초록
<P><B>Abstract</B></P> <P>Codon adaptation index (CAI) of a 1263bp long endoglucanase encoding gene from the thermophilic mould <I>Myceliopthora thermophile</I> BJA has been improved from 0.44 to 0.76 by in vitro gene synthesis. The codon optimized endoglucanase gene (<I>Mt-egl</I>) has been constitutively expressed in <I>Pichia pastoris</I> under the regulation of GAP promoter. Recombinant endoglucanase (rMt-egl), purified by size exclusion chromatography, has been confirmed to be a monomeric protein of ∼47kDa. rMt-egl is optimally active at pH 10 and 50°C, displaying stability in broad pH and temperature ranges, with a t<SUB>1/2</SUB> of 60 and 15min at 90 and 100°C, respectively. This retained ∼70% of activity after 3h incubation at pH 5–12. The K<SUB>m</SUB>, V<SUB>max</SUB>, k<SUB>cat</SUB> and k<SUB>cat</SUB>/K<SUB>m</SUB> of rMt-egl were 5mgmL<SUP>−1</SUP>, 20μmolesmin<SUP>−1</SUP> mg<SUP>−1</SUP>, 1.02×10<SUP>3</SUP> s<SUP>−1</SUP> and 204s<SUP>−1</SUP> mg<SUP>−1</SUP> mL<SUP>−1</SUP>, respectively. Homology modeling and bioinformatics analysis confirmed catalytically important role of glutamate 234 and 344. rMt-egl released high amounts of reducing sugars from wheat bran and corn cobs (421 and 382mgg<SUP>−1</SUP>), thus making it a useful biocatalyst for producing bioethanol and fine chemicals from agro-residues.</P> <P><B>Highlights</B></P> <P> <UL> <LI> 1st report on constitutive expression of endoglucanase (Mt-egl) from a thermophilic mould. </LI> <LI> rMt-egl is a thermostable biocatalyst with t<SUB>1/2</SUB> of 15min at 100°C. </LI> <LI> rMt-egl is a novel alkalistable GH5 family endoglucanase in contrast to its acidic counterparts from other thermophilic molds. </LI> <LI> rMt-egl has biotechnological potential in generating cello-oligosaccharides from agro-residues. </LI> </UL> </P>