Low Temperature Cellulase Gene Cloning, Expression And Production Of Screening And Cloning Of The Amylase Of Thermophilic Bacteria

One bacteria strain DY3 with high endoglucanse activity was isolated from deep sea sediment sample ES0109.The 16s rDNA sequence of DY3 exhibits identity of 99% with those of the same genus bacteria Pseudoalteromonas citrea and Pseudoalteromonas elyakovii. Extracellular enzymes secreted by DY3 strain at 8 can hydrolyze Carboxymethylcellulose (CMC).Primers designed according to the reported cellulase sequence were used to amplify a 1.5kb fragment which was cloned into T-vector subsequently for sequencing. Analysis reveals that the fragment, 1479bp in length, is a cellulase gene belonging to family 5 glycosyl hydrolase.The gene was named as ce/X,and its protein is CelX. CleX contains 492 amino acid residues with a family 5 catalytic domain at its N terminal.The analysis of crude enzyme reveals that CelX has a optimal temperature of 40 癈 and retains 98% of relative activity after having been kept at 20 癈 for 60mins.The stability of the enzyme falls with temperature elevation , and the enzyme loses all its activity after having been kept at 55 for 60mins.CelX has a optimal pH between 6-7.The celX gene was expressed in E.co//BL21(DE3) using pET-GST expression vector. The protein purified is capable of hydrolyzing CMC to cellobiose (G2) and cellotriose (G3), hydrolyzing cellopentaose (G5) to G2 and G3, hydrolyzing cellotetraose (G4) to G2, but has no effect on G2 or G3.Tibet highland is considered as the third polar area of the earth. One thermophilic bacteria strain YBJ-1 was isolated from hot spring samples collected from Yangbajing in Tibet. The 16s r DNA sequence of YBJ-1 (151 Ibp in length) shares 98% identity with that of Thermus.scotoductus strain ITI-252T.The full-length ORF of amylase gene of YBJ-1 (YBJ-ow/T) was amplified by PCR technique and cloned into T-vector. The complete sequence of YB3-amyT is 1767bp,coding for 588 amino acids.The deduced amino acids share 99% similarity with alpha-cyclodextrinse of Bacillus sterothermophilus,96% with maltogenic amylse of Thermus.sp IM6501,and 81% with neopullulanase of Bacillus sterothermophlus.