A novel carboxylesterase from Aspergillus niger and its hydrolysis of succinimide esters

Abstract

A novel carboxylesterase (EC 3.1.1.1) from Aspergillus niger has been purified 1400-fold by ammonium sulphate fractionation, ion exchange chromatography, hydrophobic interaction chromatography and gel filtration. The enzyme consisted of two identical subunits, each with a molecular weight of 60,000. The isoelectric point was 4.5. The optimal pH for the hydrolysis of cinnamic acid ethyl ester and 2-furylacryloyl N-hydroxy succinimide ester was between 5 and 7. The enzyme, which had no lipase activity, catalyzed the hydrolysis of activated ester substrates where the alcohol moiety was N-hydroxysuccinimide, p-nitrophenol and phenol as well as, with lower rates, unactivated esters like ethyl and benzyl esters. The enzyme exhibited a preference for substrates with an acyl moiety containing an aryl group. The enzyme was inhibited by PMSF but not by Hg2+ and EDTA. It is classified as a serine carboxylesterase. INTRODUCTIONCarboxyl ester hydrolases (EC 3.1.1.-), which catalyze the hydrolysis of ester bonds, are widely distributed in nature. Based on their hydrolytic specificities, these enzymes may be divided into carboxylesterases (EC 3.1.1.1), arylesterases (EC 3.1.1.2) and lipases (EC 3.1.1.3). Lipases hydrolyse triacyl-glycerols and a number of other esters, participating in hydrolysis reactions at the water/fat interface. Arylesterases preferentially cleave aromatic esters, such as esters of phenol, naphthol and indole (4). Carboxylesterases, which are rather unspecific, catalyze the hydrolysis of ester bonds, where both the alcohol moiety and the acid moiety may be aliphatic as well as aromatic (3,5,11,14,18,19).Carboxylesterases are involved in a number of biotransformations: hydrolysis of xenobiotic esters, activation of prodrugs, and metabolism of extraneous compounds. Some blood esterases Abbreviations: Ac = acetyl; Bz = benzoyl; Bzl = benzyl; Caps = 3-(cyclohexylamino)-l-propanesulfonic acid; Ches = 2(N-cyclohexylamino)ethane sulfonic acid; Cin = cinnamoyl; DMF = N,N-dimethylformamide; DMSO = dimethylsulfoxide; EDTA = ethylenediamine tetraacetic acid; Et = ethyl; Fa = furylacryloyl; Hepes = N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid; Hex = hexanoyl; HPLC = high performance liquid chromatography; Me = methyl; Mes = 2-(N-morpholino)ethanesulfonic acid; Ph = phenyl; PMSF = phenylmethylsulfonyl fluoride; Pr = propionyl; Oct = octanoyl; ONp = p-nitrophenoxy; ONSu = succinimidooxy; SDS = sodium dodecyl sulfate; Z = carbobenzoxy.Springer-Verlag

Chitin-Lignin Material as a Novel Matrix for Enzyme Immobilization

“…The high percentage of acidic amino acids (Asp and Glu) gives the molecule a net negative charge, which is higher than the total for the positively charged residues (Arg, Lys, and His) [ 62 ]. That is why the isoelectric point of this protein is about 4 [ 63 , 64 ]. This value indicates that only at pH values below it will the surface charge (and indirectly zeta potential) be positive.…”