Literature summary extracted from

Lebbink, J.H.; Kaper, T.; Bron, P.; van der Oost, J.; de Vos, W.M.
Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus beta-glucosidase CelB by directed evolution (2000), Biochemistry, 39, 3656-3665.

Protein Variants

EC Number	Protein Variants	Comment	Organism
3.2.1.B28	A419T	159% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 109% with 4-nitrophenyl beta-D-galactoside, 53% residual activity after 1 h at 106°C	Pyrococcus furiosus
3.2.1.B28	I161V	110% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 37% with 4-nitrophenyl beta-D-galactoside, 84% residual activity after 1 h at 106°C	Pyrococcus furiosus
3.2.1.B28	I67T	110% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 97% with 4-nitrophenyl beta-D-galactoside, 100% residual activity after 1 h at 106°C	Pyrococcus furiosus
3.2.1.B28	K285R	155% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 103% with 4-nitrophenyl beta-D-galactoside, 58% residual activity after 1 h at 106°C	Pyrococcus furiosus
3.2.1.B28	K70R	175% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 147% with 4-nitrophenyl beta-D-galactoside, 1% residual activity after 1 h at 106°C	Pyrococcus furiosus
3.2.1.B28	M424V	199% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 140% with 4-nitrophenyl beta-D-galactoside, 24% residual activity after 1 h at 106°C	Pyrococcus furiosus
3.2.1.B28	N415S	259% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 64% with 4-nitrophenyl beta-D-galactoside. 7.5fold increase in the ratio of 4-nitrophenyl beta-D-glucopyranoside/p-nitrophenyl beta-D-galactopyranoside hydrolysis, no decrease in thermostability	Pyrococcus furiosus
3.2.1.B28	T371A	148% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 149% with 4-nitrophenyl beta-D-galactoside, 14% residual activity after 1 h at 106°C	Pyrococcus furiosus
3.2.1.B28	V211A	260% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 151% with 4-nitrophenyl beta-D-galactoside, 1% residual activity after 1 h at 106°C	Pyrococcus furiosus

KM Value [mM]

EC Number	KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
3.2.1.B28	0.42	-	4-nitrophenyl beta-D-glucopyranoside	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	0.53	-	4-nitrophenyl beta-D-glucopyranoside	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.57	-	4-nitrophenyl beta-D-glucopyranoside	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.7	-	4-nitrophenyl beta-D-galactopyranoside	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.92	-	4-nitrophenyl beta-D-glucopyranoside	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	1.1	-	4-nitrophenyl beta-D-glucopyranoside	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	1.2	-	4-nitrophenyl beta-D-galactopyranoside	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	1.3	-	4-nitrophenyl beta-D-glucopyranoside	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	1.3	-	4-nitrophenyl beta-D-galactopyranoside	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	1.4	-	4-nitrophenyl beta-D-glucopyranoside	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	2	3	cellobiose	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	3.4	-	4-nitrophenyl beta-D-galactopyranoside	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	4.5	-	4-nitrophenyl beta-D-galactopyranoside	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	5	-	4-nitrophenyl beta-D-galactopyranoside	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	8.5	-	4-nitrophenyl beta-D-galactopyranoside	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	11	-	cellobiose	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	14	-	cellobiose	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	16	-	cellobiose	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	20	-	cellobiose	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	25	-	cellobiose	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	38	-	cellobiose	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	52	-	lactose	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	52	-	lactose	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	83	-	lactose	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	95	-	lactose	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	97	-	lactose	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	120	-	lactose	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	220	-	lactose	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus

Organism

EC Number	Organism	UniProt	Comment	Textmining
3.2.1.B28	Pyrococcus furiosus	Q51723	-	-

Substrates and Products (Substrate)

EC Number	Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
3.2.1.B28	4-nitrophenyl beta-D-galactopyranoside + H2O	-	Pyrococcus furiosus	4-nitrophenol + D-galactopyranose	-	?
3.2.1.B28	4-nitrophenyl beta-D-glucopyranoside + H2O	-	Pyrococcus furiosus	4-nitrophenol + D-glucopyranose	-	?
3.2.1.B28	cellobiose + H2O	-	Pyrococcus furiosus	2 D-glucose	-	?
3.2.1.B28	lactose + H2O	-	Pyrococcus furiosus	D-glucose + D-galactose	-	?

Temperature Optimum [°C]

EC Number	Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
3.2.1.B28	98	-	-	Pyrococcus furiosus

Temperature Stability [°C]

EC Number	Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
3.2.1.B28	106	-	wild-type, 1 h, no loss of activity	Pyrococcus furiosus

Turnover Number [1/s]

EC Number	Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
3.2.1.B28	4.2	-	4-nitrophenyl beta-D-galactopyranoside	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	9.1	-	4-nitrophenyl beta-D-galactopyranoside	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	11	-	4-nitrophenyl beta-D-galactopyranoside	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	12	-	4-nitrophenyl beta-D-galactopyranoside	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	13	-	lactose	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	13	-	4-nitrophenyl beta-D-glucopyranoside	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	14	-	4-nitrophenyl beta-D-galactopyranoside	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	15	-	lactose	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	15	-	cellobiose	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	16	-	cellobiose	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	17	-	lactose	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	18	-	cellobiose	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	19	-	cellobiose	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	20	-	cellobiose	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	21	-	lactose	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	21	-	lactose	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	26	-	4-nitrophenyl beta-D-glucopyranoside	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	40	-	4-nitrophenyl beta-D-glucopyranoside	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	40	-	4-nitrophenyl beta-D-glucopyranoside	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	47	-	4-nitrophenyl beta-D-glucopyranoside	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	240	-	cellobiose	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	530	-	lactose	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	670	-	cellobiose	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	1000	-	4-nitrophenyl beta-D-galactopyranoside	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	1300	-	lactose	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	1400	-	4-nitrophenyl beta-D-glucopyranoside	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	1600	-	4-nitrophenyl beta-D-glucopyranoside	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	2400	-	4-nitrophenyl beta-D-galactopyranoside	wild-type, pH 4.8, 90°C	Pyrococcus furiosus

kcat/KM [mM/s]

EC Number	kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
3.2.1.B28	0.18	-	lactose	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.22	-	lactose	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.22	-	lactose	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.25	-	lactose	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.33	-	lactose	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.69	-	cellobiose	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	0.75	-	cellobiose	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	1	-	cellobiose	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	1.1	-	cellobiose	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	1.4	-	cellobiose	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	2.4	-	lactose	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	3.1	-	4-nitrophenyl beta-D-galactopyranoside	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	3.4	-	4-nitrophenyl beta-D-galactopyranoside	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	3.5	-	4-nitrophenyl beta-D-galactopyranoside	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	8.2	-	4-nitrophenyl beta-D-galactopyranoside	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	11	-	lactose	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	13	-	4-nitrophenyl beta-D-galactopyranoside	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	25	-	4-nitrophenyl beta-D-glucopyranoside	wild-type, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	31	-	4-nitrophenyl beta-D-glucopyranoside	mutant A419T, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	43	-	4-nitrophenyl beta-D-glucopyranoside	mutant M424V, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	43	-	4-nitrophenyl beta-D-glucopyranoside	mutant N4515S, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	45	-	4-nitrophenyl beta-D-glucopyranoside	mutant T371A, pH 4.8, 20°C	Pyrococcus furiosus
3.2.1.B28	120	-	4-nitrophenyl beta-D-galactopyranoside	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	480	-	4-nitrophenyl beta-D-galactopyranoside	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	1000	-	cellobiose	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	1000	-	4-nitrophenyl beta-D-glucopyranoside	mutant N4515S, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	2400	-	cellobiose	wild-type, pH 4.8, 90°C	Pyrococcus furiosus
3.2.1.B28	3900	-	4-nitrophenyl beta-D-glucopyranoside	wild-type, pH 4.8, 90°C	Pyrococcus furiosus