Journal
PROCESS BIOCHEMISTRY
Volume 45, Issue 4, Pages 451-456Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.procbio.2009.11.001
Keywords
Recombinant rhamnosidase; Clostridium stercorarium; His-tag; IMAC; Entrapped; Naringin; Kinnow juice
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Funding
- Council of Scientific and Industrial Research (CSIR), India [EMR38/1133/EMR-II]
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alpha-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 degrees C respectively. At 50 degrees 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. (C) 2009 Elsevier Ltd. All rights reserved.
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