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E157G
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
E313G
site-directed mutagenesis, altered kinetics compared to the wild-type enzyme
E229D
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site-directed mutagenesis, enzyme BgaS7a, the mutant shows slightly increased activity compared to the wild-type enzyme
E229D/G803D
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site-directed mutagenesis, inactive mutant
E229D/V405A
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site-directed mutagenesis, enzyme BgaS7, the mutant shows similar thermal optima and thermostabilities as BgaS, but shows a 2.5fold increase in catalytic activity at 15°C and hydrolyzes 80% of lactose in skim milk in less than half the time of BgaS at 2.5°C
E229D/V405A/G803D
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site-directed mutagenesis, enzyme BgaS6, the mutant shows similar thermal optima and thermostabilities as BgaS, the mutant shows slightly increased activity compared to the wild-type enzyme
G803D
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site-directed mutagenesis, inactive mutant
V405A
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site-directed mutagenesis, enzyme BgaS7b, the mutant shows slightly increased activity compared to the wild-type enzyme
V405A/G803D
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site-directed mutagenesis, inactive mutant
E229D
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site-directed mutagenesis, enzyme BgaS7a, the mutant shows slightly increased activity compared to the wild-type enzyme
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E229D/G803D
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site-directed mutagenesis, inactive mutant
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G803D
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site-directed mutagenesis, inactive mutant
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V405A/G803D
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site-directed mutagenesis, inactive mutant
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S259F/P261T/L262S/G263S
mutant shows slightly reduced affinity for substrate (Km: 3.76 mM), 8.5fold reduction in inhibition by galactose, maximal activity lower than wild-type, pH stability lower than wild-type
N140C
the mutant shows improved activity compared to the wild type enzyme
N140C/W806F
the mutant shows improved activity compared to the wild type enzyme
W806F
the mutant shows improved activity compared to the wild type enzyme
N140C
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the mutant shows improved activity compared to the wild type enzyme
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N140C/W806F
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the mutant shows improved activity compared to the wild type enzyme
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W806F
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the mutant shows improved activity compared to the wild type enzyme
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E359A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The E359A mutant exhibits no activity
F349A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The F349A, R111A, and W187A mutant enzyme in the presence of galactose shows remaining activity of 65%
F349S
mutant exhibits the highest activity in the presence of galactose relative to the activity measured in the absence of galactose among the tested mutant enzymes at position 349. The Ki-value for galactose of the F349S mutant (160 mM), which is 13fold that of the wild-type enzyme, is the highest among the reported values of beta-galactosidase. The wild-type enzyme hydrolyzes 62% of 100 g lactose/l with the addition of 30 g galactose/l, whereas the F349S mutant hydrolyzes more than 99%
H362A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The H362A mutant in the presence of galactose exhibits remaining activity below 30%
I44A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The I44A mutant in the presence of galactose exhibits remaining activity below 30%
N149A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The N149A mutant in the presence of galactose exhibits remaining activity below 30%
R111A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The F349A, R111A, and W187A mutant enzyme in the presence of galactose shows remaining activity of 46%
S41A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The S41A mutant in the presence of galactose exhibits remaining activity below 30%
W187A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The F349A, R111A, and W187A mutant enzyme in the presence of galactose shows remaining activity of 44%
W319A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The W319A mutant in the presence of galactose exhibits remaining activity below 30%
Y281A
30 g galactose/l. The wild-type enzyme exhibits 13% remaining activity relative to the activity observed in the absence of galactose. The Y281A mutant exhibits no activity
D429A
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the mutant has wild type activity
E354A
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the activity of the E354A mutant with nonactivated natural substrates is 1100fold lower than that of the wild type enzyme, while its activity is only 10fold lower when assayed with 2,4-dinitrophenyl 2-acetamido-2-deoxy-alpha-D-galactopyranosyl-(1-3)-[alpha-L-fucopyranosyl-(1-2)]-beta-D-galactopyranoside
E467A
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the mutant has wild type activity
E416Q
the mutant enzyme does not have a Mg2+ bound at the active site even at high Mg2+ concentrations (200 mM), low catalytic activity and the pH profile is very different from that of the native enzyme
E416V
the mutant enzyme does not have a Mg2+ bound at the active site even at high Mg2+ concentrations (200 mM), low catalytic activity and the pH profile is very different from that of the native enzyme
H418E
the mutant shows low catalytic activity
H418F
the mutant shows low catalytic activity, the variant has reduced stability, binds Mg2+ weakly and cannot be crystallized
H418N
the mutant shows low catalytic activity
N460T
analysis of rate constants and activation thermodynamics to demonstrate the valuable mechanistic details of enzymes that can be obtained from studies of this type
E416Q
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the mutant enzyme does not have a Mg2+ bound at the active site even at high Mg2+ concentrations (200 mM), low catalytic activity and the pH profile is very different from that of the native enzyme
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E416V
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the mutant enzyme does not have a Mg2+ bound at the active site even at high Mg2+ concentrations (200 mM), low catalytic activity and the pH profile is very different from that of the native enzyme
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R109K
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the mutant shows decreased specific activity towards 4-nitrophenyl-beta-D-galactopyranoside compared to the wild type enzyme, the apparent Km values toward the natural substrates lactose and lactosucrose are strongly increased
R109V
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the mutant shows increased specific activity towards 4-nitrophenyl-beta-D-galactopyranoside compared to the wild type enzyme, the apparent Km values toward the natural substrates lactose and lactosucrose are strongly increased
R109W
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the mutant shows increased specific activity towards 4-nitrophenyl-beta-D-galactopyranoside compared to the wild type enzyme, the apparent Km values toward the natural substrates lactose and lactosucrose are strongly increased
R109K
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the mutant shows decreased specific activity towards 4-nitrophenyl-beta-D-galactopyranoside compared to the wild type enzyme, the apparent Km values toward the natural substrates lactose and lactosucrose are strongly increased
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R109V
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the mutant shows increased specific activity towards 4-nitrophenyl-beta-D-galactopyranoside compared to the wild type enzyme, the apparent Km values toward the natural substrates lactose and lactosucrose are strongly increased
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R109W
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the mutant shows increased specific activity towards 4-nitrophenyl-beta-D-galactopyranoside compared to the wild type enzyme, the apparent Km values toward the natural substrates lactose and lactosucrose are strongly increased
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D332N
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mutation seriously reduces catalytic activity
R148S
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mutation has no effect on catalytic activity
E491A
the mutant shows wild type optimal temperature, but displays an optimal pH at 6.5-7.0
L317F
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maximal velocity deviates from that of wild-type enzyme only at lower temperatures, in 2 mM Mg2+. This temperature-dependent effect can be suppressed by increasing the Mg2+ concentration
E173N
mutant enzyme shows no activity
E389N
mutant enzyme shows no activity
E446N
beta-fucosidase activity is 26.49% compared to wild-type activity, beta-glucosidase activity is 21.37% compared to wild-type activity, beta-galactosidase activity is 15.7% compared to wild-type activity
N172A
beta-fucosidase activity is 18.38% compared to wild-type activity, beta-glucosidase activity is 17.65% compared to wild-type activity, beta-galactosidase activity is 13.4% compared to wild-type activity
T29A/V213I/L217M/N277H/I387V/R491C/N496D
large range mutant screening after direct chemical evolution, the mutant variant YG6762 has nucleotide changes in 13 sites: T30C, A85G, C621A, G637A, T649A, A829V, T912G, C924T, A1159G, G1242C, C1471T, A1486G, and C1497A, the mutant YG6762 shows 4.9-7.5fold increased activity, dependent on the reaction temperature, but decreased thermostability, compared to the wild-type enzyme, overview, revertion of the mutation at each single of the 8 mutation sites results in decreased activity of the altered mutant, especially when reverting residue 277 to Asn, overview
F359Q
compared to wild-type enzyme the mutant enzyme is less stable at 75°C, the optimum is similar to that of wild-type enzyme. Km of the mutant enzyme is increased about 2fold and the kcat is increased by 32.3%
F359Q
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compared to wild-type enzyme the mutant enzyme is less stable at 75°C, the optimum is similar to that of wild-type enzyme. Km of the mutant enzyme is increased about 2fold and the kcat is increased by 32.3%
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E184A
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site-directed mutagenesis, the mutant functions as an efficient thioglycoligase, which synthesises thiogalactosides with linkages to the 3 and 4 positions of glucosides and galactosides in high yields, the mutant shows reduced galactohydrolase activity compared to the wild-type enzyme
E184Q
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site-directed mutagenesis, the mutant does not function as a thioglycoligase, the mutant shows reduced galactohydrolase activity compared to the wild-type enzyme
F441Y
compared to wild-type enzyme the mutant enzyme is less stable at 75°C, the optimum is similar to that of wild-type enzyme. Km of the mutant enzyme is increased about 2fold and the kcat is increased by 36.6%
F441Y
mutant enzyme shows good thermostability
F441Y
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compared to wild-type enzyme the mutant enzyme is less stable at 75°C, the optimum is similar to that of wild-type enzyme. Km of the mutant enzyme is increased about 2fold and the kcat is increased by 36.6%
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F441Y
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mutant enzyme shows good thermostability
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additional information
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gene bgaS3 encoding a loss of function variant is subjected to random mutagenesis to restore activity and discover potential interactions important in cold activity
additional information
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gene bgaS3 encoding a loss of function variant is subjected to random mutagenesis to restore activity and discover potential interactions important in cold activity
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additional information
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construction of a hybrid mutant enzyme exchanging domains from Kluyveromyces lactis and Aspergillus niger enzymes, addition of a secretory signal in the N-terminus of the recombinant protein, Kluyveromyces lactis enzyme optimization for industrial productions by fusion with extracellular and more stable enzyme from Aspergillus niger, overview, kinetics of growth and secretion
additional information
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construction of a mutant strain H26-10-7 from parental wild-type strains H26 and H-10-7, the enzyme from mutant strain H26-10-7 shows a 5fold increased activity and enhanced thermostability compared to the wild-type strain enzyme, overview
additional information
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cross-linking of the enzyme with concanavalin A as insoluble complex using glutaraldehyde, and entrappment into calcium alginate beads, the entrapped complex cross-linking enhances the enzyme stability during activity and storage, method development, overview
additional information
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construction of LacA and GalA knockout mutants, the increased beta-galactosidase activity generated in response to the addition of galactan is eliminated by inactivating lacA or galA but unaffected by the inactivation of yesZ, overview
additional information
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a peptide of 27 amino acids, representing the G-H loop amino acid sequence of foot-and mouth disease virus VP1 protein is inserted into different sites of protein lACZ, a pseudo-wild-type beta-galactosidase lacking the 8 N-terminal amino acids
additional information
engineering the Escherichia coli beta-galactosidase for the screening of antiviral protease inhibitors. Identification of a new accomodation site between amino acids 581 and 582, in a solvent-exposed and flexible beta-turn of domain III. the replacement of the model peptide reproducing the matrix-capsid (p17/p24) gag cleavage sequence renders a highly active and efficiently digested chimeric construct. The use of this insertion site, that increases the cleavage potential of this reporter enzyme, can improve the sensitivity and dynamic range of the antiviral drug assay
additional information
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engineering the Escherichia coli beta-galactosidase for the screening of antiviral protease inhibitors. Identification of a new accomodation site between amino acids 581 and 582, in a solvent-exposed and flexible beta-turn of domain III. the replacement of the model peptide reproducing the matrix-capsid (p17/p24) gag cleavage sequence renders a highly active and efficiently digested chimeric construct. The use of this insertion site, that increases the cleavage potential of this reporter enzyme, can improve the sensitivity and dynamic range of the antiviral drug assay
additional information
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modulation of the catalytic properties of the enzyme using different supports and immobilization strategies, e.g. bearing glyoxyl, epoxy, or BrCN groups or by glutaraldehyde crosslinking on matrices containing primary amino groups, different derivatives exhibit very different synthetic activity/hydrolytic activity ratios in transglycosylation reactions strongly depending on the experimental conditions, overview
additional information
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expression in Escherichia coli under control of araBD promoter. The addition of D-fucose causes an improvement in specific beta-galactosidase production, although beta-galactosidase is produced as an inclusion body. The addition of D-fucose after induction leads to an increase in the specific activity of beta-galactosidase inclusion bodies and causes a changes in the structure of beta-galactosidase inclusion bodies, with higher enzyme activity
additional information
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single enzyme molecule assays performed on the native enzyme as well as recombinant enzyme with and without His-tag reveal significant differences in the average combined turnover number indicating that in vivo and in vitro produced enzymes are not identical and the presence of a C-terminal His-tag may alter the activity of beta-galactosidase. Average combined turnover numbers of 35800 per min and 31700 per min are obtained respectively for the in vitro synthesized enzyme from strain 35321 with the absence and presence of a C-terminal His6-tag
additional information
an overproducing mutant is isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contain 25fold higher enzyme levels than the parent
additional information
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an overproducing mutant is isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contain 25fold higher enzyme levels than the parent
additional information
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an overproducing mutant is isolated by UV mutagenesis and screening on agar plates containing X-Gal substrate. Cytoplasmic extracts of the mutant contain 25fold higher enzyme levels than the parent
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additional information
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transfection of beta-galactosidase gene to fibroblast cells in culture using liposomes. 24 h after transfection, treated cells show a higher specific enzyme activity than untreated cells. Cells maintained in culture for 7 days show values similar to those of untreated patients with GM1 gangliosidosis
additional information
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construction of a hybrid mutant enzyme exchanging domains from Kluyveromyces lactis and Aspergillus niger enzymes, addition of a secretory signal in the N-terminus of the recombinant protein, enzyme optimization for industrial productions by fusion with extracellular and more stable enzyme from Aspergillus niger, overview, kinetics of growth and secretion
additional information
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construction of a hybrid mutant enzyme exchanging domains from Kluyveromyces lactis and Aspergillus niger enzymes, addition of a secretory signal in the N-terminus of the recombinant protein, enzyme optimization for industrial productions by fusion with extracellular and more stable enzyme from Aspergillus niger, overview, kinetics of growth and secretion
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additional information
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introduction of a second catalytic domain A to give mutant FMNP01Gal-2. Mutant shows almost 3fold increase in specific activity
additional information
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introduction of a second catalytic domain A to give mutant FMNP01Gal-2. Mutant shows almost 3fold increase in specific activity
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additional information
disruption of gene mbgA in the bacterial chromosome results in loss of lactose-inducible beta-galactosidase production, base substitutions within two partially overlapping catabolite-responsive elements CRE-I and/or CRE-II cause partial relief from catabolite repression
additional information
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in-frame gene fusion between galactose dehydrogenase, beta-galactosidase and galactokinase, the two major enzyme forms of hybrid protein consist of 4 and 8 subunits, each subunit is made up of one monomer each of galactose dehydrogenase, EC 1.1.1.48, beta-galactosidase, EC 3.2.1.23, and galactokinase, EC 2.7.1.6
additional information
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isolation of three phenotypically stable mutants by screening on activity against 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside, isolation of the insertion sequence ISC1217 from the mutant enzymes, ISC1217 contains terminal inverted repeats and is flanked by a direct repeat of 6 bp, overview
additional information
the bgaC deletion mutation does not significantly attenuate the virulence of Streptococcus pneumoniae in vivo, but the bgaC mutant strain shows relatively low numbers of viable cells compared to the wild type after 24 h of infection in vivo. The mutant shows higher colonization levels at 6 and 24 h postinfection in vivo
additional information
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the bgaC deletion mutation does not significantly attenuate the virulence of Streptococcus pneumoniae in vivo, but the bgaC mutant strain shows relatively low numbers of viable cells compared to the wild type after 24 h of infection in vivo. The mutant shows higher colonization levels at 6 and 24 h postinfection in vivo
additional information
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enzyme immobilization on acid-cleaned, silanized support