NDLI: Selective removal of enzymes from substrate and products. An alternative to immobilization for enzymes acting on macromolecular or solid substrates

Content Provider	Springer Nature Link
Author	Brena, Beatriz M. Lidholm, Jonas Batista Viera, Francisco Carlsson, Jan
Copyright Year	1999
Abstract	A new approach for the control and interruption of enzymatic reactions via selective enzyme immobilization has been developed. The technique was exemplified by the use of three model enzymes with the corresponding macromolecular substrates: α-amylase/starch, trypsin/ insoluble collagen, and alkaline phosphatase/plasmid DNA. Prior to incubation with its substrate, each enzyme was provided withde novo thiol-groups by a two-step reaction involvingN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and DTT. The chemical modification was achieved such that at least 80% of the native enzyme activity was preserved in all cases.In order to interrupt rapidly the reactions in which the enzymes were used, the modified enzyme was immobilized by reaction via its thiol groups on a thiolsulfinate-agarose derivative. The gel-bound enzyme could then be easily removed from unreacted substrate and product by filtration or centrifugation. Comparative studies showed that the immobilized enzymes had much lower activities in the reactions studied than the corresponding soluble ones. The potential for enzyme reuse was also demonstrated with the a-amylase derivatives, which were quantitatively released and eluted in fully active form from the agarose. We have shown that it is possible to achieve practically complete enzyme immobilization in short times and thus to control the progress of the reactions. Because of its simplicity and high efficiency, this approach may represent an interesting alternative for biotechnological processes involving macromolecular or solid substrates.
Starting Page	323
Ending Page	341
Page Count	19
File Format	PDF
ISSN	02732289
Journal	Applied Biochemistry and Biotechnology
Volume Number	75
Issue Number	2-3
e-ISSN	15590291
Language	English
Publisher	Humana Press
Publisher Date	1999-01-01
Publisher Place	Totowa
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Biotechnology Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Environmental Engineering Biochemistry Bioengineering Applied Microbiology and Biotechnology Biotechnology

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