Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity

Abstract

Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme.

Ribonuclease A modification with poly[N‐(2‐hydroxypropyl)methacrylamide] copolymers: new route of synthesis and purification

“…The reactive groups of the copolymer are on the sides of the chain hence, the availability of the aldehyde groups depends strongly on the conformational arrangement. This result is opposite to a common PEGylation reaction, where the modification yield is better using low molecular weight of polyethylene glycol (PEG) . Nevertheless, PEG polymers are activated at the end of the chain, which is less sterically hindered.…”

Catalytic Activation of Esterases by PEGylation for Polyester Synthesis

“…[13] Regarding the methodology used to PEGylate, it is likely that the amine residue of the N ‐terminus had been also PEGylated. This assumption is based on the use of acidic pH during the reaction, which led to the activation of the N ‐terminus …”

PEGylation Greatly Enhances Laccase Polymerase Activity

“…The role of PEG as a template of MTX polymerization was also evaluated. To achieve this, α‐chymotrypsin was PEGylated with mPEG (5 kDa) and further used for MTX polymerization . From the results obtained, no differences in DP and amount of polymer were observed after polymerization with PEGylated α‐chymotrypsin (Table ; 1 a) and 1 b)).…”