کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
2020233 1542318 2016 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Different recombinant forms of polyphenol oxidase A, a laccase from Marinomonas mediterranea
موضوعات مرتبط
علوم زیستی و بیوفناوری بیوشیمی، ژنتیک و زیست شناسی مولکولی زیست شیمی
پیش نمایش صفحه اول مقاله
Different recombinant forms of polyphenol oxidase A, a laccase from Marinomonas mediterranea
چکیده انگلیسی


• A recombinant membrane polyphenol oxidase was expressed as soluble protein.
• The kinetic properties on catechol were higher than for a number of laccases.
• A high thermostability and a strong resistance to NaCl and DMSO were apparent.
• The kinetic and stability properties are suitable for biotechnological applications.

Polyphenol oxidase from the marine bacterium Marinomonas mediterranea (MmPPOA) is a membrane-bound, blue, multi-copper laccase of 695 residues. It possesses peculiar properties that distinguish it from known laccases, such as a broad substrate specificity (common to tyrosinases) and a high redox potential. In order to push the biotechnological application of this laccase, the full-length enzyme was overexpressed in Escherichia coli cells with and without a C-terminal His-tag. The previous form, named rMmPPOA-695-His, was purified to homogeneity by HiTrap chelating chromatography following solubilization by 1% SDS in the lysis buffer with an overall yield of ≈1 mg/L fermentation broth and a specific activity of 1.34 U/mg protein on 2,6-dimethoxyphenol as substrate. A truncated enzyme form lacking 58 residues at the N-terminus encompassing the putative membrane binding region, namely rMmPPOA-637-His, was successfully expressed in E. coli as soluble protein and was purified by using the same procedure set-up as for the full-length enzyme. Elimination of the N-terminal sequence decreased the specific activity 15-fold (which was partially restored in the presence of 1 M NaCl) and altered the secondary and tertiary structures and the pH dependence of optimal stability. The recombinant rMmPPOA-695-His showed kinetic properties on catechol higher than for known laccases, a very high thermal stability, and a strong resistance to NaCl, DMSO, and Tween-80, all properties that are required for specific, targeted industrial applications.

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ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Protein Expression and Purification - Volume 123, July 2016, Pages 60–69
نویسندگان
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