Extraction and purification of lipase from Enterococcus faecium MTCC5695 by PEG/phosphate aqueous-two phase system (ATPS) and its biochemical characterization

Ramakrishnan, Venkat M.; Goveas, Louella Concepta; Suralikerimath, Niranjan; Chandrasekhar, J.; Halami, Prakash M.; Bhaskar, N.

doi:10.1016/j.bcab.2016.02.005

Cited by 69 publications

(41 citation statements)

References 38 publications

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“…Other constraints of these traditional purification techniques are laborious procedures and low yields [79, 80]. Ultrafiltration and hydrophobic interaction chromatography are also multi-step, costly and low-yielding purification techniques.…”

Section: Applications Of Aqueous Two-phase System (Atps)mentioning

confidence: 99%

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Aqueous two-phase system (ATPS): an overview and advances in its applications

et al. 2016

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Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications.

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Section: Applications Of Aqueous Two-phase System (Atps)mentioning

confidence: 99%

“…Microbial lipases are ubiquitous enzymes that are considered to be very important biocatalysts because of their substrate specifities and diversified properties [80, 85]. These enzymes are essential for a number of biotechnological applications.…”

Section: Applications Of Aqueous Two-phase System (Atps)mentioning

confidence: 99%

Aqueous two-phase system (ATPS): an overview and advances in its applications

et al. 2016

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“…The recombinant enzyme Egls was partitioned to the bottom phase (salt-rich phase) of this system. It has been found that some enzymes would tend to partition to the bottom phase when it was purified with two-phase system (Gu et al 2012;Loc et al 2013;Ramakrishnan et al 2016). An increase of pH and NaCl concentration in the two-phase system leads to the decrease of recovery rate of Egls.…”

Section: Expression and Purification Of The Recombinant Enzymementioning

confidence: 99%

“…It is one of low cost and high-efficient purification methods (Asenjo and Andrews 2011). Recently, many enzymes (α-amylase, α-galactosidase, lipase, proteinase), recombinant proteins, and antibody have been purified using this method (Azevedo et al 2009;Porfiri et al 2011;Gu et al 2012;Loc et al 2013;Ramakrishnan et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

Liu

Guo

et al. 2018

Bioresour. Bioprocess.

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Background: Cellulases played an important role in the production of bioenergy and bio-products. Cellulases from bacteria with some special characteristics drew great attention due to its fast growth speed, wide adaption to harsh environment, and production of multi-function cellulases.Results: An endoglucanase gene egls from Bacillus velezensis A4 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant enzyme Egls was partially purified using aqueous two-phase system. The highest recovery rate of the enzyme was 90.39% at PEG 4000 (25% w/w), phosphate buffer 8.08% (w/w) (pH 6.0), and NaCl (5% w/w). The enzyme molecular weight was 55 KD estimated by zymogram. The optimal pH and temperature of recombinant enzyme Egls were pH 6.0 and 55 °C, respectively. The enzyme was stable at pH range of 5.0-7.0 at 55 °C for 60 min. The enzyme exhibited K m , V max , K cat values as 63.38 mg/ml, 55.6 mg/min, and 3.93 × 10 3 /S, respectively. The addition of 10 mM of Mg 2+ , Mn 2+ , or 5% (w/w) of Triton-X 100 in the reaction system enhanced the enzyme activity significantly. The enzyme showed both endoglucanase and exoglucanase activity. Conclusions:An endoglucanase gene egls from B. velezensis A4 was cloned and expressed in E. coli BL21 (DE3). The recombinant enzyme Egls was purified by aqueous two-phase system and characterized. The enzyme can be applied for the efficient pretreatment of lignocellulosic biomass for bioenergy and bio-products production.

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“…Ketnawa et al [1] partitioned bromelain from pineapple peels using the system containing 18% (w/w) PEG6000 and 17% (w/w) MgSO 4 . Ramakrishnan et al [26] observed that the enzyme recovery of lipase from Enterococcus faecium MTCC5695 was increased with the increase of PEG molecular weight from 1000 to 8000. The purified sample from the top phase (PEG-rich) was subjected to SDS-PAGE analysis.…”

Section: Aqueous Two-phase Systemmentioning

confidence: 99%

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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Extraction and purification of lipase from Enterococcus faecium MTCC5695 by PEG/phosphate aqueous-two phase system (ATPS) and its biochemical characterization

Cited by 69 publications

References 38 publications

Aqueous two-phase system (ATPS): an overview and advances in its applications

Aqueous two-phase system (ATPS): an overview and advances in its applications

Purification and characterizations of a novel recombinant Bacillus velezensis endoglucanase by aqueous two-phase system

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

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