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