کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8288552 | 1536257 | 2018 | 11 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Structural and functional insights into RHA-P, a bacterial GH106 α-L-rhamnosidase from Novosphingobium sp. PP1Y
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کلمات کلیدی
E. coliGHSSDSABSORFα-L-rhamnosidaseIPTGTLCGTsBSA - BSASite-directed mutagenesis - mutagenesis مواجه با سایتbovin serum albumin - آلبومین سرم بوینEscherichia coli - اشریشیا کُلیisopropyl β-D-1-thiogalactopyranoside - ایزوپروپیل β-D-1-thiogalactopyranosideAbsorbance - جذبRoom temperature - دمای اتاقsodium dodecyl sulphate - سدیم دودسیل سولفاتFlavonoid - فلاونوئیدopen reading frame - قاب خواندن بازLuria Bertani - لوریا برتانیHomology modeling - مدل سازی همگراGlycosyl hydrolases - هیدرولیزهای گلیسوزیلoptical density - چگالی نوریthin layer chromatography - کروماتوگرافی لایه نازکGlycosyl transferases - گلیکوزیل ترانسفراز
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شیمی
پیش نمایش صفحه اول مقاله
چکیده انگلیسی
α-L-Rhamnosidases (α-RHAs, EC 3.2.1.40) are glycosyl hydrolases (GHs) hydrolyzing terminal α-l-rhamnose residues from different substrates such as heteropolysaccharides, glycosylated proteins and natural flavonoids. Although the possibility to hydrolyze rhamnose from natural flavonoids has boosted the use of these enzymes in several biotechnological applications over the past decades, to date only few bacterial rhamnosidases have been fully characterized and only one crystal structure of a rhamnosidase of the GH106 family has been described. In our previous work, an α-l-rhamnosidase belonging to this family, named RHA-P, was isolated from the marine microorganism Novosphingobium sp. PP1Y. The initial biochemical characterization highlighted the biotechnological potential of RHA-P for bioconversion applications. In this work, further functional and structural characterization of the enzyme is provided. The recombinant protein was obtained fused to a C-terminal His-tag and, starting from the periplasmic fractions of induced recombinant cells of E. coli strain BL21(DE3), was purified through a single step purification protocol. Homology modeling of RHA-P in combination with a site directed mutagenesis analysis confirmed the function of residues D503, E506, E644, likely located at the catalytic site of RHA-P. In addition, a kinetic characterization of the enzyme on natural flavonoids such as naringin, rutin, hesperidin and quercitrin was performed. RHA-P showed activity on all flavonoids tested, with a catalytic efficiency comparable or even higher than other bacterial α-RHAs described in literature. The results confirm that RHA-P is able to hydrolyze both α-1,2 and α-1,6 glycosidic linkages, and suggest that the enzyme may locate different polyphenolic aromatic moities in the active site.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Archives of Biochemistry and Biophysics - Volume 648, 15 June 2018, Pages 1-11
Journal: Archives of Biochemistry and Biophysics - Volume 648, 15 June 2018, Pages 1-11
نویسندگان
Francesca Mensitieri, Federica De Lise, Andrea Strazzulli, Marco Moracci, Eugenio Notomista, Valeria Cafaro, Emiliano Bedini, Matthew Howard Sazinsky, Marco Trifuoggi, Alberto Di Donato, Viviana Izzo,