Table 2. Kinetic Parameters of TxGH116 and Its Mutants for Hydrolysis of 4NP-Glycosides and Oligosaccharides.

		kinetic parameters (60 °C)			temperature
protein	substrate	KM (mM)	kcat (s–1)	kcat/KM (mM–1 s–1)	optimum	Tm
WT without N-term taga	4NP β-d-glucoside	0.18 ± 0.008	49.0 ± 0.8	272	75 °C	-
	4NP β-d-galactoside	16.3 ± 0.94	255 ± 5	15.6
	cellobiose	0.25 ± 0.016	44.4 ± 0.7	178
	laminaribiose	0.27 ± 0.018	41.4 ± 0.7	153
WT with N-term tagb	4NP β-d-glucoside	0.21 ± 0.012	37.6 ± 0.6	179	75 °C	81.3 °C
D508H	4NP β-d-glucoside	110 ± 3	3.47 ± 0.17	0.031	65 °C	66.5 °C
D508N	4NP β-d-glucoside	41.0 ± 2.5	30.2 ± 0.71	0.74	60 °C	67.2 °C
R544W	4NP β-d-glucoside	0.26 ± 0.011	48.2 ± 0.53	186	60 °C	76.2 °C
R786H	4NP β-d-glucoside	15.7 ± 0.50	193 ± 2.4	12.3	70 °C	73.9 °C

^aWild type without the N-terminal tag is used for comparison to R544W and R786H, which were purified in the same way as the crystallized protein.

^bWild type with the N-terminal tag is used for comparison to D508H and D508N, which could not be digested with enterokinase without internal cleavage of the protein. Temperature optimum curves are shown in SI Figure S8A. Melting temperatures (T_m) for all mutant proteins are those for the fusion protein without removal of the N-terminal tag. All T_m values were determined based on the change in circular dichroism at 220 nm upon heating. The melting curves are provided in SI Figure S8B.