Increasing the thermostability of sucrose phosphorylase by multipoint covalent immobilization.
Sucrose phosphorylase from Bifidobacterium adolescentis was recombinantly expressed in Escherichia coli and purified by use of a His-tag. Kinetic characterization of the enzyme revealed an optimal temperature for phosphorolytic activity of 58°C, which is surprisingly high for an enzyme from a mesophilic source. The temperature optimum could be further increased to 65°C by multipoint covalent immobilization on Sepabeads EC-HFA. The optimal immobilization conditions were determined by surface response design. The highest immobilization yield (72%) was achieved in a phosphate buffer of 0.04 mM at pH 7.2, irrespective of the temperature. The immobilized enzyme was able to retain 65% of its activity after 16 h incubation at 60°C. Furthermore, immobilization of the enzyme in the presence of its substrate sucrose, increased this value to 75%. The obtained biocatalyst should, therefore, be useful for application in carbohydrate conversions at high temperatures, as required by the industry.
Cerdobbel
An
A
Centre of Expertise for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Biosciences Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. An.Cerdobbel@ugent.be
Desmet
Tom
T
De Winter
Karel
K
Maertens
Jo
J
Soetaert
Wim
W
D001644:Bifidobacterium / Q000201:enzymology / Q000235:genetics; D019149:Bioreactors; D004795:Enzyme Stability; D004800:Enzymes, Immobilized / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005964:Glucosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D016014:Linear Models; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D004800:Enzymes, Immobilized; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D005964:Glucosyltransferases; C025901:sucrose phosphorylase