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E134A/E138A
-
no enzymic activity, but reaction occurs in presence of sodium azide, E138 is the general acid-base catalyst
N207D
mutant displays better thermotolerance than the wild type but also reduced activity
A98W
-
active-site variant
E105Q/E109Q
-
catalytically impaired
E131Q
-
active-site variant
E63D
-
active-site variant
E63Q
-
active-site variant
H99D
-
active-site variant
K48A
-
123% of wild-type activity, thermostability and halostability are enhanced. Catalytic efficiency of mutants is enhanced as a result of the increase in substrate affinity. The half-lives of the mutant increases up to about 7fold at 60-70°C
K48L
-
137% of wild-type activity, thermostability and halostability are enhanced. Catalytic efficiency of mutants is enhanced as a result of the increase in substrate affinity. The half-lives of the mutant increases up to about 7fold at 60-70°C
N26A
-
active-site variant
R65A
-
active-site variant
S90A
-
active-site variant
W184Y
-
active-site variant
W192A
-
active-site variant
Y123A
-
active-site variant
Y123F
-
active-site variant
Y24A
-
active-site variant
Y24F
-
active-site variant
Y24W
-
active-site variant
K48A
-
123% of wild-type activity, thermostability and halostability are enhanced. Catalytic efficiency of mutants is enhanced as a result of the increase in substrate affinity. The half-lives of the mutant increases up to about 7fold at 60-70°C
-
K48L
-
137% of wild-type activity, thermostability and halostability are enhanced. Catalytic efficiency of mutants is enhanced as a result of the increase in substrate affinity. The half-lives of the mutant increases up to about 7fold at 60-70°C
-
K48A
mutation enhances catalytic efficiency, thermostability and halostability
K48L
mutation enhances catalytic efficiency, thermostability and halostability
T113S
mutant displays improved thermal stability
T113S
-
mutant displays improved thermal stability
-
E46P/S43E/H205P/S40E
-
64% increase in specific activity, 20°C increase in optimal enzymatic temperature and a 13.8 °C rise in protein melting temperature compared to wild-type
K20S/N31C/S40E/S43E/E46P/P102C/K117S/N125C/K165S/T187C/H205P
mutant with increased catalytic activity and thermostability
N31C/T187C
-
the mutations significantly enhance the protein thermostability
P102C/N125C
-
the mutations significantly enhance the protein thermostability
E46P/S43E/H205P/S40E
-
64% increase in specific activity, 20°C increase in optimal enzymatic temperature and a 13.8 °C rise in protein melting temperature compared to wild-type
-
N31C/T187C
-
the mutations significantly enhance the protein thermostability
-
P102C/N125C
-
the mutations significantly enhance the protein thermostability
-
233Stop
28.4% of wild-type activity
D202L
shows a 1.2fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
D202N
exhibits a 1.8fold increase in catalytic efficiency (kcat/KM) compared to the wild-type
D206M
shows a 1.1fold increase in catalytic efficiency (kcat/KM) compared to the wild-type
D206N
exhibits a 1.5fold increase in catalytic efficiency (kcat/KM) compared to the wild-type
D206R
exhibits the highest relative activity at 50°C over 10 min, shows a 1.2fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
D58A
-
no enzymatic activity
D58E
-
dramatic decrease in kcat, substrate affinity similar to wild type
D58N
-
dramatic decrease in kcat, substrate affinity similar to wild type
E11L
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 10fold decrease in specific activity, more than 2fold increase in KM-value, significant decrease in catalytic efficiency
E47I
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 2fold increase in KM-value
E56A
-
no enzymatic activity
E56D
-
dramatic decrease in kcat, substrate affinity similar to wild type
E56Q
-
no enzymatic activity
E60A
-
no enzymatic activity
E60D
-
dramatic decrease in kcat, substrate affinity similar to wild type
E60Q
-
no enzymatic activity
E85D
has 5fold lower kcat/Km ratios than the wild-type
E85I
has 5fold lower kcat/Km ratios than the wild-type
F205L
shows a 3.8fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
F40I
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 10fold decrease in specific activity, significant decrease in catalytic efficiency
G201S
shows a 1.5fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
G207N
shows a fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
G63A
-
decrease in thermostability
K200F
is the most heat-sensitive enzyme, retains 72% of activity at 45°C for 10 min, shows a 1.2fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
K200M
shows a 1.1fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
K64A
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure
K64M
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 2fold increase in KM-value
L62G
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure
N139A
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 2fold increase in KM-value, significant decrease in catalytic efficiency
N208G
shows a fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
N44L
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure
N44Q
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 2fold increase in KM-value
N72A
has 11fold lower kcat/Km ratios than the wild-type
N72Q
has 17fold lower kcat/Km ratios than the wild-type
Q70A
has 299fold lower kcat/Km ratios than the wild-type
Q70D
has 62fold lower kcat/Km ratios than the wild-type
Q70E
has 106fold lower kcat/Km ratios than the wild-type
Q70I
has 499fold lower kcat/Km ratios than the wild-type
Q70N
has 63fold lower kcat/Km ratios than the wild-type
Q70R
has 35fold lower kcat/Km ratios than the wild-type
Q81I
has 2fold lower kcat/Km ratios than the wild-type
Q81N
has 2.5fold lower kcat/Km ratios than the wild-type
R137M
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 10fold decrease in specific activity, more than 2fold increase in KM-value, significant decrease in catalytic efficiency
R137Q
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 10fold decrease in specific activity, more than 2fold increase in KM-value, significant decrease in catalytic efficiency
R209M
shows a 1.1fold increase, in catalytic efficiency (kcat/KM) compared to the wild-type
T204F
shows a 2.2fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
W105F
-
significant decrease in thermostability
W105H
-
significant decrease in thermostability
W141F
-
5-7-fold increase in KM-value for lichenan compared to wild type, decrease in kcat-value, no significant change in thermal stability
W141H
-
5-7-fold increase in KM-value for lichenan compared to wild type, decrease in kcat-value, no significant change in thermal stability
W148F
-
decrease in kcat-value, no significant change in thermal stability
W165F
-
after incubation at pH 3.0, 1 h, 3-7-fold higher activity than wild type
W165H
-
significant decrease in thermostability
W186F
-
increase in kcat-value, no significant change in thermal stability
W198F
-
significant decrease in thermostability
W54F
-
decrease in kcat-value, no significant change in thermal stability
W54Y
-
decrease in kcat-value, no significant change in thermal stability
Y42L
mutant of truncated beta-glucanase catalytic domain, residues 1-243. No discernible changes in secondary structure, more than 10fold decrease in specific activity
A79P
-
decrease in thermal stability
F85Y
-
decrease in thermal stability
G44R
-
decrease in thermal stability
H300P
-
increase in thermal stability
K23R
-
decrease in thermal stability
M298K
-
thermal stabiltiy similar to wild type
N290H
-
slight increase in thermal stability
D70V
-
thermostable mutant, similar katalytic efficiency as wild type
C263S
-
random mutagenesis, the mutant shows an acidic shift in the pH optimum and altered substrate specificity compared to the wild-type enzyme
D221G2
-
random mutagenesis, the mutant shows unaltered properties compared to the wild-type enzyme
D56G
-
random mutagenesis, the mutant shows an acidic shift in the pH optimum and altered substrate specificity compared to the wild-type enzyme
D56G/D221G/C263S
-
random mutagenesis, mutant PtLic16AM2 shows an acidic shift in the pH optimum and altered substrate specificity compared to the wild-type enzyme. Mutation D221G alone does not lead to altered enzyme properties
E113A
-
inactive mutant. In the structure of E113A/1,3-1,4-beta-glucotriose complex, the sugar bound to the -1 subsite adopts an intermediate-like (alpha-anomeric) configuration
W108A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W108F
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W108Y
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W253A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W253F
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W253Y
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E113A
inactive mutant. In the structure of E113A/1,3-1,4-beta-glucotriose complex, the sugar bound to the -1 subsite adopts an intermediate-like (alpha-anomeric) configuration
W108A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W108F
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W108Y
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W253A
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W253F
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
W253Y
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
E113A
-
inactive mutant. In the structure of E113A/1,3-1,4-beta-glucotriose complex, the sugar bound to the -1 subsite adopts an intermediate-like (alpha-anomeric) configuration
-
W108A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
W108F
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
W108Y
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
W253A
-
site-directed mutagenesis, the mutant shows reduced activity compared to the wild-type enzyme
-
D105K
-
activity is reduced to less than 1%
D105N
-
activity is reduced to less than 1%
E103D
-
activity is reduced to less than 1%
E103Q
-
activity is reduced to less than 1%
E107D
-
activity is reduced to less than 1%
E107H
-
activity is reduced to less than 1%
E107Q
-
activity is reduced to less than 1%
W101F
-
activity is reduced to less than 1%
W101Y
-
activity is reduced to less than 1%
D100A
-
36% relative activity compared to the wild type enzyme
D106A
-
34% relative activity compared to the wild type enzyme
D195A
-
36% relative activity compared to the wild type enzyme
D220A
-
32% relative activity compared to the wild type enzyme
D258A
-
35% relative activity compared to the wild type enzyme
D314A
-
99% relative activity compared to the wild type enzyme
D317A
-
31% relative activity compared to the wild type enzyme
E196A
-
39% relative activity compared to the wild type enzyme
E242A
-
32% relative activity compared to the wild type enzyme
E262A
-
36% relative activity compared to the wild type enzyme
E323A
-
32% relative activity compared to the wild type enzyme
D195A
-
36% relative activity compared to the wild type enzyme
-
D258A
-
35% relative activity compared to the wild type enzyme
-
D314A
-
99% relative activity compared to the wild type enzyme
-
E323A
-
32% relative activity compared to the wild type enzyme
-
D156A
almost complete loss of activity
Q95A
almost complete loss of activity
C17R/Q87T/D152G/Y307H/V330A/N344D
E149D/V370E
the mutant shows 59.9% activity at pH 5.5 and 50°C compared to the wild type enzyme
K12R/F30L/N114D/N252D/T348S/N380S
the mutant shows 62.1% activity at pH 5.5 and 50°C compared to the wild type enzyme
N4S
the mutant shows 75.3% activity at pH 5.5 and 50°C compared to the wild type enzyme
S11R/T210S
the mutant shows 231.3% activity at pH 5.5 and 50°C compared to the wild type enzyme
Y389F/ K395R
the mutant shows 81% activity at pH 5.5 and 50°C compared to the wild type enzyme
K142N/Q203L/N214D
mutant isolated by error-prone PCR, 48.6 increase in catalytic activity compared to wild-type. The optimal pH of the mutated enzyme is 5.0, which is lower than the parent enzyme, but thermal stability is almost the same
K142N/Q203L/N214D
mutation leads to increased activity and decreased optimal pH, while the same thermostability is maintained
K142N/Q203L/N214D
-
mutant isolated by error-prone PCR, 48.6 increase in catalytic activity compared to wild-type. The optimal pH of the mutated enzyme is 5.0, which is lower than the parent enzyme, but thermal stability is almost the same
-
K142N/Q203L/N214D
-
mutation leads to increased activity and decreased optimal pH, while the same thermostability is maintained
-
E134A
-
catalyzes condensation reaction with alpha-fluorido-substrates
E134A
-
inactive mutant, analysis of substrate binding
E134A
-
mutant devoid of hydrolase activity but efficiently catalyzing transglycosylation
M44V/N53H
mutant displays best ability to tolerate an acid environment and improved thermal stability
M44V/N53H
mutant displays improved thermal stability
M44V/N53H
-
mutant displays best ability to tolerate an acid environment and improved thermal stability
-
M44V/N53H
-
mutant displays improved thermal stability
-
N31C/T187C/P102C/N125C
-
introduction of disulfide pairs N31C-T187C and P102C-N125C, 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature, catalytic properties are similar to wild-type with a decrease in optimal pH
N31C/T187C/P102C/N125C
-
the mutant with wild type activity shows a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature compared to the wild type enzyme
N31C/T187C/P102C/N125C
-
introduction of disulfide pairs N31C-T187C and P102C-N125C, 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature, catalytic properties are similar to wild-type with a decrease in optimal pH
-
N31C/T187C/P102C/N125C
-
the mutant with wild type activity shows a 48.3% increase in half-life value at 60°C and a 4.1°C rise in melting temperature compared to the wild type enzyme
-
M27D/M39R
0.1% of wild-type activity
M27D/M39R
site-directed mutagenesis, almost inactive mutant
M27R/M39D
0.2% of wild-type activity
M27R/M39D
site-directed mutagenesis, almost inactive mutant
M39F
-
5-fold increase in km value
M39F
92.8% of wild-type activity
M39F
site-directed mutagenesis, 7% reduced activity compared to wild-type
S71F
loss of activity
S71F
site-directed mutagenesis, inactive mutant
S84D
80.9% of wild-type activity
S84D
site-directed mutagenesis, 19% reduced activity compared to wild-type
V18Y
104% of wild-type activity. Increase in thermostability by 2 degrees
V18Y
site-directed mutagenesis, 4% reduced activity compared to wild-type
V18Y/W203Y
134.3% of wild-type activity
V18Y/W203Y
site-directed mutagenesis, 34% increased activity compared to wild-type
V61F
20.9% of wild-type activity
V61F
site-directed mutagenesis, 79% reduced activity compared to wild-type
W203F
-
increase in kcat-value, no significant change in thermal stability
W203F
exhibits a 2.4fold increase in catalytic efficiency (kcat/KM) compared to the wild-type
W203F
87.6% of wild-type activity
W203F
mutant of truncated beta-glucanase catalytic domain, residues 1-243. mutant has increased hydrolytic activity. Residue W203 is stacked with the glucose product of cellotriose. Two extra calcium ions and a Tris molecule bind to the mutant structure. A Tris molecule, bound to the catalytic residues of E56 and E60, is found at the position normally taken by substrate binding at the -1 subsite. A second Ca2+ ion is found near the residues F152 and E154 on the protein's surface, and a third one near the active site residue D202
W203F
site-directed mutagenesis, 13% reduced activity compared to wild-type
W203F
-
site-directed mutagenesis, truncated and mutated 1,31,4-beta-D-glucanase, no activity with laminarin
W203R
-
5-7-fold increase in KM-value for lichenan compared to wild type, decrease in kcat-value, after incubation at pH 3.0, 1 h, 3-7-fold higher activity than wild type, no significant change in thermal stability
W203R
exhibits a 207fold decrease in catalytic efficiency (kcat/KM) compared to the wild-type
W203Y
130.2% of wild-type activity
W203Y
site-directed mutagenesis, 30% increased activity compared to wild-type
C17R/Q87T/D152G/Y307H/V330A/N344D
2.1fold increase in specific activity
C17R/Q87T/D152G/Y307H/V330A/N344D
the mutant shows 200.8% activity at pH 5.5 and 50°C compared to the wild type enzyme
N197D/M322V
1.2fold increase in specific activity
N197D/M322V
the mutant shows 144.7% activity at pH 5.5 and 50°C compared to the wild type enzyme
E269S
site-directed mutagenesis, nucleophile replacement, inactive mutant
E269S
-
site-directed mutagenesis, nucleophile replacement, inactive mutant
-
additional information
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construction of diverse enzyme truncation mutants for domain functional analysis, overview
additional information
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generation of transgenic Nicotiana tabacum plants expressing LicB from Clostridium thermocellum, no altered phenotype, overview. Expression of bacterial beta-1,3-1,4-glucanase gene exerts no significant influence on tobacco plant metabolism, while the expression of bacterial beta-1,3-glucanase affects plant metabolism only at early stages of growth and development. By contrast, the expression of bacterial beta-1,4-glucanase has a significant effect on transgenic tobacco plant metabolism
additional information
construction of a fusion gene, encoding beta-1,3-1,4-glucanase both from Bacillus amyloliquefaciens and Clostridium thermocellum, via end-to-end fusion and expression in Escherichia coli. The catalytic efficiency of the fusion enzyme for oat beta-glucan is 2.7- and 20fold higher than that of the parental Bacillus amyloliquefaciens and Clostridium thermocellum enzymes, respectively, and the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bacillus amyloliquefaciens and Clostridium thermocellum enzymes are both less than 30%
additional information
-
construction of a fusion gene, encoding beta-1,3-1,4-glucanase both from Bacillus amyloliquefaciens and Clostridium thermocellum, via end-to-end fusion and expression in Escherichia coli. The catalytic efficiency of the fusion enzyme for oat beta-glucan is 2.7- and 20fold higher than that of the parental Bacillus amyloliquefaciens and Clostridium thermocellum enzymes, respectively, and the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bacillus amyloliquefaciens and Clostridium thermocellum enzymes are both less than 30%
additional information
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construction of hybrid genes encoding circularly permutated lichenase variants with integrated small peptides, i.e. NC-L-53, NC-L-99, NC-L-53-99, and NC-L-140, method overview. Generation of a thermostable lichenase gene variant encoding only the enzyme's catalytic domain LicBM3. Thermostabilities of the mutant constructs, overview
additional information
end-to-end fusion, circular permutation, domain insertion
additional information
end-to-end fusion, circular permutation, domain insertion
additional information
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end-to-end fusion, circular permutation, domain insertion
-
additional information
-
construction of diverse enzyme truncation mutants for domain functional analysis, overview
-
additional information
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construction of hybrid beta-glucanase enzyme H1 which contains the 107 amino-terminal residues of mature Bacillus amyloliquefaciens beta-glucanase and the 107 carboxyl-terminal amino acid residues of Bacillus beta-glucanase. Hybrid enzyme H2 consists of the 105 amino-terminal residues from the Bacillus macerans enzyme and the carboxyl-terminal 107 amino acids from Bacillus amyloliquefaciens. Hybrid enzyme H1 exhibits increased thermostability especially in an acidic environment compared to both parental enzymes. Hybrid enzyme H2 is more thermolabile than the naturally occuring beta-glucanases
additional information
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hybrid enzymes containing 16, 36, 78, or 152 amino acid N-terminal sequence derived from Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan glucanohydrolase followed by a C-terminal segment derived from Bacillus macerans 1,3-1,4-beta-D-glucan glucanohydrolase, expression in Escherichia coli
additional information
construction of a fusion gene, encoding beta-1,3-1,4-glucanase both from Bacillus amyloliquefaciens and Clostridium thermocellum, via end-to-end fusion and expression in Escherichia coli. The catalytic efficiency of the fusion enzyme for oat beta-glucan is 2.7- and 20fold higher than that of the parental Bacillus amyloliquefaciens and Clostridium thermocellum enzymes, respectively, and the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bacillus amyloliquefaciens and Clostridium thermocellum enzymes are both less than 30%
additional information
-
construction of a fusion gene, encoding beta-1,3-1,4-glucanase both from Bacillus amyloliquefaciens and Clostridium thermocellum, via end-to-end fusion and expression in Escherichia coli. The catalytic efficiency of the fusion enzyme for oat beta-glucan is 2.7- and 20fold higher than that of the parental Bacillus amyloliquefaciens and Clostridium thermocellum enzymes, respectively, and the fusion enzyme can retain more than 50% of activity following incubation at 80°C for 30 min, whereas the residual activities of Bacillus amyloliquefaciens and Clostridium thermocellum enzymes are both less than 30%
additional information
-
modifying five (out of 12) epsilon-amino groups in lysine residues with nitrous acid improves thermostablility and enzymatic activity of the enzyme, optimizing the condition of chemical modification, overview. Compared to the wild-type enzyme, the optimally-modified enzyme has higher specific activity and T50 value, which are 3370 U/mg and 70°C, respectively. Its half-life values at 50 and 60°C are extended and reach 58.5 and 49.5 min, respectively
additional information
-
covalent linkage between the N- and C-termini of a polypeptide chain to create circular variants of the enzyme by an intein-driven protein splicing approach, method, overview. Two circular variants, LicAC1 and LicA-C2, which have connecting loops of 20 and 14 amino acids, respectively, show catalytic activities that are approximately two and three times higher, respectively, compared to that of the linear LicA, LicA-L1. Also the thermal stability of the circular variants is significantly increased compared to the linear form. The circular proteins contain a thrombin recognition site and can be linearized by cleavage with thrombin
additional information
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subsite +1 mutants, kinetic analysis
additional information
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the truncated gene product, devoid of cellulose-binding domain, shows 60% of activity and binds to avicel
additional information
construction of a chimeric bifunctional laccase/beta-1,3-1,4-glucanase mutant enzyme by insertion fusion of the bglS and cotA genes, protein CotA, UniProt ID P07788, the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface increase catalytic activities,molecular dynamics simulations, overview. The laccase efficiency with substrate 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) is higher in the chimera, while the glucanase activity with lichenan shows a Kcat/KM value increased by 26%, a lower Km anf kcat. The beta-1,3-1,4-glucanase hydrolyzes plant cell wall beta-glucans, and the copper-dependent oxidase laccase catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The mutant chimeric enzyme shows synergistic sugar release from milled sugarcane bagasse
additional information
-
construction of a chimeric bifunctional laccase/beta-1,3-1,4-glucanase mutant enzyme by insertion fusion of the bglS and cotA genes, protein CotA, UniProt ID P07788, the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface increase catalytic activities,molecular dynamics simulations, overview. The laccase efficiency with substrate 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) is higher in the chimera, while the glucanase activity with lichenan shows a Kcat/KM value increased by 26%, a lower Km anf kcat. The beta-1,3-1,4-glucanase hydrolyzes plant cell wall beta-glucans, and the copper-dependent oxidase laccase catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The mutant chimeric enzyme shows synergistic sugar release from milled sugarcane bagasse
additional information
end-to-end fusion by cyclization with SpyTag/SpyCatcher, and oligomerization by Foldon
additional information
end-to-end fusion by cyclization with SpyTag/SpyCatcher, and oligomerization by Foldon
additional information
end-to-end fusion by cyclization with SpyTag/SpyCatcher, and oligomerization by Foldon
additional information
end-to-end fusion by cyclization with SpyTag/SpyCatcher, and oligomerization by Foldon
additional information
-
end-to-end fusion by cyclization with SpyTag/SpyCatcher, and oligomerization by Foldon
-
additional information
-
construction of a chimeric bifunctional laccase/beta-1,3-1,4-glucanase mutant enzyme by insertion fusion of the bglS and cotA genes, protein CotA, UniProt ID P07788, the approximation of the two catalytic domains in the chimeric enzyme, and the formation of an inter-domain interface increase catalytic activities,molecular dynamics simulations, overview. The laccase efficiency with substrate 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid) is higher in the chimera, while the glucanase activity with lichenan shows a Kcat/KM value increased by 26%, a lower Km anf kcat. The beta-1,3-1,4-glucanase hydrolyzes plant cell wall beta-glucans, and the copper-dependent oxidase laccase catalyzes the oxidation of aromatic compounds with simultaneous reduction of oxygen to water. The mutant chimeric enzyme shows synergistic sugar release from milled sugarcane bagasse
-
additional information
-
the truncated gene product, devoid of cellulose-binding domain, shows 60% of activity and binds to avicel
-
additional information
rational design of disulfide bonds in the enzyme by site-directed mutagenesis
additional information
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rational design of disulfide bonds in the enzyme by site-directed mutagenesis
-
additional information
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truncated form of enzyme containing the catalytic domain from amino acid 1-258, higher thermal stability and enzymatic activity than wild type protein, crystal structure
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construction of mutants based on catalytic domain, residues 1-243, with higher thermostability and specific activity
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engineering of dual-functional hybrid glucanases from a truncated and mutated 1,3-1,4-beta-D-glucanase gene TFsW203F from Fibrobacter succinogenes, and a 1,3-beta-D-glucanase gene TmLam from hyperthermophilic Thermotoga maritima used as target enzymes, by ligating substrate-binding domains (TmB1 and TmB2) and the catalytic domain (TmLamCD) of TmLam to the N- or C-terminus of TFsW203F to create four hybrid enzymes, TmB1-TFsW203F, TFsW203F-TmB2, TmB1-TFsW203F-TmB2 and TFsW203F-TmLamCD, creation of desirable hybrid enzymes with economic benefits for industrial applications. Improved thermal tolerance of the hybrid enzyme TFsW203FTmLamCD, fluorescence and circular dichroism spectrometric analyses, overview. Kinetic properties of parental TFsW203F and mutant hybrid glucanases
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kinetic and thermostability analysis of the mutant enzymes, overview
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end-to-end fusion, site-directed mutagenesis
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end-to-end fusion, site-directed mutagenesis
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protein-engineered, thermostable (1,3-1,4)-beta-glucanase: the codons for hybrid H(A12-M)DELTAY13 are modified to match those of the gene encoding barley (1,3-1,4)-beta-glucanase isoenzyme EII
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screening of the random mutant library, mutations D56G, D221G, and C263S have only minor effects on specific activity and pH stability
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construction of hybrid beta-glucanase enzyme H1 which contains the 107 amino-terminal residues of mature Bacillus amyloliquefaciens beta-glucanase and the 107 carboxyl-terminal amino acid residues of Bacillus beta-glucanase. Hybrid enzyme H2 consists of the 105 amino-terminal residues from the Bacillus macerans enzyme and the carboxyl-terminal 107 amino acids from Bacillus amyloliquefaciens. Hybrid enzyme H1 exhibits increased thermostability especially in an acidic environment compared to both parental enzymes. Hybrid enzyme H2 is more thermolabile than the naturally occuring beta-glucanases
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hybrid enzymes containing 16, 36, 78, or 152 amino acid N-terminal sequence derived from Bacillus amyloliquefaciens 1,3-1,4-beta-D-glucan glucanohydrolase followed by a C-terminal segment derived from Bacillus macerans 1,3-1,4-beta-D-glucan glucanohydrolase, expression in Escherichia coli
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end-to-end fusion
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truncated mutants, which comprise 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type, show complete loss of enzymatic activity
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UV irradiation leads to mutants TC2 and TC5 with increased activity against barley beta-glucan during growth on solka floc
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deletion of the N-terminal sequence up to residue Ser38. The mutant is devoid of the helix-turn motif interacting with residues of the substrate-binding cleft at the level of subsite -3. The mutation uncovers the enzyme cleft at the -3 subsite and turns the enzyme into an endo-beta(1,4)-glucanase
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deletion of the N-terminal sequence up to residue Ser38. The mutant is devoid of the helix-turn motif interacting with residues of the substrate-binding cleft at the level of subsite -3. The mutation uncovers the enzyme cleft at the -3 subsite and turns the enzyme into an endo-beta(1,4)-glucanase
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