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Expression, purification, and characterization of a recombinant stem bromelain from ananas comosus

Amid, Azura and Ismail, Nurul Azira and Yusof, Faridah and Mohd. Salleh, Hamzah (2011) Expression, purification, and characterization of a recombinant stem bromelain from ananas comosus. Process Biochemistry, 46 (12). pp. 2232-2239. ISSN 1359-5113

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Abstract

Commercially available bromelain is prepared by performing a tedious and costly purification method that provides multiple degrees of purified bromelain. In the current study, a gene encoding stem bromelain from Ananas comosus was amplified using polymerase chain reaction (PCR). This bromelain gene was initially cloned into pENTR/TEV/D-TOPO before being sub-cloned into the expression vector pDEST17. DNA sequencing of the amplified products exhibited a high level of homology to the corresponding gene from the NCBI public database. Protein expression was conducted in the Escherichia coli, BL21-AI. The recombinant bromelain was then purified in a single step using a Ni-NTA spin column, an example of immobilized metal affinity chromatography. Purified recombinant bromelain was detected by Western blotting. In addition, the purified enzyme exhibited hydrolytic activity towards gelatin and a synthetic substrate, LNPE. The purified recombinant bromelain exhibited optimum activity at pH 4.6 and 45oC.

Item Type: Article (Journal)
Additional Information: 3688/2374
Uncontrolled Keywords: Bromelain; cloning; protein expression; protease; recombinant enzyme
Subjects: Q Science > Q Science (General)
Kulliyyahs/Centres/Divisions/Institutes (Can select more than one option. Press CONTROL button): Kulliyyah of Engineering > Department of Biotechnology Engineering
Depositing User: Dr Azura Amid
Date Deposited: 15 Sep 2011 14:09
Last Modified: 19 Apr 2012 11:45
URI: http://irep.iium.edu.my/id/eprint/2374

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