Biotechnology Letters 23: 1625–1627, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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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, tomatopectinase

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) andpurifications of 10-fold (Aspergillus niger) and 9-fold (tomato) were obtained. The final purified enzyme enzymefrom tomato showed a single band on SDS-PAGE with a molecular weight of 46 kDa.

Introduction

Pectinases have a variety of applications in foodprocessing 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 purificationof pectinases. In this technique, the aqueous crudeextract of the enzyme is mixed with tert-butanol inthe presence of ammonium sulphate. The enzyme ap-pears as an interfacial precipitate between the loweraqueous and upper tert-butanol phases. In practice,the separation of desired protein in the interfacial pre-cipitate is made by varying the ammonium sulphateconcentration, tert-butanol volume and temperature(Dennison & Lovrein 1997, Sharma et al. 2000). Inthis work, we have employed three-phase partitioningfor a one-step purification of pectinase activity fromAspergillus niger (commercial preparation) and fromtomato extract.

Materials and methods

Fresh ripe tomatoes were purchased from a local mar-ket. Polygalacturonic acid was from Sigma ChemicalCo. Pectinex 3 XL (pectinase from Aspergillus niger)was a product of Novo Nordisk. All other chemicalsused 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 theamount of enzyme which liberates 1 µmole of reduc-ing groups (calculated as galacturonic acid) per minat 30 ◦C. Protein content was estimated by the dyebinding method.

Preparation of the pectinase extract from tomato

The crude extract of tomato pectinase was preparedfrom 200 g fresh tomatoes according to the proceduredescribed 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 fromAspergillus niger and tomato extract at 25 ◦C, mixedgently to dissolve the salt followed by addition oftert-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

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Table 1. Purification of pectinase from pectinex 3 XL with three-phase partitioning. The ammonium sulphate (30%, w/v) was addedto the crude extract of pectinase (1 ml containing 38 U). This was followed by addition of tert-butanol in a ratio of 1:1 (v/v). Threephases formed were collected separately. The enzyme and protein activity were estimated in the interfacial precipitate after dissolvingthe precipitate in 1 ml of 100 mM acetate buffer, pH 5.

Steps Activity Protein Specific activity Yield Purification

(units) (mg) (U mg−1) (%) fold

Crude 38 11 3 100 1

Precipitate 29 1 29 76 10

All the experiments were run in duplicate and the difference in the readings in duplicates was less than ±5%.

Table 2. Purification of pectinase from tomato by subjecting to three-phase partitioning. The ammonium sulphate (20%, w/v) was addedto the crude extract of tomato pectinase (1 ml containing 6 U). This was followed by addition of tert-butanol in a ratio of 1:2 (crudeextract:tert-butanol). Three phases formed were collected separately. Activity was estimated in the interfacial precipitate. The loweraqueous phase was subjected to second phase of three-phase partitioning in an identical way. Interfacial precipitate was tested for enzymeactivity.

Steps Activity Protein Specific activity Yield Purification

(units) (µg) (U mg−1) (%) fold

Crude 6 70 86 100 1

Precipitate-1 1 51 20 17 –

Precipitate-2 11 15 733 183 9

All the experiments were run in duplicate and the difference in the readings in duplicates was less than ±5%.

used for precipitating the protein in the interfacialphase. This concentration should be less than the onewhich causes ‘salting out’ of any protein. Generally,one starts with a minimum salt concentration of 20%(w/v) and optimizes this so as to obtain the maximumamount of the desired protein in the interfacial precip-itate (Dennison & Lovrein 1997). Table 1 shows thatpectinases activity from a commercial preparation ofthe enzyme from Aspergillus niger can be separatedby three-phase partitioning. The recovery of 76% en-zyme activity and 10-fold purification indicated thatthis may be a useful approach for separation of theenzyme from crude sources.

Table 2 shows the results of purification of tomatopectinase from the crude extract. The tomato en-zyme, however, behaved differently and only 17%activity was found in the interfacial precipitate. How-ever, when the aqueous phase was subjected to secondround of three-phase partitioning, bulk of the enzymeactivity appeared in the interfacial precipitate. Thisshowed 9-fold purification. The unusual enzyme re-covery of 183% needs explanation. It has been oftenobserved (Dennison & Lovrein 1997) that three-phasepartitioning leads to simultaneous activation of the en-zyme which (if the enzyme recovery is high) resultsto such an apparently observed value of 183% yield.Recently, we have found that enzyme activation fre-

quently observed during three-phase partitioning maybe the result of increased flexibility in the enzymemolecule. X-Ray diffraction studies show three-phasepartitioning treated proteinase K has an unusually highB-factor (Singh et al. 2001).

The final purified enzyme preparation showeda single band on SDS-PAGE after staining withCoomassie Brilliant Blue R-250, with a molecularweight of 46 kDa which is in agreement with the valuereported earlier (Moshrefi & Luh 1983).

The procedure described here is simple and scal-able and should be applicable to pectinases from othersources as well. In view of the applications of pecti-nases in food industry (Alkorta et al. 1998), this maybe found useful as a bioseparation strategy for theenzyme.

Acknowledgement

This work was supported by project funds fromthe Council for Scientific and Industrial Research(TMOP&M project, Govt. of India organization).

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References

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Dennison C, Lovrein R (1997) Three-phase partitioning: concen-tration and purification of proteins. Protein Exp. Purif. 11:149–161.

Man B, Mauhudzi I (1996) Use of cross linked mucilage fromreuredzo (Dicerocaryum Zanguebarum) in the purification ofpolygalacturonase extracted from tomato. Food Chem. 56: 433–467.

Moshrefi M, Luh BS (1983) Carbohydrate composition and elec-trophoretic properties of tomato polygalacturonase isoenzymes.J. Biochem. 135: 511–514.

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Singh RK, Gourinath S, Sharma S, Roy I, Gupta MN, Betzel Ch,Srinivasan A, Singh TP (2001) Enhancement of enzyme activitythrough three-phase partitioning: crystal structure of a modifiedserine proteinase at 1.5 Å resolution. Protein Eng. 14: 307–313.