Cloning, Identification And Expression Of Cellulase Gene Of Uncultured Microorganism From Bovine Rumen

A metagenomic library of uncultured microorganism from bovine rumen fluid content was constructed, which contained 12,000 clones and the capacity of inserted DNA of which was 4.2x108 bp. Ten clones expressing β-glucosidase activity and ten clones expressing β-1,4-endoglucanase activity were isolated. Furthermore two of the ten expressing β-l,4-endoglucanase activity clones designated pGXN1035 and pGXN1067 also expressed xylanase activity. Clone pGXN1009 expressing β-glucosidase activity,pGXN1035 and pGXN1067 were chosen for further study. By subcloning and sequencing, gene umbg13A, umcel5C and umcel5D were obtained.An ORF umbg13A was 1956 bp, encoding product had a molecular weight of 70 133 Da. UmbgBA shared 63% identity and 79% similarity with a β-glucosidase of uncultured murine large bowel bacterium. UmbgBA possessed a domain of glycosyl hydrolase family 3 and a domain of glycosyl hydrolase family 3C. Phylogentic analysis of UmbgBA indicated umbgBA may come from a species of Cytophaga.An ORF umcel5C was 1314 bp, encoding product had a molecular weightof 48 675 Da. Umcel5C shared 45% identities and 62% positives with a xylanase oi Prevotella ruminicola, and shares 38% identities and 55% positives with a β-1,4-endoglucanase of Prevotella ruminicola. Umcel5C possessed a domain of glycosyl hydrolase family 5.The gene umcel5D contained an ORF of 1641 bp, encoding product had a molecular weight of 60674 Da. Umcel5D shared 51% identities and 65% positives with a xylanase of Prevotella ruminicola, shared shares 45% identities and 59% positives with a β-1,4-endoglucanase oi Prevotella bryanti. Umcel5D possessed a signal peptide and a domain of glycosyl hydrolase family 5. Umcel5C shared 71% identities and 80% positives with umcel5D. And phylogentic analysis indicated umcel5C and umcel5D come from a species of Prevotella.An expressing plasmid was constructed by cloning the coding region of umcel5D into vecter pQE-30, and transformed into E coli M15/pREP4. Most of the expressed product was soluble induced by IPTG The enzyme properties were characterized. The optimum pH was 3.0, and the optimum temperature was 35℃.