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One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis

Paper ID Volume ID Publish Year Pages File Format Full-Text
35770 45106 2010 6 PDF Available
Title
One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis
Abstract

α-l-Rhamnosidase (EC 3.2.1.40) is an enzyme that catalyzes the cleavage of terminal rhamnoside groups from naringin to prunin and rhamnose. In this study, a His-tag was genetically attached to the rhamnosidase gene ramA from Clostridium stercorarium to facilitate its purification from Escherichia coli BL21 (DE3) cells containing the pET-21d/ramA plasmid. Immobilized metal-chelate affinity chromatography (IMAC) resulted in one-step purification of N-terminally His-tagged recombinant rhamnosidase (N-His-CsRamA) which was immobilized in Ca2+ alginate (3%) beads. The optimum pH levels of the free and immobilized recombinant rhamnosidase were found to be 6.0 and 7.5, and the optimum temperature 55 and 60 °C respectively. At 50 °C, the free enzyme was relatively stable and exhibited a less than 50% reduction in residual activity after 180 min of incubation. The free and immobilized enzymes achieved 76% and 67% hydrolysis of the naringin in Kinnow juice respectively. Immobilization of recombinant rhamnosidase enabled its reutilization up to 9 hydrolysis batches without an appreciable loss in activity. This result indicated that the His-tagged thermostable rhamnosidase could be prepared as described and may serve to illustrate an economical and commercially viable process for industrial application.

Keywords
Recombinant rhamnosidase; Clostridium stercorarium; His-tag; IMAC; Entrapped; Naringin; Kinnow juice
First Page Preview
One-step purification and immobilization of His-tagged rhamnosidase for naringin hydrolysis
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 45, Issue 4, April 2010, Pages 451–456
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering