Catalytic Activities of Pyrococcus horikoshii Mutant Endoglucanase
Hyperthermophilic enzyme β-1,4 endoglucanase
(EGPh) from Pyrococcus horikoshii shows great promise as a
candidate for use in cellulose hydrolysis at high temperatures. A
single amino acid mutation (Y245G) in the active site of the
endoglucanase from Acidotermus cellulolyticus (EGAc) improved
the release of glucose by 13%. Based on these findings, we
postulated that the catalytic activity of endoglucanase from P.
horikoshii (EGPh) might as be improved by a similar mutation of
the homologous tyrosine to glycine (Y304G). We then cloned and
mutated the EGPh gene to produce a new variant named EGPhm.
With successful mutation confirmed by sequencing, this gene
along with EGAc was expressed in BL21-Codon plus (DE3)-RIL
cells. Purification was carried out using ionic exchange
chromatography and gel filtration. Further kinetic analysis using
p-nitrophenyl-β-D-Cellobioside (PNPC) was carried out.
Substrate binding of EGPh and EGPhm remained unchanged
with a similar Km for both enzymes (1.44mM). The Vmax values
for EGPh and EGPhm were 7.72E-05mM/second and 1.06E-
4mM/second respectively. The Turnover number (TON) for EGPh
was 2382/minute whereas in the case of EGPhm it was
3271/minute. The 37.4% increase in TON value proves that the
tyrosine to glycine (Y304G) mutation improves the catalytic rate