Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase
Nakai, H. ; Petersen, B.O. ; Westphal, Y. ; Dilokpimol, A. ; Hachem, M.A. ; Duus, J.O. ; Schols, H.A. ; Svensson, B. - \ 2010
Protein Engineering, Design & Selection 23 (2010)10. - ISSN 1741-0126 - p. 781 - 787.
thermoanaerobacter-brockii - enzymatic-synthesis - crystal-structure - enzymes - cloning - brevis - gene - oligosaccharides - glucoamylase - purification
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.
Production of glucose syrups in highly concentrated systems
Veen, M.E. van der; Goot, A.J. van der; Boom, R.M. - \ 2005
Biotechnology Progress 21 (2005)2. - ISSN 8756-7938 - p. 598 - 602.
enzymatic-hydrolysis - alpha-glucosidase - kinetic-model - wheat-starch - glucoamylase - optimization - mechanism - extruder - reactor
We have investigated the hydrolysis of maltodextrins in a high concentration (up to 70%), by means of enzymatic and acid catalysis. The study revealed that the equilibrium compositions of the catalyzed reactions were kinetically determined by the selectivity of the catalyst, the substrate concentration and the reaction time. A model comprising a set of two kinetic equations was used to describe the hydrolysis and condensation reactions of glucoamylase-catalyzed reactions, even to highly concentrated systems. Increased substrate concentration resulted in the formation of more condensation products. The enzyme inhibition was low and was found to be independent of the substrate concentration.
Oligosaccharide synthesis by the hyperthermostable b-glucosidase from Pyrococcus furiosus: kinetics and modelling
Bruins, M.E. ; Strubel, M. ; Lieshout, J.F.T. van; Janssen, A.E.M. ; Boom, R.M. - \ 2003
Enzyme and Microbial Technology 33 (2003)1. - ISSN 0141-0229 - p. 3 - 11.
escherichia-coli - enzymatic-synthesis - bacillus-circulans - hydrolysis - galactosidase - lactose - temperature - disaccharides - glycosidases - glucoamylase
Oligosaccharides can be synthesised from monosaccharides or disaccharides, using glycosidases as a catalyst. To investigate the potential of this synthesis with beta-glycosidase from Pyrococcus furiosus we determined kinetic parameters for substrate conversion and product formation from cellobiose, lactose, glucose and galactose. The obtained parameters for initial rate measurements of disaccharide conversion were also used for the interpretation of experiments in time. The model for cellobiose gave a good description of the experiments. The enzyme was found to be uncompetitively inhibited by cellobiose and competitively inhibited by glucose. Lactose conversion however, could not be modelled satisfactorily; apparently additional reactions take place. Monosaccharide condensation also yielded oligosaccharides, but much slower. The use of a hyperthermostable, enzyme was found to be positive. More substrate could be dissolved at higher temperatures, which benefited all reactions.