Application | Comment | Organism |
---|---|---|
biotechnology | controlled mono-PEGylation of A1-III alginate lyase mutant A53C produces a conjugate with wild type levels of activity. The PEGylated mutant exhibits enhanced solution phase kinetics with bacterial alginate. In vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer show that the PEGylated enzyme is substantially less immunoreactive. More than 90% of adherent, mucoid, Pseudomonas aeruginosa biofilms are removed from abiotic surfaces following a one h treatment with the PEGylated mutant, whereas the wild type enzyme removes only 75% of biofilms in parallel studies | Sphingomonas sp. |
medicine | controlled mono-PEGylation of A1-III alginate lyase mutant A53C produces a conjugate with wild type levels of activity. The PEGylated mutant exhibits enhanced solution phase kinetics with bacterial alginate. In vitro binding studies with both enzyme-specific antibodies, from immunized New Zealand white rabbits, and a single chain antibody library, derived from a human volunteer show that the PEGylated enzyme is substantially less immunoreactive. More than 90% of adherent, mucoid, Pseudomonas aeruginosa biofilms are removed from abiotic surfaces following a one h treatment with the PEGylated mutant, whereas the wild type enzyme removes only 75% of biofilms in parallel studies | Sphingomonas sp. |
medicine | site-specific mono-PEGylation of genetically engineered A1-III alginate lyase yields an enzyme with enhanced performance relative to therapeutically relevant metrics. Over 90% of adherent, mucoid, Pseudomonas aeruginosa biofilms are removed from abiotic surfaces following a one h treatment with the PEGylated variant, whereas the wild-type enzyme removes only 75% of biofilms | Sphingomonas sp. |
Cloned (Comment) | Organism |
---|---|
expression in T7 driven pET vector system | Sphingomonas sp. |
expression of C-terminally His-tagged wild-type and mutant enzymes in Escherichia coli | Sphingomonas sp. |
Protein Variants | Comment | Organism |
---|---|---|
A270C | decrease in both KM value and maximum reaction velocity | Sphingomonas sp. |
A270C | site-directed mutagenesis, codon optimization, the mutant shows reduced activity compared to the wild-type enzyme | Sphingomonas sp. |
A328C | decrease in both KM value and maximum reaction velocity | Sphingomonas sp. |
A328C | site-directed mutagenesis, codon optimization, the mutant shows reduced activity compared to the wild-type enzyme | Sphingomonas sp. |
A41C | low maximum reaction velocities | Sphingomonas sp. |
A41C | site-directed mutagenesis, codon optimization, the mutant shows reduced activity compared to the wild-type enzyme | Sphingomonas sp. |
A53C | maximum reaction velocities similar to wild-type, decrease in KM value. Application of mutant to produce lyase-PEG conjugates with enhanced catalytic function and reduced immunoreactivity | Sphingomonas sp. |
A53C | site-directed mutagenesis, codon optimization, the mutant maintains Vmax values similar to the wild-type enzyme. Subsequent PEGylation produces a 60% increase in Vmax restoring the variant's maximum reaction velocity to wild-type levels while not altering the reduced Km value. The result is an enzyme-PEG conjugate with a 2fold improved catalytic efficiency compared to wild-type | Sphingomonas sp. |
additional information | construction of rationally designed, site-specific PEGylation variants from codon optimized enzyme by orthogonal maleimide-thiol coupling chemistry, the genetically engineered alginate lyase-PEG conjugates exhibit enhanced solution phase kinetics with bacterial alginate,enhanced catalytic function and reduced immunoreactivity. In contrast to random PEGylation of the enzyme by NHS-ester mediated chemistry, controlled mono-PEGylation of A1-III alginate lyase produces a conjugate that maintains wild-type levels of activity towards a model substrate. Over 90% of adherent, mucoid, Pseudomonas aeruginosa biofilms are removed from abiotic surfaces following a one h treatment with the PEGylated variant, whereas the wild-type enzyme removes only 75% of biofilms | Sphingomonas sp. |
S32C | decrease in both KM value and maximum reaction velocity | Sphingomonas sp. |
S32C | site-directed mutagenesis, codon optimization, the mutant shows highly reduced activity compared to the wild-type enzyme | Sphingomonas sp. |
KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
---|---|---|---|---|---|
additional information | - |
additional information | KM value of wild-type, substrate alginate, 0.08 mg/ml | Sphingomonas sp. | |
additional information | - |
additional information | Michaelis-Menten kinetics of wild-type and mutant enzymes, overview | Sphingomonas sp. |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Sphingomonas sp. | Q75WP3 | - |
- |
Sphingomonas sp. | Q9KWU1 | - |
- |
Purification (Comment) | Organism |
---|---|
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli by metal affinity chromatography | Sphingomonas sp. |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
alginate | - |
Sphingomonas sp. | ? | - |
? | |
sodium alginate | brown seaweed alginate (BSWA) as model substrate or baterial alginate purified from the mucoid Pseudomonas aeruginosa clinical isolate FRD1 | Sphingomonas sp. | ? | - |
? |
Synonyms | Comment | Organism |
---|---|---|
A1-III | - |
Sphingomonas sp. |
alginate lyase | - |
Sphingomonas sp. |