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

Amid, Azura; Ismail, Nurul Azira; Yusof, Faridah; Salleh, Hamzah Mohd.

doi:10.1016/j.procbio.2011.08.018

Cited by 55 publications

(66 citation statements)

References 36 publications

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“…Besides, the processing of pineapple crop to produce bromelain generates bulk waste that eventually leads to environmental issues. Considering the significance of economic, efficient production and downstream processing of bromelain, recombinant bromelain was successfully expressed in various hosts, including Escherichia coli BL21-A1 [3], E.coli BL21 DE3pLysS [4] and Brassica rapa [5]. Our research group has successfully cloned the stem bromelain from Ananas comosus into the pENTR/TEV/D-TOPO vector and subcloned it into the pDEST17 expression vector.…”

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confidence: 99%

“…Our research group has successfully cloned the stem bromelain from Ananas comosus into the pENTR/TEV/D-TOPO vector and subcloned it into the pDEST17 expression vector. Later, E. coli BL21-A1 was transformed with the expression vector [3] and was cultivated in a batch process using auto-induction media [6]. The recombinant bromelain is an intracellular enzyme and remains abundantly in the cytoplasm of the E. coli cells.…”

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“…Commonly, recombinant bromelain was purified using affinity chromatography under native condition [3,8]. An instrument such as AKTA prime plus® (GE Healthcare, USA) provides a quick design to facilitate affinity chromatography purification as the plasmid vector of recombinant bromelain has a unique polyhistidine (His6) tag [3].…”

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confidence: 99%

“…An instrument such as AKTA prime plus® (GE Healthcare, USA) provides a quick design to facilitate affinity chromatography purification as the plasmid vector of recombinant bromelain has a unique polyhistidine (His6) tag [3]. It was reported that, the recombinant bromelain purified from E. coli BL21-A1 has a molecular weight of 50 -55 kDa and exhibited optimum proteolytic activity at pH 4.6 [3]. In the majority of food industry, the enzyme is added in bulk quantity.…”

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confidence: 99%

“…In the majority of food industry, the enzyme is added in bulk quantity. The enzyme purified by Amid et al [3] was only produced in a small amount (~200 µl) and could not be used for large scale food-related processes. Therefore, there is a need to develop a purification protocol that accompanies higher recovery, lower cost, and shorter processing time.…”

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

Arshad¹,

Amid²,

Yusof³

et al. 2017

MJAS

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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 IntroductionBromelain is a plant protease that is commercially obtained from stem, fruit, and waste of pineapple plant [1]. Bromelain is notable for being used in multitude industrial applications such as meat tenderization, food, beverages, and pharmaceuticals, hence increases its market demand [2]. However, there is a rising argument whether pineapple

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

Arshad¹,

Amid²,

Yusof³

et al. 2017

MJAS

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Stability, purification, and applications of bromelain: A review

Novaes

Jozala

Lopes

et al. 2015

Biotechnol Progress

123

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Bromelain is a cysteine protease found in pineapple tissue. Because of its anti-inflammatory and anti-cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid-liquid extractions by aqueous two-phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up-to-date compilation of the data reported on this enzyme.

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