Aglycone specificity of Thermotoga neapolitana β-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis

Table 3 Michaelis-Menten constants.

Enzyme	K_M (mM)	k_cat (s^-1)	k_cat/K_M (s^-1 mM^-1)
p NPGlc-hydrolysis
Wild-type	0. 24 ± .04	485 ± 31	2000
N221S	0.43 ± 0.11	1170 ± 152	2720
N221S/P342L	0.89 ±0.19	1710 ± 237	1910
N221S/P165L/M278I	0.66 ±0.18	1070 ± 171	1630
F219L/K214R	0.24 ±0.04	594 ± 36	2510
F219L/P165L/M278I	0.18 ±0.006	253 ± 3	1440
G222Q/V203M/K214R	0.10 ± .008	154 ± 3	1470
G222Q/P165L/M278I	0.27 ± .05	181 ± 15	678
G222M	0.17 ±0.03	254 ± 16	1470
Q4'-hydrolysis*
Wild type	0.06 ±0.03	7.2 ± 1.2	122
N221S/P342L	0.02 ±0.015	6.2 ± 0.95	281
Q3-hydrolysis*
Wild type	0.13 ±0.06	7.2 ± 1.8	55
N221S/P342L	0.05 ±0.02	7.1 ± 1.1	154

*Data from Lindahl et al [17].
For p NPGlc hydrolysis measurements were made at pH 5.6, 80°C, and for the quercetin glucoside hydrolysis (wt and one mutant) at pH 5.0, 90°C.

ISSN: 1471-2091