Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor

Saxena, Lalit; Iyer, Bharti K.; Ananthanarayan, Laxmi

doi:10.1016/j.procbio.2006.09.016

Cited by 58 publications

(33 citation statements)

References 10 publications

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“…Ammonium sulfate precipitation is a cheap and extensively used method for protein purification (Saxena et al 2007). Ammonium sulfate precipitation resulted in 2.81 purification fold of bromelain (Devakate et al 2009).…”

Section: Other Methodsmentioning

confidence: 99%

Bromelain: Methods of Extraction, Purification and Therapeutic Applications

Manzoor

Nawaz

Mukhtar

et al. 2016

Braz. arch. biol. technol.

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Section: Other Methodsmentioning

confidence: 99%

Bromelain: Methods of Extraction, Purification and Therapeutic Applications

Manzoor

Nawaz

Mukhtar

et al. 2016

Braz. arch. biol. technol.

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“…After mixing crude enzyme extract and selected salt from previous step, the pH of the system was adjusted to each pH values (6,7,8,9, and 10) by addition of 2M HCl or NaOH. Next, the selected organic solvent was added to the mixture with the ratios of 1: 0.5 (v/v).…”

Section: Effect Of Phs On Proteases Partitioningmentioning

confidence: 99%

“…TPP is a concentrating or dewatering step and some enzymes have enhanced catalytic activities in these conditions within short periods of time (about 1 h) [4]. TPP has been widely used to separate and purify various enzymes such as Aspergillus oryzae [4], protease/amylase inhibitor from wheat germ [5] and ragi (Eleusine coracana) [6], phospholipase D from Dacus carota [7], α-galactosidase form tomato [8], and pepino (Solanum muricatum) [9], invertase from baker's yeast [10], and tomato [11], proteases from papaya peels [12], Calotropis procera latex [13] and giant catfish viscera [14], inulinase from Aspergillus niger [15]. Rawdkuen et al [14] showed the best condition for separating of alkaline proteases from farmed giant catfish viscera that was consisted of the crude enzyme extract to t-butanol ratio of 1.0: 0.5 in the presence of 50% (NH 4 ) 2 SO 4 .…”

Section: Introductionmentioning

confidence: 99%

Three-phase partitioning and proteins hydrolysis patterns of alkaline proteases derived from fish viscera

Ketnawa

Benjakul

Martínez-Álvarez

et al. 2014

Separation and Purification Technology

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In this study, the recovery alkaline proteases from farmed giant catfish viscera were isolated by using three-phase partitioning (TPP). Factors affecting partitioning efficiency such as salts, solvent types, pH, and incubation temperatures were studied. Furthermore, the application of extracted alkaline proteases on proteins hydrolysis was also determined. The system consisted of crude enzyme extract: t-butanol 1: 0.5 (w/v), 50% sodium citrate, pH 8.0 with incubation temperature of 25ºC provided the highest enzyme recovery (220%). The smear protein bands with molecular weight of 20, 24, and 215 kDa of TPP fractions appeared on the protein stained gel. Two major clear zones (24 and 130 kDa) in the interphase were observed on casein-substrate gel electrophoresis. Extracted alkaline proteases showed relatively high effective in protein hydrolysis. As a result, TPP provided high enzyme recovery and could be applied to other enzymes. The obtained alkaline proteases can be further applied in preparation of protein hydrolysates.

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“…This reason requires the second cycle TPP with the use of first cycle TPP's aqueous phase as the starting material [29]. Fig.…”

Section: First Cycle Of Tppmentioning

confidence: 99%

“…Chaiwut et al [18] reported highest enzymatic activity and proteolytic activity (253.5%) of protease in the bottom phase. Wang et al [28] and Saxena et al [29] reported purification of α-amylase inhibitor and amylase/ protease inhibitor (81 and 80%, respectively) by TPP. They also found the most of the target protein content in aqueous phase and optimized this phase by adding of further t-butanol or salt concentrations.…”

Section: Second Cycle Of Tppmentioning

confidence: 99%

Purification and Recovery of Invertase from Potato Tubers (Solanum tuberosum) by Three Phase Partitioning and Determination of Kinetic Properties of Purified Enzyme

Duman¹

2014

Turk J Bioch

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Objective: Three phase partitioning (TPP) is a simple and economical purification and recovery technique for proteins and also enzymes. In this technique; proteins are separated in the interfacial or lower phase while contaminant parts of crude extract partitioning occur in the upper organic phase. In this study invertase was purified and recovered from potato by TPP. Methods:The highest enzyme recovery of 121% and purification fold 5.67 was seen in the aqueous phase at the 20% ammonium sulfate saturation with 1.0: 1.0 t-butanol ratio at the first cycle of partitioning. The TPP was optimized by adding ammonium sulfate to final concentration at 25% with the constant t-butanol ratio to starting material. Results: Purification fold and activity recovery were found as 12.8 and 156%, respectively. The Km value, optimal temperature and optimal pH of purified enzyme were also determined. Conclusion:The present study showed that, TPP can be an attractive and effective technique for the extraction of invertase from potato. Key Words: TPP, invertase, recovery, purification, potato tuber. Conflict of Interest:The authors declare no conflict of interest. ÖZETAmaç: Üçlü faz ayrılması proteinleri ve enzimlerin geri kazanılması ve saflaştırılması için ekonomik ve basit bir yöntemdir. Bu yöntemde proteinler sulu ya da ara fazda toplanırken; ham enzimin kontamine içeriği üst kısımdaki organik fazda toplanma eğilimindedir. Bu çalışmada patates yumrularında üçlü faz ayrılması ile invertaz saflaştırılması ve geri kazanımı gerçekleş-tirilmiştir. Metod: Üçlü faz ayrımı iki döngüde gerçekleştirilmiştir. birinci döngüde enzim %121 geri kazanım ve 5.67 saflaştırma katsayısı ile %20 amonyum sülfat doygunluğunda ve1 ). The reaction was incubated at 37 ºC for 30 min then was stopped by adding 0.1 ml of DNS (3,5-Dinitrosalicylic acid) reagent. The mixture was heated in a boiling water bath for 5 min. It was then cooled in ice bath and the amount of reducing sugars was measured spectrophotometrically at 540 nm [22]. The data presented for all invertase activity determinations are mean values of duplicate assay. One unit of invertase activity was defined as the amount of enzyme which released 1 µmole of glucose from sucrose per minute at pH 4.7 and 37 ºC. Protein concentrationConcentration of the protein was determined by Bradford method [23] using Coomassie brilliant blue G-250 dye as a reagent and bovine serum albumin (BSA) as standard, by measuring the absorbance at 595nm at 25 ºC. Assays were performed in duplicate and the averages were used in calculations. Three-phase partitioning Effect of t-butanolTPP experiments were carried out by employing various t-butanol ratios (crude extract:t-butanol; 1.0:0.5, 1.0:1.0, 1.0:1.5, 1.0:2.0) with a constant ammonium sulfate saturation at 20% (w/v). The mixture was mixed gently and then allowed to stand for 30 min at 37 ºC. Afterwards the mixture was centrifuged at 5000 rpm for 10 min at +4 ºC to facilitate the separation of phases. The lower aqueous phase and the interfacial phase were coll...

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Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor

Cited by 58 publications

References 10 publications

Bromelain: Methods of Extraction, Purification and Therapeutic Applications

Bromelain: Methods of Extraction, Purification and Therapeutic Applications

Three-phase partitioning and proteins hydrolysis patterns of alkaline proteases derived from fish viscera

Purification and Recovery of Invertase from Potato Tubers (Solanum tuberosum) by Three Phase Partitioning and Determination of Kinetic Properties of Purified Enzyme

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