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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Record number 401448
Title Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase
Author(s) Nakai, H.; Petersen, B.O.; Westphal, Y.; Dilokpimol, A.; Hachem, M.A.; Duus, J.O.; Schols, H.A.; Svensson, B.
Source Protein Engineering, Design & Selection 23 (2010)10. - ISSN 1741-0126 - p. 781 - 787.
DOI http://dx.doi.org/10.1093/protein/gzq055
Department(s) Food Chemistry Group
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2010
Keyword(s) thermoanaerobacter-brockii - enzymatic-synthesis - crystal-structure - enzymes - cloning - brevis - gene - oligosaccharides - glucoamylase - purification
Abstract Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (a/a)6-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with ß-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (a/a)6-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413–Glu421, His413–Ile418 and His413–Glu415 from loop 3, that include His413 and Glu415 presumably recognising the a-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426–Ala431 in TP and Thr419–Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (a1, a1)- and a-(1,2)-regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.
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