7/27/2019 Purification, Peptide Sequencing , Modelling and Characterisation of Extracellular Laccase Secreted by Pleurotus S

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urification, peptide sequencing, modelling and

aracterization of extracellular laccase secreted by

eurotus sajor-caju MTCC 141

ntik Bose

ffilations I Contributions I Corresponding authors

extracellular laccase which is secreted by Pleurotus sajorcaju has been purified using

monium sulphate precipitation, DEAE-cellulose, Sephadex G-100, ConA-Sepharose 4B and

090A HPLC fitted with Vydac C-18 Reverse- Phase chromatography with 722.2 fold of

fication and yield of 1.64%. 12% SDS-PAGE showed a monomer of 90KD. Western Blotting was

e with monoclonal Lcc Cbr2 rabbit antibody raised against copper binding region II of laccase

m Agaricus bisporus. Peptide sequencing of the purified laccase showed 529 amino acids long

tide with a 23 amino acids long signal peptide. Three Plastocyanin like domains were observed (59, 170-312 and 380-499). Residues 57, 239, 282 and 465 of the extracellular laccase showed N-

ed N-acetyl D-glucoseamine residues. The enzyme showed KM of 36M, 216pM, 380pM, 370pM

260pM with 2,6 dimethoxyphenol, Catechol, m-cresol, pyrogallol and syringaldazine

ectively. CuSo4, BaCl2, MgCl2, FeCL3, ZnCl3, Urea, PCMB, DTT, -mercaptoethanol had no effect

enzyme activity while HgCl2 and MnCl2 were weak inhibitors and SDS, Sodium azide were strong

bitors. Crystallization data, collection, synchrotron radiation and

cessing have been carried out. Orthorhombic crystals of space group P2 12 121 w A, b= 85.0 A, c= 91.5

A 3/Da, assuming one molecule per asymmetric unit. Diffraction data was collected using Beamline BW7B of Synchroton De

tonen Synchroton (DESY) at room temperature. Data was processed and scaled with programs DENZO and SCALEPACK of Hkl suit. For 1.9-20 A resoluti

data were 99.7% complete and scale with an overall R sym of 0.063 and Rsym of 0.38 for data in highest resolution -

- A resolution

t of 0.434 was obtained. A2F

0Fe electron density map revealed clearly all four copper sites . Repeated rounds of refinement with REFMAC resul

plete and well-defined model with an Rwork A resolution. Only 4 out of total 499 amino acids were in ge

wed region of Ramachandran Plot.

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