Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis

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Item Type:	Article
Title:	Purification and characterization of the constitutive form of laccase from the basidiomycete Coriolus hirsutus and effect of inducers on laccase synthesis
Creators Name:	Koroljova-Skorobogatko, O.V., Stepanova, E.V., Gavrilova, V.P., Morozova, O.V., Lubimova, N.V., Dzchafarova, A.N., Jaropolov, A.I. and Makower, A.
Abstract:	An isolate of Coriolus hirsutus constitutively expresses substantial amounts of extracellular laccase on a defined growth medium. The most efficient inducer of extracellular laccase synthesis was syringaldazine, which increased the enzyme yield by 1000% at a concentration of 0.11 microM. The constitutive form of the enzyme was purified 312-fold. Laccase from C. hirsutus, with an estimated molecular mass of 55 kDa and pI of 4.0, is a monomeric glycoprotein containing 12% carbohydrate consisting of mannose and N-acetylglucosamine. The laccase was found to contain 3.9-4.1 copper atoms per molecule. The absorption spectrum shows a maximum at 610 nm and a shoulder at 330 nm, which is typical of laccase possessing type 1 and type 3 copper atoms. The parameters of the first type of copper were determined by EPR as g perpendicular=2.046 and g parallel=2.200, A parallel=8.103 x 10(-3) cm-1. Laccase was found to be a pH-stable and thermostable enzyme. With organic substrates it exhibits a pH optimum of 4.5, but with the inorganic substrate K4[Fe(CN)6] this decreased to 3.5. The highest efficiency of catalysis was observed with sinapinic acid as the substrate. The kinetic constants kcat and Km of this reaction were 578 s-1 and 24 microM respectively. It was established that the kinetics of the assayed reaction shows a Ping Pong mechanism.
Keywords:	Amino Acids, Basidiomycota, Coumaric Acids, Electron Spin Resonance Spectroscopy, Enzyme Induction, Fungal Proteins, Glycoproteins, Hydrazones, Hydrogen-Ion Concentration, Isoenzymes, Kinetics, Laccase, Metalloproteins, Oxidoreductases, Spectrophotometry, Substrate Specificity
Source:	Biotechnology and Applied Biochemistry
ISSN:	0885-4513
Publisher:	Portland Press
Volume:	28
Number:	Pt 1
Page Range:	47-54
Date:	1 August 1998
Official Publication:	http://www.babonline.org/bab/028/bab0280047.htm
PubMed:	View item in PubMed

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