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Increasing the thermostability of sucrose phosphorylase by multipoint covalent immobilization

Paper ID Volume ID Publish Year Pages File Format Full-Text
24200 43504 2010 6 PDF Available
Title
Increasing the thermostability of sucrose phosphorylase by multipoint covalent immobilization
Abstract

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.

Keywords
SPase, sucrose phosphorylase; α-d-G1P, α-d-glucose-1-phosphate; PGM, phosphoglucomutase; G6P-DH, glucose-6-phosphate dehydrogenaseSucrose phosphorylase; Immobilisation; Sepabeads; Thermostability; BCA (reducing sugars)
First Page Preview
Increasing the thermostability of sucrose phosphorylase by multipoint covalent immobilization
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Biotechnology - Volume 150, Issue 1, 1 October 2010, Pages 125–130
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering