A new procedure based on column chromatography to purify bromelain by ion exchange plus gel filtration chromatographies

Author

Helber B.CostaabPatricia M.B.FernandesaWandersonRomãobcJosé A.Venturaad

a

Núcleo de Biotecnologia, Universidade Federal do Espírito Santo, 29040-090, Vitória, ES, Brazil

b

Laboratório de Petroleômica e Química Forense, Departamento de Química, Universidade Federal do Espírito Santo (UFES), Avenida Fernando Ferrari, 514, Goiabeiras, Vitória, ES, CEP: 29075-910, Brazil

c

Instituto Federal de Educação, Ciência e Tecnologia do Espírito Santo, 29040-780, Vila Velha, ES, Brazil

d

Instituto Capixaba de Pesquisa Assistência Técnica e Extensão Rural - Incaper, 29052-010, Vitória, ES, Brazil

Received 13 February 2014, Revised 17 April 2014, Accepted 23 April 2014, Available online 31 May 2014.

Highlights

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A powerful isolation method to obtain bromelain was developed.

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The new method couples two liquid chromatographies: ionic exchange and gel filtration.

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The methodology developed presents a higher yield of proteolytic activity.

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The methodology has a lower cost than other existing methodologies.

Abstract

Bioproducts separation and purification processes are an important segment of the biotechnical industry. Bromelain is an enzyme which has great commercial value and is of wide interest in the pharmaceutical, alimentary, and textile industries, among others. The goal of this study was to develop a new method for bromelain purification from the stem residues resulting from agricultural processing of the pineapple plant. Bromelain was purified using two liquid chromatography steps, ion exchange plus gel filtration chromatography. Use of the methodology which was developed produced an enzyme with a molecular weight of 30 kDa (confirmed by SDS-PAGE), high recovery of enzymatic activity (89%), and with a purification factor of 16.93, a result superior to the methodologies described in the literature. HPLC showed the presence of two peaks in the ion exchange chromatogram and only one protein in the gel filtration chromatogram. Results indicate that, depending on the destination of the bromelain, the process can be stopped after the first purification step. The MALDI-TOF MS provided the peptide mass fingerprint of bromelain and MALDI-MS/MS the fragmentation profile and sequencing of the ions of m/z 951 and 1584. Thus, the connectivity and chemical structure of bromelain was confirmed. Moreover, besides its superiority to other methodologies, it can be applied to take advantage of the agricultural and industrial pineapple plant residues.

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