Biotechnology Letters 23: 1625–1627, 2001.
© 2001 Kluwer Academic Publishers. Printed in the Netherlands.
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: firstname.lastname@example.org)
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
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.
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
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
C. Protein content was estimated by the dye
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
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