Cloning And Expression Of A Novel Endo-1, 4-β-d-glucanase Gene Umcel5k And Characterization Of The Translated Product

In this study, total DNA of uncultured microorganisms from the enrichment cultures of the contents of buffalo rumens was isolated . A metagenomic cosmid library that contained approximate 8×10³ clones was constructed. By functional screening, a clone expressing both exoglucanase and endoglucanase activity was found from the library, and subcloning and sequencing analysis identified one gene (designated umcel5K) that contained an open reading frame (ORF) encoding a protein of 333 amino acid residues. The encoded products of the gene shared 53% identities and 68% similarities with a cellulase CelA(ABA02176) of glycosyl hydrolase family 5 from a uncultured bacterium. The ORF of umcel5K amplified by PCR was clonded into vecter pET30a(+), and over-expressed in Escherichia coli. The Ni-NTA purified expressed product was characterized. The optimum pH of the recombinant enzyme was 4.5～5.0, and optimum temperature was 50℃for endoglucanase activity. The enzyme kinetics experiment determined that the values for Km and Vmax of Umcel5K is 3.843mg/ml and 152.4 U/ mg. Certain ions such as Fe³⁺,Cr²⁺ and Cu²⁺ had inhibitory effect on the activity of the enzyme, while K⁺,Li⁺ showed little influence.