One-step purification and immobilization of his-tagged rhamnosidase for naringin hydrolysis
Data(s) |
01/04/2010
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Resumo |
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 <i>ram</i>A from <i>Clostridium stercorarium </i>to facilitate its purification from <i>Escherichia coli </i>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 Ca<sup>2+</sup> 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 <i>Kinnow juice </i>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. |
Identificador | |
Idioma(s) |
eng |
Publicador |
Elsevier Ltd |
Relação |
http://dro.deakin.edu.au/eserv/DU:30023625/puri-onesteppurificationand-2010.pdf http://dx.doi.org/10.1016/j.procbio.2009.11.001 |
Direitos |
2009, Elsevier Ltd. |
Palavras-Chave | #recombinant rhamnosidase #Clostridium stercorarium #His-tag #entrapped #naringin #kinnow juice |
Tipo |
Journal Article |