Vol. 8 | No.2 |186 -193 | April - June | 2015 ISSN: 0974-1496 | e-ISSN: 0976-0083 | CODEN: RJCABP http://www.rasayanjournal.com http://www.rasayanjournal.co.in ADSORPTION OF PEPSIN WITH POLYETHYLENEIMINE Bidisha Ghosh et. al ADSORPTION OF PEPSIN WITH POLYETHYLENEIMINE Bidisha Ghosh 1 , Rutuja Vaze 1 , Annamma Anil 1* and Arvind Lali 1 1 DBT-ICT Centre for Energy Biosciences, Institute of Chemical Technology, Nathalal Parikh Marg, Matunga (E), Mumbai -400 019, India. *E-mail: a.annamma@ictmumbai.edu.in ABSTRACT Polyethyleneimine is being increasingly applied for immobilizing enzymes employing its primary, secondary and tertiary ionisable amino groups. In this study, we attempt to immobilize pepsin on Sepabeads functionalized with Polyethyleneimine (PEI) polymers of varying molecular sizes (M w 600, 1200, 10,000 gmol -1 ). The stability and activity of pepsin bound to PEI was found to be dependent on PEI molecular weight and also on the ionic strength of medium, used for immobilization and protein hydrolysis. Immobilized pepsin exhibited good storage and operational stability which was investigated for generating F (ab’) 2 fragments from hydrolysis of immunoglobulins. Digestion of IgG by soluble pepsin and immobilized pepsin showed the same electrophoresis profile (of F (ab') 2 and Fc fragments) indicating no modification in pepsin specificity after immobilization on PEI activated Sepabeads support. The above method of immobilization presents an efficient means to immobilize pepsin onto a solid support wherein the said preparation would be free from autolysis and can be reused for multiple cycles. Key words: Polyethyleneimine, Pepsin, Sepabeads, Immobilization ©2015 RASYAN. All rights reserved INTRODUCTION Pepsin (EC 3.4.23.1), has generated lot of interest due its ability to remain active at acidic pH. This animal protease has potential industrial applications in cheese industry, wine industry and digestion of IgG to produce F (ab´) 2 fragments which has several therapeutic uses 1-4 . Pepsin belongs to protease group of enzymes which have the inherent propensity to undergo autolysis causing self destruction thus decreasing their potential as an industrial enzyme. Immobilization of pepsin stabilizes the enzyme by increasing its half life leading to reutilization of the biocatalyst 5 .Modified catalyst would have advantage of ease of handling i.e. simple filtration for separation of products from reaction mixture 6 and enzyme recycling. Additionally, use of biocatalyst i.e. enzymes eliminates the use of hazardous chemicals such as acid (which is conventionally used for protein hydrolysis) thus empowering green chemistry which is the need of the time 7 , enabling the formation of products via safer route 8 . In order to produce F (ab´) 2 fragments for therapeutic purposes it is necessary to obtain them in active form which can be done using enzymes, due to their regio-selectivity and mild operating conditions 9 . Earlier, Pepsinogen was immobilized and converted to active pepsin by refolding it on hydrophobic or hydrophilic polymeric surfaces; this entire renaturation process takes 300hr, making it unfeasible 10 . Frýdlová et al., have derivatized Sepharose with -acetyl phenylalanine and iodinated tyrosine that binds to pepsin 11 . This approach involves multiple preparative steps, making it unsuitable for large-scale production. Covalent immobilization chemistries like glutaraldehyde activation and divinylsulphone methods are effective in neutral or alkaline conditions, however, X-ray diffraction analysis have shown pepsin to denature under alkaline conditions restricting the methodologies that can be used for pepsin immobilization 12 . Ionic adsorptions of proteins on matrices are preferred alternative when harsher irreversible covalent techniques cannot be applied 13, 14 . Direct modification with polymer prior to enzyme attachment preserves the functionality of protein 15 . PEI is widely used as enzyme carriers for monomeric as well as multimeric enzymes like penicillin G acylase, lipases QL and CALB glutarylacylase, glutamate, dehyrogenase etc. 16 . The hydrophilic microenvironment and multipoint attachment sites provided by PEI has a positive