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

Munish Puri, Aneet Kaur, Ram Singh, Wolfgang Schwarz, Amrit Kaur

    Research output: Contribution to journalArticle

    19 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)451-456
    Number of pages6
    JournalProcess Biochemistry
    Volume45
    Issue number4
    DOIs
    Publication statusPublished - Apr 2010

    Keywords

    • Clostridium stercorarium
    • Entrapped
    • His-tag
    • IMAC
    • Kinnow juice
    • Naringin
    • Recombinant rhamnosidase

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