์ฝ˜ํ…์ธ ๋กœ ๊ฑด๋„ˆ๋›ฐ๊ธฐ
Merck

Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site-directed mutagenesis.

Microbial biotechnology (2013-07-03)
Mohammed Sherif, Debbie Waung, Bihter Korbeci, Valentina Mavisakalyan, Robert Flick, Greg Brown, Mamdouh Abou-Zaid, Alexander F Yakunin, Emma R Master
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Multicopper oxidases can act on a broad spectrum of phenolic and non-phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,6-dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where kcat and Km values at 60ยฐC were 8.1 (ยฑโ€‰0.8) sโปยน and 0.9 (ยฑโ€‰0.3) mM respectively. SLAC had a broad pH optimum for activity (between pHโ€‰4 and 8) and temperature optimum at 60-70ยฐC. It demonstrated remarkable thermostability with a half-life of over 10โ€‰h at 80ยฐC and over 7โ€‰h at 90ยฐC. Site-directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10-fold increase in activity compared with the wild-type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta-position of aromatic substrates.

MATERIALS
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Sigma-Aldrich
Laccase from Rhus vernicifera, crude acetone powder, โ‰ฅ50 units/mg solid
Sigma-Aldrich
Laccase from Trametes versicolor, powder, light brown, โ‰ฅ0.5 U/mg
Sigma-Aldrich
Laccase from Agaricus bisporus, powder, deep brown, โ‰ฅ4 U/mg