www.fems-microbiology.org

FEMS Yeast Research 5 (2005) 859–865

Pectinolytic enzymes secreted by yeasts from tropical fruits

Evania Geralda da Silva a, Maria de Fatima Borges b, Clara Medina c,Roberta Hilsdorf Piccoli a, Rosane Freitas Schwan a,*

a Biology Department, Federal University of Lavras, Cx. Postal 37, 37.200-000, Lavras, MG, Brazilb EMBRAPA/CNPAT, Rua dos Tabajaras, 11–Praia de Iracema, Caixa Postal 3761, Fortaleza, CE, CEP 66060-510, Brazil

c CORPOICA- Calle Apartado Aereo A. 100, Centro de Investigacion La Selva, Rionegro, Antioquia, Colombia

Received 9 November 2004; received in revised form 15 January 2005; accepted 15 February 2005

First published online 30 March 2005

Abstract

Three hundred yeasts isolated from tropical fruits were screened in relation to secretion of pectinases. Twenty-one isolates wereable to produce polygalacturonase and among them seven isolates could secrete pectin lyase. None of the isolates was able to secretepectin methylesterase. The pectinolytic yeasts identified belonged to six different genera. Kluyveromyces wickerhamii isolated fromthe fruit mangaba (Hancornia speciosa) secreted the highest amount of polygalacturonase, followed by K. marxianus and Stepha-

noascus smithiae. The yeast Debaryomyces hansenii produced the greatest decrease in viscosity while only 3% of the glycosidic link-ages were hydrolysed, indicating that the enzyme secreted was an endo-polygalacturonase. The hydrolysis of pectin bypolygalacturonase secreted by S. smithiae suggested an exo-splitting mechanism. The other yeast species studied showed low poly-galacturonase activity.� 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: Pectinases; Polygalacturonases; Pectin lyases; Tropical fruits; Kluyveromyces wickerhamii; Stephanoascus smithiae

1. Introduction

Pectin substances are complex structural polysaccha-rides of plant origin that contain a large proportion ofpartially methyl-esterified galacturonic acid subunitslinked by a-1,4-glycosidic linkages [1]. These D-galact-uronic acid residues present in the backbone of thepectin chain are interrupted by L-rhamnose residues, towhich arabinose and galactose residues can be attached[1]. Pectic or pectinolytic enzymes are a complex ofenzymes that degrade pectic polymers and there are sev-eral classes of enzymes involved in the degradation ofpectin: lyases (EC 4.2.2.10), pectate lyases (EC 4.2.2.2),and polygalacturonases (EC 3.2.1.15 and EC 3.2.1.67)

1567-1356/$22.00 � 2005 Federation of European Microbiological Societiesdoi:10.1016/j.femsyr.2005.02.006

* Corresponding author. Fax: +55 35 829 1100.E-mail address: rschwan@ufla.br (R.F. Schwan).

[1]. Several organisms are able to produce pectin-degrading enzymes, and these include plants [2], filamen-tous fungi [3,4], bacteria [5] and some yeasts [6,7].Production of pectinases has been widely reported andis thoroughly studied in bacteria and filamentous fungibecause they are thought to play an important role inplant pathology [5,8–10]. Pectinases are of major impor-tance in the beverage industry due to their ability to im-prove pressing and clarification of concentrated fruitjuices and they are extensively used in the food industryin the processing of fruits and vegetables, in the produc-tion of wine, the extraction of olive oil and fermentationof tea, coffee and cocoa [1,11,12].

Pectinases used in the food industry are commer-cially produced by Aspergillus niger [13,14]. This fungalspecies produces various pectinases, including pectinmethylesterase (PME), polygalacturonase (PG) and

. Published by Elsevier B.V. All rights reserved.

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pectin lyase (PL) [15]. However, there are cases whereparticular pectinases are used for specific purposes.High levels of PG are used for example to soften babyfood products and to stabilise the cloudiness in orangejuice. Many commercial pectinases from A. niger showlow PG activity and high PL and PME activity. Thehydrolysis of pectin by PME, if left in the pectinolyticdigest, produces the toxic alcohol methanol.

Filamentous fungi have been used for more than 50years in the production of industrial enzymes, and mostof them produce various enzymes simultaneously [16].Yeasts present an alternative source for the large-scaleproduction of commercial enzymes [6,13,17–19]. Yeastshave advantages compared to filamentous fungi with re-gard to the production of pectinases, because they areunicellular, the growth is relatively simple, and thegrowth medium does not require an inducer. In addi-tion, gene cloning and gene manipulation may improveenzyme production, thus suggesting that commercial en-zyme production by yeasts should be possible [13]. Inrelation to the production of pectinase, yeasts usuallydo not secret PME and, therefore, their pectinases canbe used to clarify fruit juice and wine without releasingmethanol [12,20].

In Brazil a large variety of fruits occurs and most ofthem are commercialised in their natural state or as fruitjuices [21–23]. In general, fruits represent an importantmicrohabitat for a variety of yeast species due to thehigh concentration of sugars, low pH and intense visita-tion by insect vectors [24,25].

Yeasts have a great potential for the production ofmicrobial enzymes for the food industry and they offeran alternative source of these enzymes. The main aimof this study was to select and identify the yeasts, presenton the surface of tropical fruits, which are able to secretepectinases, and to characterise some of the properties ofthe enzymes produced.

2. Materials and methods

2.1. Isolation of yeasts

Yeasts obtained from the Culture Collection of theMicrobial Physiology Laboratory at DBI/UFLA, Lav-ras, MG Brazil had been isolated from the followingtropical fruits: acerola (Malghia glabra), ata-pinha(Annona squamosa), bacuri (Platonia insignis), cocoa(Theobroma cacau), caja (Spondias lutea), ciriguela(Spondias purpurea), cupuacu (Theobroma grandiflorum

schum), graviola (Annoma muricata), lulo (Solanumquitoense), mangaba (Hancornia speciosa), passionfruit (Passiflora edulis var. edulis), pseudolulo (Sola-num pseudolulo), and umbu-caja (Spondias tuberosa).Fresh fruit and fruit pulp from CEPLAC-BA, Fort-

aleza (EMBRAPA) and Corpoica-Rionegro (Colom-bia) were microbiologically analysed. The fruits wereobtained from trees and/or from the ground andstored in sterile plastic bags. The fruits were placedin flasks containing sterile peptone water. An aliquotwas serially diluted and plated in three differentmedia: Bacto W L Nutrient Medium Dehydrated(DIFCO, Detroit, Mich.), YW nutrient medium con-taining yeast extract 3.0 g l�1; malt extract 3.0 g l�1;soy peptone 5.0 g l�1 and glucose 10.0 g l�1 [26] (pH3.5) containing 60 lg of tetracycline or chlorampheni-col per ml and fruit-agar medium (1% fruit pulp, 1%yeast extract, 25% agar), incubated at 25 �C for 24–48 h. The yeasts isolated were maintained at 4 �C onYW agar slopes.

2.2. Selection for pectinolytic activity

Pectinolytic activity was detected according to themethod described by Schwan [7]. The yeasts isolatedwere grown in plates with mineral medium containingpolygalacturonic acid (MP5) for polygalacturonase(PG) activity and mineral medium containing pectin(MP7) for pectin lyase activity [27]. Enzyme activitywas indicated by the formation of a clear halo aroundthe colonies after precipitation of polygalacturonic acidwith 1% cetyl trimethyl ammonium bromide (CTAB).The yeast Kluyveromyces marxianus CCT 3172 [7] wasused as the positive control.

2.3. Identification of pectinase-secreting yeasts

Pectinase-secreting yeast isolates were identified tothe species level by physiological and morphologicalmethods and were identified using taxonomic keysdescribed in the literature [26,28].

2.4. Enzyme assays and protein determination

For enzyme production, a volume of 500 ml of YWculture medium, pH 7.0, was dispensed in 1-l roundflat-bottomed flasks fitted with fermentation locks.Flasks were inoculated with 1.0 mg dry-weight equiva-lent of organisms from a 24-h starter culture (100 mlof medium inoculated with part of a single colony andincubated at 30 �C on an orbital shaker at200 rev min�1). Cultures were incubated at 30 �C underself-induced anaerobic conditions and stirred continu-ously with magnetic bars of 5 cm length [29]. Cultureswere sampled at intervals and growth (dry weightml�1) was determined from OD measurements at600 nm against an appropriate calibration curve. Afterremoving the cells by centrifugation at 4800 rpm for10 min at 4 �C, samples of the supernatant fluid wereused for the determination of enzyme activity.

E.G. da silva et al. / FEMS Yeast Research 5 (2005) 859–865 861

2.4.1. Polygalacturonase (PG)

Two methods were used to measure polygalacturo-nase activity in culture filtrates, namely by analyzingthe release of reducing groups and the decrease in vis-cosity of the substrate. Polygalacturonase activity wasassayed by measuring the increase in reducing groupsderived from polygalacturonate using the method de-scribed by Miller [30]. One unit of enzyme activity wasexpressed as lmol of galacturonic acid releasedmin�1 lg total protein�1. Reduction in viscosity wasmeasured according the method of Cooper and Wood[31]. To assay the decrease in viscosity, 2 ml of a suitablydiluted culture filtrate was mixed with 8 ml 3.2 % (w/v)polygalacturonic acid Na-salt in 0.1-M citrate buffer.Readings of the flow of the reaction mixture were re-corded at short intervals measured in seconds at25 ± 0.01 �C using a Technico (Ekkattuthangal, Chen-nai, Tamil Nadu, India) viscometer (size 200), in whichthe flow-through time for the standard volume of waterwas 10 s [29]. One relative viscosity unit (RVU) was de-fined as the enzyme quantity required to decrease theinitial viscosity by 50% per minute under the conditionspreviously described.

2.4.2. Pectin lyase (PL)

Pectin lyase in the supernatant of the cultures wasdetermined spectrophotometrically (A235) according tothe method described by Albersheim [32]. The reactionmixture consisted of 1 ml of 2.5% (w/v) citrus pectin(85% esterified) in 100 mM phosphate buffer, pH 6.8,and 1.5 ml of culture supernatant. The reaction mixturewas incubated for 0, 10, 15, 20 and 30 min at 40 �C. Thereaction was stopped by the addition of 4.5 ml of 0.01-NHCl. One unit of enzyme activity (U) of pectin lyase wasdefined as 1 lmol of unsaturated product min�1 [7].

2.4.3. Pectin methylesterase (PME)

For the determination of PME, yeasts were grown in50 ml of YW medium containing 1% citrus pectin (85%esterified) and incubated at 28 �C in a rotary shaker at120 rpm for 48 h. The cultures were then centrifugedand the supernatant was collected for the PME assay.

Pectin methylesterase was assayed using two meth-ods. One was by continuous titrimetric determinationof the carboxyl groups liberated from methylester bonds[7]. PME activity was expressed as microequivalents ofpolygalacturonic acid produced ml�1h�1. PME was alsomeasured as the release of methanol from pectin chainsas detected by gas chromatography. Samples were fil-tered through a cellulose nitrate membrane of 0.45 lm,and afterwards 1 ml of internal standard (202 mg of tol-uene/100 ml ethanol) was added to 0.5 ml of sample. A1-ll aliquot was then injected into a gas chromatograph(Varian CG 3800 version 4.5, Palo Alto, CA), using aCarbowax column, resulting in a retention time of11 min 47 s.

2.4.4. Total protein

Total protein determination was performed by themethod of Bradford [33], using bovine serum albumin(BSA) as the standard.

2.5. Characterisation of PG with variation of time,temperature and pH of substrate

Polygalacturonase activity was assayed by measuringthe increase of reducing sugars [7,30] using various incu-bation times (15, 30, 45 and 60 min), and different pH ofthe substrate (3.5, 4.5, 5.5 and 6.5) and incubation tem-peratures (30, 35, 40, 45 and 50 �C). The results obtainedwere extrapolated using a standard curve for galact-uronic acid.

3. Results and discussion

3.1. Screening for pectinolytic activity

Three hundred yeast isolates from tropical fruits wereevaluated for their potential to produce and secrete pec-tinase in, either, solid medium containing polygalactu-ronic acid and glucose and/or galactose as carbonsource for polygalacturonase (PG) activity, solid med-ium containing pectin for pectin lyase (PL) activity, orliquid medium for pectinmethylesterase (PME) activity.Among the 300 isolates, only 21 (7%) were positive forpolygalacturonase activity, and of these, seven were po-sitive for pectin lyase activity (Table 1). PME was notdetected in any of these yeast cultures filtrates. The iso-lates IC-50 and IC-54 secreted polygalacturonase onlywhen glucose was the source of carbon, while the isolateSL-140 secreted the enzyme when galactose was the car-bon source (Table 1). The yeast SL-140 did not grow inMP5-glucose culture medium, but did grow in culturemedium when the source of carbon was substituted withgalactose. It has been reported that galactose was a bet-ter source of carbon than glucose using strains of Sac-charomyces cerevisiae which were genetically modifiedto produce polygalacturonase [13]. The results obtainedin this study indicate that the yeast Debaryomyces hanse-

nii (SL-140) also secreted b-galactosidase (data notshown).

The yeasts Stephanoascus smithiae (isolates FT-01,168, 36 and 147), Pichia sp. (FT-28), Pichia anomala

(SL-125) and K. wickerhamii (185) (Table 1) were alsocapable of secreting pectin lyase in MP7 medium (pH7.0) containing pectin (85% esterified). Although it hasbeen reported by several authors that yeasts secretedmainly PG, Gainvors [34] also has detected pectin lyaseactivity in the yeast Saccharomyces cerevisiae isolatedduring wine fermentations.

A great diversity occurs among the yeast species asso-ciated with fruits [35,36] or fruit pulp [22]. It is likely

Table 1Polygalacturonase (PG) and pectin lyase (PL) secretion by tropical yeasts

Strain Yeast isolates MP5-glucosea (PG) MP5-galactosea (PG) MP7-pectina (PL)

185 Kluyveromyces wickerhamii + + +166 Kluyveromyces marxianus + + �168 Stephanoascus smithiae + + +162 Pichia angusta + + �CH-144A Zygosacchoromyces fermentati + + �36 Stephanoascus smithiae + + +IC-50 Kluyveromyces wickerhamii + � �IC-54 Stephanoascus smithiae + � �CH-142A Candida krusei + + �SL-125 Pichia anomala + + +SL-140 Debaryomyces hansenii � + �CH-156A Pichia guilliermondii + + �CH-146A Zygosaccharomyces cidri + + �147 Stephanoascus smithiae + + +FT-01 Stephanoascus smithiae + + +FT-175 Candida pseudoglaebosa + + �53CO Debaryomyces polymorphus + + �FT20 Debaryomyces hansenii + + �FT-28 Pichia sp. + + +FT-35 Candida intermedia + + �IC-38 Pichia guilliermondii + + �Enzyme activity was detected by the presence of a clear zone around a colony in an otherwise opaque medium, indicating degradation of eitherpolygalacturonic acid or pectin.a Schwan et al., 1997 [7].

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that some strains developed pectinolytic activity to opti-mise their growth [34]. Secretion of pectinolytic activityby yeasts may be a means to increase survival when theyhave to consume simpler sources of carbon [22], butother authors [18,19] have reported that the functionof these enzymes in yeasts is unknown. Yeasts isolatedfrom cocoa secreted polygalacturonase even if they werenot capable to utilise pectin or galacturonic acid as asole carbon source [7]. It is possible that yeasts presenton some tropical fruits produce pectinases to degradepectin present in fruit pulp in order to assimilate the sug-ars occurring on the side chains of pectin such as rham-nose, galactose and arabinose. The utilisation of thesesugars by the yeasts isolated was demonstrated in bio-chemical tests utilised in the identification at the specieslevel. All 21 yeasts able to produce pectinase could uti-lise at least one of these three sugars present in thechains of pectin.

3.2. Yeast identification

The yeast strains that showed pectinolytic activitywere identified at the species level. Six genera, compris-ing thirteen species of yeasts, were found capable to se-crete pectinases (Tables 1 and 2). The thirteen specieswere, with their numbers identified in parentheses: K.wickerhamii (2), S. smithiae (5), K. marxianus (1), P. an-gusta (1), P. anomala (1), P. guilliermondii (2), Zygosac-choromyces fermentati (1), Z. cidri (1), Candida krusei

(1), C. pseudoglaebosa (1), C. intermedia (1), D. hansenii

(2), D. polymorphus (1) and one unidentified speciesbelonging to the genus Pichia. One third of the identifiedpectinolytic yeasts were isolated from the surface of thetropical fruit mangaba (H. speciosa). It is possible thatthese results are due to the pectin concentration foundin this fruit, which is approximately 1.2%. In contrast,the other fruits presented pectin contents varying from0.2% to 0.7% (data not shown).

Some species of Pichia and Candida isolated fromtropical environments (soil, water, insect and plantmaterials) also showed pectinolytic activity [37]. Thespecies identified in this work (Tables 1 and 2) occurin other environments as well, such as flowers, cactiand other fruits [22,24,35,38].

3.3. Production of pectinolytic enzymes by tropical yeasts

The quantity of enzyme secreted varied considerablyamong the isolates (Table 2). Significant differences wereobserved among the yeast isolates in the determinationof polygalacturonase activity secreted in liquid mediumusing Tukey�s test at the 5%-probability level. K. wick-erhamii strain 185 showed the highest enzyme activitywith the conditions tested (viz. 24.0 lmol galacturonicacid released min�1 lg protein�1). K. marxianus andS. smithiae also showed high enzyme activities of 14.2and 12.9 lmol of galacturonic acid releasedmin�1 lg protein�1, respectively. These values were,however, approximately 50% of the enzyme activity se-creted by K. wickerhamii strain 185.

Table 2Extracellular polygalacturonase (PG) activity and decrease of relative viscosity (RVU) due to polygalacturonase secreted by various tropical yeasts*

Strain Fruit Yeast PG activity** RVU***

185 Mangaba Kluyveromyces wickerhamii 24.0d**** 44.44166 Mangaba Kluyveromyces marxianus 14.2c 5.0168 Mangaba Stephanoascus smithiae 12.9c 0162 Mangaba Pichia angusta 3.3b 33.33CH-144A Cocoa Zygosacchoromyces fermentati 3.1b 3.8036 Caja Stephanoascus smithiae 1.7a b 11.11IC-50 Bacuri Kluyveromyces wickerhamii 0.96a 0.095IC-54 Mangaba Stephanoascus smithiae 0.87a 7.14CH-142A Cocoa Candida krusei 0.66a 0SL-125 Pseudo-lulo Pichia anomala 0.62a 100.0SL-140 Pseudo-lulo Debaryomyces hansenii 0.61a 5000.0CH-156A Cocoa Pichia guilliermondii 0.53a 10.0CH-146A Cocoa Zygosaccharomyces cidri 0.46a 6.46147 Mangaba Stephanoascus smithiae 0.46a 3.50FT-01 Ciriguela Stephanoascus smithiae 0.36a 5.13FT-175 Mangaba Candida pseudoglaebosa 0.34a 11.1153-CO Lulo Debaryomyces polymorphus 0.29a 4.40FT-20 Umbu–caja Debaryomyces hansenii 0.21a 6.46FT-28 Umbu–caja Pichia sp. 0.16a 0FT-35 Caja Candida intermedia 0.15a 0IC-38 Bacuri Pichia guilliermondii 0.098a 0

* Cultures were grown in a medium containing 1 % (w/v) glucose + 1% (w/v) pectin under self-induced anaerobic conditions for 3 days.** PG activity is expressed as lmol of galacturonic acid released min�1 lg total protein�1.*** Relative viscometric unit (RVU) is defined as the enzyme quantity required to decrease 50% of the initial viscosity per minute. The decrease inviscosity was observed using a 3.2% (w/v) polygalacturonic acid solution at 25 �C.**** Means of PG activity followed by same letter did not significantly differ using Tukey test (5%).

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The assay to determine pectin lyase activity in the cul-ture media MF pH 5.0, YW pH 7.0 and MP7 pH 7.0,and with pectin as an inducer, did not demonstrateany activity of this enzyme by the isolates tested. Thisis in agreement with the observed rare production ofpectin lyase by yeasts [7,19].

3.4. Properties of polygalacturonase secreted by tropical

yeasts

PGactivitywas determined for those yeastswhichwereconsidered asmajor pectinases producers, viz.K.wickerh-amii strain 185 and K. marxianus strain 166. The enzymeactivity was determined after incubation times of 15, 30,45 and 60 min, with pH values of 3.5, 4.5, 5.5 and 6.5and incubation temperatures of 30, 35, 40, 45 and 50 �C.The highest PG activity, viz. 24.0 lmol of polygalactu-ronic acidmin�1 lg protein�1, occurred inK.wickerhamii

strain 185 after 15 min incubation, at pH 4.5 and at a tem-perature of 35 �C (Fig. 1(a)). For the isolateK. marxianus

strain 166, the highest activity was at pH 5.5 after 15 minincubation at 35 �C (Fig. 1(b)). PG secreted by theseyeasts showed an activity between pH 4.0 and 6.0, whichis typical of PG secreted by yeasts [7,39–41].

3.5. Mechanism of enzyme action

Determination of polygalacturonase activity by the le-vel of sugar reduction can detect exo-PG as well as endo-

PG activity. Exo-PG releases small fragments from thepolymer and does not reduce viscosity significantly[15,19]. Endo-PG activity is characterised by a substan-tial decrease in viscosity (in general 50%), resulting fromthe release of reducing groups (1–3%), whereas an exo-PG must hydrolyse more than 20% of the glycosidic link-age bonds in order to obtain a decrease in viscosity of50% [42]. The decrease in viscosity differed substantiallyamong the yeasts tested (Table 2 and Fig. 2). The resultsshowed that the enzyme secreted by D. hansenii strainSL-140 produced the greatest decrease (viz. 50%) in vis-cosity in 10 min (5000 RVU; Table 2), while only 3% ofthe glycosidic linkages of the substrate were hydrolysed.This reduction in viscosity was greater than the resultsobtained by Schwan [7] for K. marxianus, which yieldeda 50% decrease in viscosity in 18 min. This decrease inviscosity due to the enzyme secreted from D. hansenii

strain SL-140 was unexpected because only smallamounts of galacturonic acid were released (Table 2).It is possible that the action of endo-polygalacturonaseliberates fragments of galacturonic acid, which may notbe detectable by the method of liberation of reducinggroups. P. anomala strain SL-125, K. wickerhamii strain185 and C. intermedia strain FT �35 showed a 50% de-crease in viscosity after a 25-35 min of reaction, whichrepresented 100, 44.44 and 33.33 relative viscosity units(RVU), respectively (Table 2). The results suggestedthe presence of a random mechanism of hydrolysis,and the enzyme secreted by these yeasts is a poly

03.5 4.5 5.5 6.5

3.5 4.5 5.5 6.5

5

10

15

20

25

30

pH

Po

lyg

alac

turo

nas

e a

ctiv

ity

Po

lyg

alac

turo

nas

e a

ctiv

ity

0

5

10

15

20

25

30

pH

(a)

(b)

Fig. 1. Effect of pH and temperature of incubation on PG activitysecreted by yeasts. PG activity was measured at different pH values anddifferent temperatures of incubation: (r) 30 �C, (h) 35 �C, (d) 40 �C,(*) 45 �C. (a) Kluyveromyces wickerhamii (strain185), (b) Kluyveromy-

ces marxianus (strain 166). PG activity was defined as lmol ofgalacturonic acid released min�1lg total protein�1.

0

10

20

30

40

50

60

0 10 20 30 40 50 60

Time (min)

Vis

cosi

ty (

% z

ero-

time

valu

e)

Fig. 2. Decrease in viscosity produced by endo-PG secreted from (s)Kluyveromyces wickerhamii (strain185), (n) Debaryomyces hansenii

(strain SL-140) and (.·.) Pichia anomala (strain SL-125).

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a-1,4-D-galacturonide glycanohydrolase (EC 3.21.15) oran endo-PG. The yeast S. smithiae strain 168 showed arelatively high polygalacturonase activity of 12.9 lmol

of galacturonic acid min�1 lg of protein�1 (Table 2) asmeasured by the release of reducing groups, but therewas no decrease in viscosity of a 3.2% (w/v) polygalactu-ronic acid solution after 120 min of incubation at 25 �C.This behaviour strongly suggests that the PG secreted bythis isolate acts by an exo-splitting mechanism [15]. Theother yeasts studied showed low polygalacturonaseactivity as determined by both methods.

It has already been demonstrated that the yeast K.

marxianus has considerable economic advantages overAspergillus as a source of endo PG even without geneticimprovement of the strain [7,13,19,43]. The use of PGfrom yeasts could be highly advantageous for the wine,cider and fruit juice industries and in the softening ofvegetables for the preparation of baby foods. Basedon the data obtained in the present study, it can beconcluded that yeasts from tropical fruits have a greatpotential for the production of pectinolytic enzymes,which could be used in the food industry. More de-tailed studies on the secretion of enzymes from yeastsoccurring on tropical fruits are needed to further pro-vide support for their potential utilisation in the foodindustry.

Acknowledgements

This work was supported by a European UnionINCO-DC Project Grant (IC18 CT97 0182) and byCAPES (Coordenacao de Aperfeicoamento de Pessoalde Nıvel Superior). We thank the technical staff atCEPLAC for isolation of yeasts from cocoa and Mrs.Maria Aparecida Gomes Souza-Dias for her help inthe yeast identification.

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