Literature summary for 3.2.1.73 extracted from

Huang, J.W.; Cheng, Y.S.; Ko, T.P.; Lin, C.Y.; Lai, H.L.; Chen, C.C.; Ma, Y.; Zheng, Y.; Huang, C.H.; Zou, P.; Liu, J.R.; Guo, R.T.
Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (2012), Appl. Microbiol. Biotechnol., 94, 111-121.

Application

Application	Comment	Organism
agriculture	1,3-1,4-beta-D-glucanase are widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems	Fibrobacter succinogenes

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli and Pichia pastoris	Fibrobacter succinogenes
expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), heterologous expression of extracellular wild-type and mutant enzymes in Pichia pastoris	Fibrobacter succinogenes

Crystallization (Commentary)

Crystallization (Comment)	Organism
apo-form and substrate complex structures of mutant V18Y/W203Y, to 1.53 A resolution	Fibrobacter succinogenes
purified recombinant mutant V18Y/W203Y alone and in complex with product cellotetraose , from 0.1 M TrisHCl, pH 7.5, 0.3 M calcium acetate, and 29% PEG 5000 MME for the free mutant, and 0.15 M Tris, pH 8.5, 0.4 M calcium acetate and 33% PEG 5000 MME plus soaking in mother liquor with 5 mM cellotetraose for 1 h for the complexed mutant, X-ray diffraction structure determination and analysis at 1.53 A resolution, molecular replacement method	Fibrobacter succinogenes

Crystallization (Comment)

Organism

apo-form and substrate complex structures of mutant V18Y/W203Y, to 1.53 A resolution

Fibrobacter succinogenes

purified recombinant mutant V18Y/W203Y alone and in complex with product cellotetraose , from 0.1 M TrisHCl, pH 7.5, 0.3 M calcium acetate, and 29% PEG 5000 MME for the free mutant, and 0.15 M Tris, pH 8.5, 0.4 M calcium acetate and 33% PEG 5000 MME plus soaking in mother liquor with 5 mM cellotetraose for 1 h for the complexed mutant, X-ray diffraction structure determination and analysis at 1.53 A resolution, molecular replacement method

Fibrobacter succinogenes

Protein Variants

Protein Variants	Comment	Organism
233Stop	28.4% of wild-type activity	Fibrobacter succinogenes
M27D/M39R	0.1% of wild-type activity	Fibrobacter succinogenes
M27D/M39R	site-directed mutagenesis, almost inactive mutant	Fibrobacter succinogenes
M27R/M39D	0.2% of wild-type activity	Fibrobacter succinogenes
M27R/M39D	site-directed mutagenesis, almost inactive mutant	Fibrobacter succinogenes
M39F	92.8% of wild-type activity	Fibrobacter succinogenes
M39F	site-directed mutagenesis, 7% reduced activity compared to wild-type	Fibrobacter succinogenes
additional information	construction of mutants based on catalytic domain, residues 1-243, with higher thermostability and specific activity	Fibrobacter succinogenes
additional information	kinetic and thermostability analysis of the mutant enzymes, overview	Fibrobacter succinogenes
S71F	loss of activity	Fibrobacter succinogenes
S71F	site-directed mutagenesis, inactive mutant	Fibrobacter succinogenes
S84D	80.9% of wild-type activity	Fibrobacter succinogenes
S84D	site-directed mutagenesis, 19% reduced activity compared to wild-type	Fibrobacter succinogenes
V18Y	104% of wild-type activity. Increase in thermostability by 2 degrees	Fibrobacter succinogenes
V18Y	site-directed mutagenesis, 4% reduced activity compared to wild-type	Fibrobacter succinogenes
V18Y/W203Y	134.3% of wild-type activity	Fibrobacter succinogenes
V18Y/W203Y	site-directed mutagenesis, 34% increased activity compared to wild-type	Fibrobacter succinogenes
V61F	20.9% of wild-type activity	Fibrobacter succinogenes
V61F	site-directed mutagenesis, 79% reduced activity compared to wild-type	Fibrobacter succinogenes
W203F	87.6% of wild-type activity	Fibrobacter succinogenes
W203F	site-directed mutagenesis, 13% reduced activity compared to wild-type	Fibrobacter succinogenes
W203Y	130.2% of wild-type activity	Fibrobacter succinogenes
W203Y	site-directed mutagenesis, 30% increased activity compared to wild-type	Fibrobacter succinogenes

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism	Structure
additional information	-	additional information	2.25 mg/ml for wild-type enzyme, 2.44 mg/ml for mutant V18Y, 3.67 mg/ml for mutant W203Y, and 3.29 mg/ml for mutant V18Y/W203Y, with lichenan, pH 5.0, 50°C, calculated as reducing sugar released from substrate	Fibrobacter succinogenes

Organism

Organism	UniProt	Comment	Textmining
Fibrobacter succinogenes	P17989	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, dialysis, tag cleavage and again nickel affinity chromatography, recombinant extracellular wild-type and mutants V18Y, W203Y, and V18Y/W203Y enzymes from Pichia pastoris cell culture supernatant by dialysis and anion exchange chromatography	Fibrobacter succinogenes

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg]	Specific Activity Maximum [µmol/min/mg]	Comment	Organism
5694	-	wild-type, expressed in Escherichia coli, pH 5.0, 50°C	Fibrobacter succinogenes
6520	-	mutant V18Y, expressed in Escherichia coli, pH 5.0, 50°C	Fibrobacter succinogenes
9263	-	mutant W203Y, expressed in Escherichia coli, pH 5.0, 50°C	Fibrobacter succinogenes
9967	-	mutant V18Y/W203Y, expressed in Escherichia coli, pH 5.0, 50°C	Fibrobacter succinogenes

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
1,3-1,4-beta-D-glucan + H2O	-	Fibrobacter succinogenes	?	-	?
barley beta-glucan + H2O	-	Fibrobacter succinogenes	?	-	?
lichenan + H2O	-	Fibrobacter succinogenes	?	-	?

Synonyms

Synonyms	Comment	Organism
1,3-1,4-beta-D-glucanase	-	Fibrobacter succinogenes

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
50	-	assay at	Fibrobacter succinogenes

Temperature Stability [°C]

Temperature Stability Minimum [°C]	Temperature Stability Maximum [°C]	Comment	Organism
57	-	Tm of wild-type enzyme	Fibrobacter succinogenes
59	-	Tm of mutant V18Y	Fibrobacter succinogenes

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
2.25	-	1,3-1,4-beta-D-glucan	wild-type, expressed in Escherichia coli, pH 5.0, 50°C	Fibrobacter succinogenes
2.44	-	1,3-1,4-beta-D-glucan	mutant V18Y, expressed in Escherichia coli, pH 5.0, 50°C	Fibrobacter succinogenes
3.29	-	1,3-1,4-beta-D-glucan	mutant V18Y/W203Y, expressed in Escherichia coli, pH 5.0, 50°C	Fibrobacter succinogenes
3.67	-	1,3-1,4-beta-D-glucan	mutant W203Y, expressed in Escherichia coli, pH 5.0, 50°C	Fibrobacter succinogenes
2924	-	lichenan	pH 5.0, 50°C, wild-type enzyme, calculated as reducing sugar released from substrate	Fibrobacter succinogenes
3224	-	lichenan	pH 5.0, 50°C, mutant V18Y, calculated as reducing sugar released from substrate	Fibrobacter succinogenes
5003	-	lichenan	pH 5.0, 50°C, mutant W203Y, calculated as reducing sugar released from substrate	Fibrobacter succinogenes
5090	-	lichenan	pH 5.0, 50°C, mutant V18Y/W203Y, calculated as reducing sugar released from substrate	Fibrobacter succinogenes

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
5	-	assay at	Fibrobacter succinogenes