抄録
From the culture supernatant of a thermophilic actinomycete KP1246,
which grows at 45°C to 66°C, two electrophoretically homogenous
p-nitrophenyl-β-D-glucopyranoside (pNPG)-degrading β-glucosidases
(BGL1, BGL2) were obtained by the combination of an ion -exchange and
gel-filtration chromatography. The molecular weight, Stokes radius, and
sedimentation coefficient of BGL1 were found to be 51,000, 2.92, and 4.4S,
respectively, which were similar to those of BGL2 (50,000, 3.06, and 4.2S,
respectively). However, differences were observed in the isoelectric point
(pI) (5.4 for BGL1 and 4.2 for BGL2) and in the optimum pH for activity
(6.6 for BGL1 and 5.5 for BGL2). The optimum temperature for activity
was found to be 70°C for BGL1 and 55°C for BGL2. In the study on the
retention of heat resistance after
30 min of processing, BGL1 was
demonstrated to retain 100% of the original activity over a wide range of pH, from 5.0 to 11.0. Meanwhile,
the stability range of pH was found to be narrower, i.e., 7 to 9.5, for BGL2.
For both BGL1 and BGL2, pNPG, as well as cellobios e and salicin, were
found to be good substrates. Neithe r BGL1 nor BGL2 reacted with Avicel,
carboxymethylcellulose (CM-cellulose), or maltose. Enzymatic reaction of
BGL1 was not at all inhibited by glucose; however, in the enzymatic r eaction
of BGL2, glucose acted as a competitive inhibitor (Ki = 408 mM).
Cellobiose acted as a noncompetitive inhibitor to BGL1 (Ki = 50 mM) and
showed a similar inhibition format to BGL2 (Ki = 15.6 mM). Addition of 2
mM EDTA, Pb2 + , Fe 2 + , Mn2 + , or Mg2 + did not at all inhibit the activity of
BGL1; however, these resulted in 26.1%, 2 5.2%, 22.3%, 17.5%, and 10.5%
inhibition of BGL2 activity, respectively. pCMB (p-chloromercuribennzoate)
was found to inhibit BGL1 activity by 100%; however,
the level of inhibition was only 32.5% for BGL2. The results of this kinetic
study, which is the first of its kind, on the thermophilic actinomycete
T. curvata suggest that at least 2 different types of heat-resistant BGLs function in the final stage of the cellulase degradation system of the
KP1246 strain and that the strain can be classified as T. curvata.
