Isolation and Cloning of Cellulase Gene from Bovine Rumen Bacteria.

Abstract

Cellulases are the enzymes that hydrolize cellulosic biomass and are produced by the microorganisms that grow over cellulosic matters. The objective of this research was to isolate and clone cellulase gene from cellulose-degrading bacteria of bovine rumen. Cellulose-degrading bacteria were isolated from rumen fluid using a selective medium. Total RNA was isolated from selected colony having cellulose degrading activity and it was used as a template for cDNA construction using reverse transcriptase polymerase chain reaction (RT-PCR) technique. The resulted cDNA was used as a template for PCR amplification of cellulose gene using specific primers. The cellulose gene candidate obtained was cloned into the pGEM-T-Easy vector followed by determination of its nucleotide sequence. The nucleotide sequence generated was aligned with sequences of cellulase genes from GenBank. A number of isolates of rumen bacteria showed cellulase activity and the isolate CR-8 was selected for further analysis. Total RNA was successfully isolated from isolate CR-8 indicated by the presence of two intense bands of ribosomal RNA (23S and 16S). The reverse transcription process was successful and amplification of cellulase gene using the specific primer F1 and R1 resulted in a DNA fragment of 1900 bp as a candidate of cellulase gene. The fragment was successfuly cloned into the pGEM-T-Easy vector, and the resulted recombinant plasmid was successfully introduced into the E. coli cells. Nucleotide sequence analysis suggested that the isolated cellulase gene shares 99% homology with the endo-1,6-beta-glucanase of T. harzianum.