Malaysian Journal of Analytical Sciences Vol 21 No 4 (2017): 958 - 971

DOI: https://doi.org/10.17576/mjas-2017-2104-23

 

 

 

COMPARISON OF PURIFICATION METHODS TO PURIFY RECOMBINANT BROMELAIN FROM Escherichia coli BL21-A1

 

(Perbandingan Kaedah Penulenan Rekombinan Bromelain daripada Escherichia coli BL21-A1)

 

Zatul Iffah Mohd Arshad^1,2*, Azura Amid², Faridah Yusof ²,Siti Zubaidah Sulaiman ¹,

Siti Kholijah Abdul Mudalip¹, Rohaida Che Man¹, Shalyda Md Shaarani¹

 

¹Faculty of Chemical Engineering & Natural Resources,

Universiti Malaysia Pahang, 26300 Gambang, Pahang, Malaysia

²Bioprocess and Molecular Engineering Research Unit, Department of Biotechnology Engineering, Faculty of Engineering,

International Islamic University Malaysia, P.O. Box 10, 50728 Kuala Lumpur, Malaysia

 

*Corresponding author:  zatul@ump.edu.my

 

 

Received: 28 November 2016; Accepted: 5 February 2017

 

 

Abstract

Recombinant bromelain is a cysteine protease that can be exploited for its protease activity in food and pharmaceutical applications. The aim of this study was to compare different purification methods aqueous two-phase system (ATPS), ammonium sulphate precipitation, ion exchange, affinity, and gel filtration chromatography) for the recombinant bromelain purification from Escherichia coli BL21-A1. From the SDS-PAGE analysis, all methods produced band with molecular weight between 50 to 55 kDa. Among the methods used, the ATPS purification consisting of 13% (w/w) of PEG6000 and 11% (w/w) potassium phosphate at pH 7.0 was chosen as the best purification method. The method produced 16.39 ± 0.03 % of yield, purification fold of 5.35 ± 0.11, and specific activity of 3.47 ± 0.11 unit/mg of recombinant bromelain. This proposed study can be used as a platform for large-scale downstream processing of recombinant bromelain in industry.

 

Keywords:  recombinant bromelain, purification, chromatography, aqueous two-phase system

 

Abstrak

Rekombinan bromelain adalah sisteina protease yang boleh dieksploitasi dalam aplikasi makanan dan farmaseutikal. Tujuan kajian ini adalah untuk membandingkan kaedah penulenan yang berbeza dengan menggunakan sistem akueus dua fasa (ATPS), pemendakan amonium sulfat, pertukaran ion, afiniti dan gel penapisan kromatografi untuk penulenan rekombinan bromelain daripada Escherichia coli BL21-A1. Daripada analisis SDS-PAGE, semua kaedah menghasilkan jalur dengan berat molekul antara 50 hingga 55 kDa. Antara kaedah yang digunakan, sistem akueus dua fasa (ATPS) yang terdiri daripada 13% (w/w) PEG6000 dan 11% (w/w) kalium fosfat pada pH 7.0 telah dipilih sebagai kaedah penulenan yang terbaik. Kaedah ini mendapat 16.39 ± 0.03% hasil, kadar penulenan 5.35 ± 0.11, dan aktiviti rekombinan bromelain sebanyak 3.47 ± 0.11 unit/mg. Kajian ini dicadangkan boleh digunakan sebagai platform untuk pemprosesan hiliran rekombinan bromelain secara besar-besaran dalam industri.

 

Kata kunci:  rekombinan bromelain, penulenan, kromatografi, sistem akueus dua fasa

 

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