FEMS Yeast Research 5 (2005) 859–865

www.fems-microbiology.org

Pectinolytic enzymes secreted by yeasts from tropical fruits

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

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a
Biology Department, Federal University of Lavras, Cx. Postal 37, 37.200-000, Lavras, MG, Brazil
b
EMBRAPA/CNPAT, Rua dos Tabajaras, 11–Praia de Iracema, Caixa Postal 3761, Fortaleza, CE, CEP 66060-510, Brazil
c
CORPOICA- Calle Apartado Aéreo 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 were
able to produce polygalacturonase and among them seven isolates could secrete pectin lyase. None of the isolates was able to secrete
pectin methylesterase. The pectinolytic yeasts identified belonged to six different genera. Kluyveromyces wickerhamii isolated from
the 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 by
polygalacturonase 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 [1]. Several organisms are able to produce pectin-

Pectin substances are complex structural polysaccha-
rides of plant origin that contain a large proportion of
partially methyl-esterified galacturonic acid subunits
linked by a-1,4-glycosidic linkages [1]. These D-galact-
uronic acid residues present in the backbone of the
pectin chain are interrupted by L-rhamnose residues, to
which arabinose and galactose residues can be attached
[1]. Pectic or pectinolytic enzymes are a complex of
enzymes that degrade pectic polymers and there are sev-
eral classes of enzymes involved in the degradation of
pectin: 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)
*
Corresponding author. Fax: +55 35 829 1100.
E-mail address: rschwan@ufla.br (R.F. Schwan).
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 and
is thoroughly studied in bacteria and filamentous fungi
because they are thought to play an important role in
plant 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 fruit
juices and they are extensively used in the food industry
in the processing of fruits and vegetables, in the produc-
tion of wine, the extraction of olive oil and fermentation
of tea, coffee and cocoa [1,11,12].
Pectinases used in the food industry are commer-
cially produced by Aspergillus niger [13,14]. This fungal
species produces various pectinases, including pectin
methylesterase (PME), polygalacturonase (PG) and

1567-1356/$22.00
2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.femsyr.2005.02.006
860 E.G. da silva et al. / FEMS Yeast Research 5 (2005) 859–865
pectin lyase (PL) [15]. However, there are cases where
particular pectinases are used for specific purposes.
High levels of PG are used for example to soften baby
food products and to stabilise the cloudiness in orange
juice. Many commercial pectinases from A. niger show
low PG activity and high PL and PME activity. The
hydrolysis of pectin by PME, if left in the pectinolytic
digest, produces the toxic alcohol methanol.
Filamentous fungi have been used for more than 50
years in the production of industrial enzymes, and most
of them produce various enzymes simultaneously [16].
Yeasts present an alternative source for the large-scale
production of commercial enzymes [6,13,17–19]. Yeasts
have advantages compared to filamentous fungi with re-
gard to the production of pectinases, because they are
unicellular, the growth is relatively simple, and the
growth medium does not require an inducer. In addi-
tion, gene cloning and gene manipulation may improve
enzyme production, thus suggesting that commercial en-
zyme production by yeasts should be possible [13]. In
relation to the production of pectinase, yeasts usually
do not secret PME and, therefore, their pectinases can
be used to clarify fruit juice and wine without releasing
methanol [12,20].
In Brazil a large variety of fruits occurs and most of
them are commercialised in their natural state or as fruit
juices [21–23]. In general, fruits represent an important
microhabitat for a variety of yeast species due to the
high concentration of sugars, low pH and intense visita-
tion by insect vectors [24,25].
Yeasts have a great potential for the production of
microbial enzymes for the food industry and they offer
an alternative source of these enzymes. The main aim
of this study was to select and identify the yeasts, present
on the surface of tropical fruits, which are able to secrete
pectinases, and to characterise some of the properties of
the enzymes produced.
2. Materials and methods
2.1. Isolation of yeasts
Yeasts obtained from the Culture Collection of the
Microbial Physiology Laboratory at DBI/UFLA, Lav-
ras, MG Brazil had been isolated from the following
tropical fruits: acerola (Malghia glabra), ata-pinha
(Annona squamosa), bacuri (Platonia insignis), cocoa
(Theobroma cacau), cajá (Spondias lutea), cirigüela
(Spondias purpurea), cupuaçu (Theobroma grandiflorum
schum), graviola (Annoma muricata), lulo (Solanum
quitoense), mangaba (Hancornia speciosa), passion
fruit (Passiflora edulis var. edulis), pseudolulo (Sola-
num pseudolulo), and umbu-cajá (Spondias tuberosa).
Fresh fruit and fruit pulp from CEPLAC-BA, Fort-
aleza (EMBRAPA) and Corpoica-Rionegro (Colom-
bia) were microbiologically analysed. The fruits were
obtained from trees and/or from the ground and
stored in sterile plastic bags. The fruits were placed
in flasks containing sterile peptone water. An aliquot
was serially diluted and plated in three different
media: 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] (pH
3.5) containing 60 lg of tetracycline or chlorampheni-
col per ml and fruit-agar medium (1% fruit pulp, 1%
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yeast extract, 25% agar), incubated at 25 C for 24–
48 h. The yeasts isolated were maintained at 4 C on
YW agar slopes.
2.2. Selection for pectinolytic activity
Pectinolytic activity was detected according to the
method described by Schwan [7]. The yeasts isolated
were grown in plates with mineral medium containing
polygalacturonic acid (MP5) for polygalacturonase
(PG) activity and mineral medium containing pectin
(MP7) for pectin lyase activity [27]. Enzyme activity
was indicated by the formation of a clear halo around
the colonies after precipitation of polygalacturonic acid
with 1% cetyl trimethyl ammonium bromide (CTAB).
The yeast Kluyveromyces marxianus CCT 3172 [7] was
used as the positive control.
2.3. Identification of pectinase-secreting yeasts
Pectinase-secreting yeast isolates were identified to
the species level by physiological and morphological
methods and were identified using taxonomic keys
described in the literature [26,28].
2.4. Enzyme assays and protein determination
For enzyme production, a volume of 500 ml of YW
culture medium, pH 7.0, was dispensed in 1-l round
flat-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 ml
of medium inoculated with part of a single colony and
incubated at 30 C on an orbital shaker at
200 rev min 1). Cultures were incubated at 30 C under
self-induced anaerobic conditions and stirred continu-
ously with magnetic bars of 5 cm length [29]. Cultures
were sampled at intervals and growth (dry weight
ml 1) was determined from OD measurements at
600 nm against an appropriate calibration curve. After
removing the cells by centrifugation at 4800 rpm for
10 min at 4 C, samples of the supernatant fluid were
used for the determination of enzyme activity.
 E.G. da silva et al. / FEMS Yeast Research 5 (2005) 859–865
2.4.1. Polygalacturonase (PG)
Two methods were used to measure polygalacturo-
nase activity in culture filtrates, namely by analyzing
the release of reducing groups and the decrease in vis-
cosity of the substrate. Polygalacturonase activity was
assayed by measuring the increase in reducing groups
derived from polygalacturonate using the method de-
scribed by Miller [30]. One unit of enzyme activity was
expressed as lmol of galacturonic acid released
min 1 lg total protein 1. Reduction in viscosity was
measured according the method of Cooper and Wood
[31]. To assay the decrease in viscosity, 2 ml of a suitably
diluted 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 at
25 ± 0.01 C using a Technico (Ekkattuthangal, Chen-
nai, Tamil Nadu, India) viscometer (size 200), in which
the flow-through time for the standard volume of water
was 10 s [29]. One relative viscosity unit (RVU) was de-
fined as the enzyme quantity required to decrease the
initial viscosity by 50% per minute under the conditions
previously described.
2.4.2. Pectin lyase (PL)
Pectin lyase in the supernatant of the cultures was
determined spectrophotometrically (A235) according to
the method described by Albersheim [32]. The reaction
mixture 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 mixture
was incubated for 0, 10, 15, 20 and 30 min at 40 C. The
reaction was stopped by the addition of 4.5 ml of 0.01-N
HCl. One unit of enzyme activity (U) of pectin lyase was
defined as 1 lmol of unsaturated product min 1 [7].
2.4.3. Pectin methylesterase (PME)
For the determination of PME, yeasts were grown in
50 ml of YW medium containing 1% citrus pectin (85%
esterified) and incubated at 28 C in a rotary shaker at
120 rpm for 48 h. The cultures were then centrifuged
and the supernatant was collected for the PME assay.
Pectin methylesterase was assayed using two meth-
ods. One was by continuous titrimetric determination
of the carboxyl groups liberated from methylester bonds
[7]. PME activity was expressed as microequivalents of
polygalacturonic acid produced ml 1 h 1. PME was also
measured as the release of methanol from pectin chains
as 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. A
1-ll aliquot was then injected into a gas chromatograph
(Varian CG 3800 version 4.5, Palo Alto, CA), using a
Carbowax column, resulting in a retention time of
11 min 47 s.
861
2.4.4. Total protein
Total protein determination was performed by the
method 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 measuring
the increase of reducing sugars [7,30] using various incu-
bation times (15, 30, 45 and 60 min), and different pH of
the substrate (3.5, 4.5, 5.5 and 6.5) and incubation tem-
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peratures (30, 35, 40, 45 and 50 C). The results obtained
were 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 were
evaluated for their potential to produce and secrete pec-
tinase in, either, solid medium containing polygalactu-
ronic acid and glucose and/or galactose as carbon
source for polygalacturonase (PG) activity, solid med-
ium containing pectin for pectin lyase (PL) activity, or
liquid medium for pectinmethylesterase (PME) activity.
Among the 300 isolates, only 21 (7%) were positive for
polygalacturonase activity, and of these, seven were po-
sitive for pectin lyase activity (Table 1). PME was not
detected in any of these yeast cultures filtrates. The iso-
lates IC-50 and IC-54 secreted polygalacturonase only
when glucose was the source of carbon, while the isolate
SL-140 secreted the enzyme when galactose was the car-
bon source (Table 1). The yeast SL-140 did not grow in
MP5-glucose culture medium, but did grow in culture
medium when the source of carbon was substituted with
galactose. It has been reported that galactose was a bet-
ter source of carbon than glucose using strains of Sac-
charomyces cerevisiae which were genetically modified
to produce polygalacturonase [13]. The results obtained
in this study indicate that the yeast Debaryomyces hanse-
nii (SL-140) also secreted b-galactosidase (data not
shown).
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 also
capable of secreting pectin lyase in MP7 medium (pH
7.0) containing pectin (85% esterified). Although it has
been reported by several authors that yeasts secreted
mainly PG, Gainvors [34] also has detected pectin lyase
activity in the yeast Saccharomyces cerevisiae isolated
during wine fermentations.
A great diversity occurs among the yeast species asso-
ciated with fruits [35,36] or fruit pulp [22]. It is likely
862 E.G. da silva et al. / FEMS Yeast Research 5 (2005) 859–865

Table 1
Polygalacturonase (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 + + +

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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 either
polygalacturonic acid or pectin.
a
Schwan et al., 1997 [7].

that some strains developed pectinolytic activity to opti-
mise their growth [34]. Secretion of pectinolytic activity
by yeasts may be a means to increase survival when they
have to consume simpler sources of carbon [22], but
other authors [18,19] have reported that the function
of these enzymes in yeasts is unknown. Yeasts isolated
from cocoa secreted polygalacturonase even if they were
not capable to utilise pectin or galacturonic acid as a
sole carbon source [7]. It is possible that yeasts present
on some tropical fruits produce pectinases to degrade
pectin 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 these
sugars by the yeasts isolated was demonstrated in bio-
chemical tests utilised in the identification at the species
level. All 21 yeasts able to produce pectinase could uti-
lise at least one of these three sugars present in the
chains of pectin.
3.2. Yeast identification
The yeast strains that showed pectinolytic activity
were 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 species
were, 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 species
belonging to the genus Pichia. One third of the identified
pectinolytic yeasts were isolated from the surface of the
tropical fruit mangaba (H. speciosa). It is possible that
these results are due to the pectin concentration found
in this fruit, which is approximately 1.2%. In contrast,
the other fruits presented pectin contents varying from
0.2% to 0.7% (data not shown).
Some species of Pichia and Candida isolated from
tropical environments (soil, water, insect and plant
materials) also showed pectinolytic activity [37]. The
species identified in this work (Tables 1 and 2) occur
in other environments as well, such as flowers, cacti
and other fruits [22,24,35,38].
3.3. Production of pectinolytic enzymes by tropical yeasts
The quantity of enzyme secreted varied considerably
among the isolates (Table 2). Significant differences were
observed among the yeast isolates in the determination
of polygalacturonase activity secreted in liquid medium
using TukeyÕs test at the 5%-probability level. K. wick-
erhamii strain 185 showed the highest enzyme activity
with the conditions tested (viz. 24.0 lmol galacturonic
acid released min 1 lg protein 1). K. marxianus and
S. smithiae also showed high enzyme activities of 14.2
and 12.9 lmol of galacturonic acid released
min 1 lg protein 1, respectively. These values were,
however, approximately 50% of the enzyme activity se-
creted by K. wickerhamii strain 185.
 E.G. da silva et al. / FEMS Yeast Research 5 (2005) 859–865 863

Table 2
Extracellular 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 44.44
166 Mangaba Kluyveromyces marxianus 14.2c 5.0
168 Mangaba Stephanoascus smithiae 12.9c 0
162 Mangaba Pichia angusta 3.3b 33.33
CH-144A Cocoa Zygosacchoromyces fermentati 3.1b 3.80
36 Cajá Stephanoascus smithiae 1.7a b 11.11
IC-50 Bacuri Kluyveromyces wickerhamii 0.96a 0.095
IC-54 Mangaba Stephanoascus smithiae 0.87a 7.14
CH-142A Cocoa Candida krusei 0.66a 0
SL-125 Pseudo-lulo Pichia anomala 0.62a 100.0

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SL-140 Pseudo-lulo Debaryomyces hansenii 0.61a 5000.0
CH-156A Cocoa Pichia guilliermondii 0.53a 10.0
CH-146A Cocoa Zygosaccharomyces cidri 0.46a 6.46
147 Mangaba Stephanoascus smithiae 0.46a 3.50
FT-01 Cirigüela Stephanoascus smithiae 0.36a 5.13
FT-175 Mangaba Candida pseudoglaebosa 0.34a 11.11
53-CO Lulo Debaryomyces polymorphus 0.29a 4.40
FT-20 Umbu–cajá Debaryomyces hansenii 0.21a 6.46
FT-28 Umbu–cajá Pichia sp. 0.16a 0
FT-35 Cajá Candida intermedia 0.15a 0
IC-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 in
viscosity 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%).

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 demonstrate
any activity of this enzyme by the isolates tested. This
is in agreement with the observed rare production of
pectin lyase by yeasts [7,19].
3.4. Properties of polygalacturonase secreted by tropical
yeasts
PG activity was determined for those yeasts which were
considered as major pectinases producers, viz. K. wickerh-
amii strain 185 and K. marxianus strain 166. The enzyme
activity was determined after incubation times of 15, 30,
45 and 60 min, with pH values of 3.5, 4.5, 5.5 and 6.5
and incubation temperatures of 30, 35, 40, 45 and 50 C.
The highest PG activity, viz. 24.0 lmol of polygalactu-
ronic acid min 1 lg protein 1, occurred in K. wickerhamii
strain 185 after 15 min incubation, at pH 4.5 and at a tem-
perature of 35 C (Fig. 1(a)). For the isolate K. marxianus
strain 166, the highest activity was at pH 5.5 after 15 min
incubation at 35 C (Fig. 1(b)). PG secreted by these
yeasts showed an activity between pH 4.0 and 6.0, which
is 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 the
polymer and does not reduce viscosity significantly
[15,19]. Endo-PG activity is characterised by a substan-
tial decrease in viscosity (in general 50%), resulting from
the 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 of
50% [42]. The decrease in viscosity differed substantially
among the yeasts tested (Table 2 and Fig. 2). The results
showed that the enzyme secreted by D. hansenii strain
SL-140 produced the greatest decrease (viz. 50%) in vis-
cosity in 10 min (5000 RVU; Table 2), while only 3% of
the glycosidic linkages of the substrate were hydrolysed.
This reduction in viscosity was greater than the results
obtained by Schwan [7] for K. marxianus, which yielded
a 50% decrease in viscosity in 18 min. This decrease in
viscosity due to the enzyme secreted from D. hansenii
strain SL-140 was unexpected because only small
amounts of galacturonic acid were released (Table 2).
It is possible that the action of endo-polygalacturonase
liberates fragments of galacturonic acid, which may not
be detectable by the method of liberation of reducing
groups. P. anomala strain SL-125, K. wickerhamii strain
185 and C. intermedia strain FT 35 showed a 50% de-
crease in viscosity after a 25-35 min of reaction, which
represented 100, 44.44 and 33.33 relative viscosity units
(RVU), respectively (Table 2). The results suggested
the presence of a random mechanism of hydrolysis,
and the enzyme secreted by these yeasts is a poly
864 E.G. da silva et al. / FEMS Yeast Research 5 (2005) 859–865

(a) 30 of galacturonic acid min 1 lg of protein 1 (Table 2) as

measured by the release of reducing groups, but there
Polygalacturonase activity

25 was no decrease in viscosity of a 3.2% (w/v) polygalactu-

ronic acid solution after 120 min of incubation at 25 C.
20
This behaviour strongly suggests that the PG secreted by
15 this isolate acts by an exo-splitting mechanism [15]. The
other yeasts studied showed low polygalacturonase
10 activity as determined by both methods.
It has already been demonstrated that the yeast K.
5
marxianus has considerable economic advantages over
0 Aspergillus as a source of endo PG even without genetic
3.5 4.5 5.5 6.5 improvement of the strain [7,13,19,43]. The use of PG

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pH from yeasts could be highly advantageous for the wine,
cider and fruit juice industries and in the softening of
(b) 30
vegetables for the preparation of baby foods. Based
on the data obtained in the present study, it can be
Polygalacturonase activity

25 concluded that yeasts from tropical fruits have a great

potential for the production of pectinolytic enzymes,
20 which could be used in the food industry. More de-
tailed studies on the secretion of enzymes from yeasts
15
occurring on tropical fruits are needed to further pro-
10 vide support for their potential utilisation in the food
industry.
5

0
3.5 4.5 5.5
pH
Fig. 1. Effect of pH and temperature of incubation on PG activity
secreted by yeasts. PG activity was measured at different pH values and
different 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 of
galacturonic acid released min 1 lg total protein 1.
Acknowledgements
6.5
This work was supported by a European Union
INCO-DC Project Grant (IC18 CT97 0182) and by
CAPES (Coordenação de Aperfeiçoamento de Pessoal
de Nı́vel Superior). We thank the technical staff at
CEPLAC for isolation of yeasts from cocoa and Mrs.
Maria Aparecida Gomes Souza-Dias for her help in
the yeast identification.

60
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