster Mushroom, Pleurotus eryngii

Urarux Romruen, Eakaphun Bangyeekhun






Abstract


A gene encoding for cellobiohydrolase (PEcbh) from P. eryngii was cloned by using RT-PCR 3’ and 5’ RACE techniques. The result showed that the PEcbh was 1377 bp nucleotide sequence encoded for 459-deduced amino acid. Analysis of predicted protein revealed that PEcbh consisted of a glycosyl hydrolase family 7 domain but lacked of
cellulose binding domain, a calculated molecular weight of 49.3 kDa and a pI of 5.3. The PEcbh was cloned into pET28a (+) to obtain a recombinant pET/PEcbh and expressed in E. coli BL21 (DE3). The optimal conditions of PEcbh expression were 0.2 mM IPTG, 1 h induction time at 180C and 4 h post-induction time. The CMCase activity
could be detected, but at a low activity. This is probably due to a lack of the cellulose binding domain in PEcbh. Expression of PEcbh in E. coli BL21 (DE3) and Rosetta (DE3) were compared and the results indicated that CMCase activity in Rosetta (DE3) was higher than in BL21 about 2 times.
Key Words: Pleurotus eryngii; Cellobiohydrolase; Glycosyl hydrolase family 7; Protein expression; Cloning

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