Catalytic Activities of Pyrococcus horikoshii Mutant Endoglucanase

  • A. Martynova Van Kley
  • J. L. Del-Aguila
  • C. Rorex
  • A. Nalian


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
of EGPhm.

How to Cite
VAN KLEY, A. Martynova et al. Catalytic Activities of Pyrococcus horikoshii Mutant Endoglucanase. GSTF Journal of Chemical Sciences (JChem), [S.l.], v. 2, n. 2, p. 5, jan. 2019. ISSN 2339-5079. Available at: <>. Date accessed: 16 feb. 2019.