Abstract
An aqueous two phase system of polyethylene glycol (PEG) and salts was evaluated for separation and purification of alkaline proteases from chicken intestine. Among the different salts evaluated potassium phosphate and sodium citrate gave higher enzyme yield (73.5% and 69.7% respectively) and enzyme purification (5.3 and 7.4 fold) in PEG rich upper phase. Increase in concentration of sodium citrate in the system resulted in reduction in enzyme yield and enzyme purification factor, with 15% salt showing highest enzyme yield (59.8%) and purification (6.7 fold). Initial protein concentration in the system did not show any specific trend on the partition behavior of the enzyme. The temperature at which the system is incubated did not show any significant (p ≥ 0.05) effect on enzyme partition and purification. Increasing the PEG concentration in the system from 15 to 25% resulted in reduction in enzyme yield from 53.7 to 21.9% and enzyme purification from 5 fold to 1.4 fold in PEG rich upper phase. pH also had a significant (p < 0.05) effect on the partition of the enzyme to the upper phase with highest purification (3.4 fold) at pH 9.0 and higher enzyme yield (46.2%) at pH 10.
Similar content being viewed by others
References
Albertsson PA (1986) Partition of cell particles and macromolecules. Wiley, New York
Andersson EA, Johansson A, Hagerdal GB (1985) Amylase production in aqueous two phase system with Bacillus subtilis. Enzyme Microb Technol 7:733–738
Antov MG (2004) Partitioning of pectinase produced by Polyporus squamosus in aqueous two phase system polyethylene glycol 4000/crude dextran at different initial pH values. Carbohydr Polym 56:295–300
Diamond AD, Hsu JT (1992) Aqueous two phase systems for bio-molecule separation. Adv Biochem Eng 47:89–135
El-Beltagy AE, El-Adaway TA, Rahma EH (2004) Purification and characterization of an acidic protease from the viscera of bolti fish (Tilapia nilotica). Food Chem 86:33–39
Francot TT, Andrews AT, Asenjo JA (1996) Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: effect of surface hydrophobicity. Biotechnol Bioeng 49:300–308
Gaikaiwari R, Shendye A, Kulkarni N, Rao M (1996) Two phase separation of xylanases from alcalophilic thermophilic Bacillus using a polyethylene glycol-potassium phosphate system. Biotechnol Appl Biochem 23:237–241
Han JH, Lee CH (1997) Effects of salts and polyethylene glycol-palmitate on the partitioning of proteins and Bacillus subtilis neutral protease in aqueous two phase systems. Colloids Surf B 9:109–116
Jamdar SN, Harikumar P (2005) Autolytic degradation of chicken intestinal proteins. Bioresour Technol 96:1276–1284
Joo HS, Park CH, Kim KM, Paik SR, Chang CS (2001) Novel alkaline protease from the polychaeta, Periserrula leucophryna: purification and characterization. Process Biochem 36:893–900
Klomklao S, Benjakul S, Visessanguan W, Simpson BK, Kishimura H (2005) Partitioning and recovery of proteinase from tuna spleen by aqueous two phase systems. Process Biochem 40:3061–3067
Lowry OH, Fan AL, Randall RJ, Rosebrough NJ (1951) Protein measurement with Folin-Phenol reagent. J Biol Chem 193:256–275
Nitsawang S, Hatti-Kaul R, Kanasawud P (2006) Purification of papain from Carica papaya latex: aqueous two-phase extraction versus two step-salt precipitations. Enzyme Microb Technol 39:1103–1107
Oliveira LA, Sarubbo LA, Porto ALF (2002) Partition of trypsin in aqueous two phase systems of polyethylene glycol and cashew-nut tree gum. Process Biochem 38:693–699
Raghavarao KSMS, Rastogi NK, Gowthaman MK, Karanth NG (1995) Aqueous two phase extraction for downstream processing of enzymes, proteins. Adv Appl Microbiol 41:97–171
Raju AA, Rose C, Rao NM (1997) Enzymatic hydrolysis of tannery fleshings using chicken intestinal proteases. Anim Feed Sci Technol 66:139–147
Ravindra Babu B, Rastogi NK, Raghavarao KSMS (2008) Liquid-Liquid extraction of bromelin and polyphenol oxidase using aqueous two-phase system. Chem Eng J 47:83–89
Reh G, Spelzini D, Tubio G, Pico G, Farruggia B (2007) Partition features and renaturation enhancement of chymosin in aqueous two phase systems. J Chromatogr B 860:98–105
Rito-Palomares M (2004) Practical application of aqueous two phase partition to process development for the recovery of biological products. J Chromatogr B 807:3–11
Salabat A (2001) The influence of salts on the two phase composition in aqueous two phase systems: experiments and predictions. Fluid Phase Equilib 187:489–498
Schmidt AS, Ventom AM, Asenjo JA (1994) Partitioning and purification of amylase in aqueous two-phase systems. Enzyme Microb Technol 16:131–141
Shahidi F, Kamil JYVA (2002) Enzymes from fish and aquatic invertebrates and their application in the food industry. Trends Food Sci Technol 13:1–30
Sinha R, Singh SP, Ahmed S, Garg SK (1996) Partitioning of Bacillus alkaline protease in aqueous two-phase systems. Bioresour Technol 55:163–166
Spelzini D, Farruggia B, Pico G (2005) Features of protease partition in aqueous two phase systems of polyethylene glycol-phosphate: Chymosin and pepsin. J Chromatogr B 821:60–66
Statsoft (1999) Statistics for windows. Statsoft Inc, Tulsa
Su CK, Chiang BH (2006) Partitioning and purification of lysozyme from chicken egg white using aqueous two phase system. Process Biochem 41:257–263
Tanuja S, Srinivas ND, Raghavarao KSMS, Gowthaman MK (1997) Aqueous two phase extraction for downstream processing of amyloglucosidase. Process Biochem 32:635–641
Tubio G, Nerli B, Pico G (2007) Partitioning features of bovine trypsin and α-chymotrypsin in polyethylene glycol-sodium citrate aqueous two phase systems. J Chromatogr B 852:244–249
Ufuk G (2000) Partitioning of bovine serum albumin in an aqueous two phase system: optimization of partition coefficient. J Chromatogr B 743:259–262
Warda M, Mao W, Toida T, Linhardt TR (2003) Turkey intestine as a commercial source of heparin: comparative structural studies of avian and mammalian glycosaminoglycans. Comp Biochem Physiol B 134:189–197
Yue H, Yuan Q, Wang WI (2007) Purification of phenylalanine ammonia-lyase in PEG1000/Na2SO4 aqeuous two-phase system by a two-step extraction. Biochem Eng J 37:231–237
Acknowledgment
Authors thank Prakash V, Director, CFTRI for his encouragement and Raghavarao KSMS, Head, Food Engineering, CFTRI for technical discussions on separation methods employed and for suggestion during the course of study.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sarangi, B.K., Pattanaik, D.P., Rathinaraj, K. et al. Purification of alkaline protease from chicken intestine by aqueous two phase system of polyethylene glycol and sodium citrate. J Food Sci Technol 48, 36–44 (2011). https://doi.org/10.1007/s13197-010-0137-7
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13197-010-0137-7