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

Puri, Munish, Kaur, Aneet, Singh, R. S., Schwarz, Wolfgang H. and Kaur, Amrit 2010, One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis, Process biochemistry, vol. 45, no. 4, pp. 1-6, doi: 10.1016/j.procbio.2009.11.001.

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Title One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis
Author(s) Puri, MunishORCID iD for Puri, Munish
Kaur, Aneet
Singh, R. S.
Schwarz, Wolfgang H.
Kaur, Amrit
Journal name Process biochemistry
Volume number 45
Issue number 4
Start page 1
End page 6
Total pages 6
Publisher Elsevier Ltd
Place of publication Oxford, England
Publication date 2010-04
ISSN 1359-5113
Keyword(s) recombinant rhamnosidase
Clostridium stercorarium
kinnow juice
Summary α-l-Rhamnosidase (EC 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.
Language eng
DOI 10.1016/j.procbio.2009.11.001
Field of Research 100301 Biocatalysis and Enzyme Technology
Socio Economic Objective 860105 Nutraceuticals and Functional Foods
HERDC Research category C1 Refereed article in a scholarly journal
HERDC collection year 2010
Copyright notice ©2009, Elsevier Ltd.
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Created: Wed, 10 Feb 2010, 13:48:56 EST by Munish Puri

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