Biotechnology Letters 23: 1625–1627, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 1625 Purification of pectinases by three-phase partitioning Aparna Sharma & M.N. Gupta ∗ Chemistry Department, Indian Institute of Technology, Hauz Khas, New Delhi 110016, India ∗ Author for correspondence (Fax: 91-11-6581073; E-mail: mn_gupta@hotmail.com) Received 29 June 2001; Revisions requested 5 July 2001; Revisions received 26 July 2001; Accepted 26 July 2001 Key words: Aspergillus niger, pectinase, protein precipitation, protein purification, three-phase partitioning, tomato pectinase Abstract Three-phase partitioning was used to purify pectinases from Aspergillus niger and tomato by addition of tert- butanol in the presence of ammonium sulphate. The yields of 76% (Aspergillus niger) and 183% (tomato) and purifications of 10-fold (Aspergillus niger) and 9-fold (tomato) were obtained. The final purified enzyme enzyme from tomato showed a single band on SDS-PAGE with a molecular weight of 46 kDa. Introduction Pectinases have a variety of applications in food processing industries (Pifferi et al. 1993) and hy- drolysis of cellulosic biomass (Alkorta et al. 1998). This work describes the use of three-phase partition- ing (Dennison & Lovrein 1997) for the purification of pectinases. In this technique, the aqueous crude extract of the enzyme is mixed with tert-butanol in the presence of ammonium sulphate. The enzyme ap- pears as an interfacial precipitate between the lower aqueous and upper tert-butanol phases. In practice, the separation of desired protein in the interfacial pre- cipitate is made by varying the ammonium sulphate concentration, tert-butanol volume and temperature (Dennison & Lovrein 1997, Sharma et al. 2000). In this work, we have employed three-phase partitioning for a one-step purification of pectinase activity from Aspergillus niger (commercial preparation) and from tomato extract. Materials and methods Fresh ripe tomatoes were purchased from a local mar- ket. Polygalacturonic acid was from Sigma Chemical Co. Pectinex 3 XL (pectinase from Aspergillus niger) was a product of Novo Nordisk. All other chemicals used were of analytical grade. Estimation of enzyme activity and amount of protein Pectinase activity was estimated by taking polygalac- turonic acid as the substrate. One unit is defined as the amount of enzyme which liberates 1 μmole of reduc- ing groups (calculated as galacturonic acid) per min at 30 ◦ C. Protein content was estimated by the dye binding method. Preparation of the pectinase extract from tomato The crude extract of tomato pectinase was prepared from 200 g fresh tomatoes according to the procedure described by Man & Mauhudzi (1996). Three-phase partitioning of pectinase The ammonium sulphate (w/v) was added to the de- sired level to the crude extracts of pectinases from Aspergillus niger and tomato extract at 25 ◦ C, mixed gently to dissolve the salt followed by addition of tert-butanol. After 1 h, the mixtures were centrifuged (2000 g for 10 min) to facilitate separation of phases. Results and discussion One of the critical parameters in three-phase parti- tioning is the concentration of ammonium sulphate