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Abstract
A recently discovered hyperthermophilic enzyme β-1,4 endoglucanase (EGPh) from Pyrococcus horikoshii shows great promise as a candidate for use in cellulose hydrolysis at high temperatures. Although there is low homology between the endoglucanase from Acidotermus cellulolyticus (EGAc) and endoglucanase from Pyrococcus horikoshii (EGPh), Ando et al. proved that the latter has an endoglucanase activity at high temperatures. Rignall et al mutated a single amino acid (Y245G) in the active site of EGAc improving the release of glucose by 13%. Based on these findings, it was postulated that the catalytic activity of EGPh could be improved by the mutation of the homologous tyrosine to glycine (Y304G). To test this hypothesis, the EGPh gene was amplified, cloned, sequenced and mutated to produce a new gene, EGPhm. Both genes were transformed into BL21-Codon plus (DE3)-RIL cells. The expressed proteins were then purified by ionic exchange chromatography and gel filtration. Kinetic analysis of the purified enzyme was performed for using the substrate p-nitrophenyl-β-D-Cellobioside (PNPC). The affinity of the substrate for EGPh and EGPhm was not changed as both showed similar Km values (1.4mM). The Vmax values for EGPh and EGPhm were 7.72E-05mM/sec and 1.06E-4mM/sec respectively. The Turnover number (TON) for EGPh was 2382/min whereas in the case of EGPhm was 3271/min. The higher TON value of EGPhm proved that catalytic rate of EGPh was improved. Together, the present findings support the hypothesis that Tyrosine at position 304 influences the catalytic activity of Pyrococcus horikoshii b-1,4-endoglucanase.
