Literature summary for 4.2.2.3 extracted from

Lamppa, J.; Ackerman, M.; Lai, J.; Scanlon, T.; Griswold, K.
Genetically engineered alginate lyase-peg conjugates exhibit enhanced catalytic function and reduced immunoreactivity (2011), PLoS ONE, 6, e17042.

Search for more literature for 4.2.2.3

Application

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(Commentary)

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

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 [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

Organism	UniProt	Comment	Textmining
Sphingomonas sp.	Q75WP3	-	-
Sphingomonas sp.	Q9KWU1	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant C-terminally His-tagged wild-type and mutant enzymes from Escherichia coli by metal affinity chromatography	Sphingomonas sp.

Substrates and Products (Substrate)

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

Synonyms	Comment	Organism
A1-III	-	Sphingomonas sp.
alginate lyase	-	Sphingomonas sp.

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Release 2023.1 (January 2023)