Abstract:
The exploitation of plant biomass due to its inexhaustible nature is an imperative
need for the production of cellulases which show a high specific activity against
cellulose if the potential agriculture and industrial benefits of plant biomass are to
be fully realized. An acidothermophilic cellulose degrading organism called
Acidothermus cellulolyticus strain 11B produces multiple acidic thermophilic
cellulases. Out of all the cellulase producing genes, Cel5A (EI beta-1,4-
endoglucanase) was amplified by PCR using the genomic DNA as a template. The
amplified fragment was ligated in T-A cloning vector using the tailing technique.
The resultant plasmid Cel5A/pTZ57R/T was transformed into E. coli DH5α cells.
Positive colonies were screened for the presence of insert. The excised and purified
gene fragments were then ligated in NdeI/XhoI double digested pET22b(+) vector.
The recombinant construct was transformed into E. coli BL21 CodonPlus RIPL,
under the control of IPTG-inducible T7-lac promoter. SDS-PAGE analysis showed
that a protein of molecular weight 34kDa was expressed in the insoluble fraction of
the host bacterium. An expression level of approximately 28% of the total cell
protein was obtained with a minimal activity of 0.275 U. A mixture of genetically
modified plant biomass and promising cellulases whether identified from natural
sources or synthetically enhanced may be required in combination for the final
critical breakthrough in this area of research.
