Pectinolytic enzymes secreted by yeasts from tropical fruits

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 Stephanoascus smithiae. The yeast Debaryomyces hansenii produced the greatest decrease in viscosity while only 3% of the glycosidic linkages 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 polygalacturonase activity.

Pectinases, Polygalacturonases, Pectin lyases, Tropical fruits, Kluyveromyces wickerhamii, Stephanoascus smithiae

1 Introduction

Pectin substances are complex structural polysaccharides of plant origin that contain a large proportion of partially methyl-esterified galacturonic acid subunits linked by α-1,4-glycosidic linkages [1]. These d-galacturonic 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 several 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) [1]. Several organisms are able to produce pectin-degrading enzymes, and these include plants [2], filamentous 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 importance in the beverage industry due to their ability to improve 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 production of wine, the extraction of olive oil and fermentation of tea, coffee and cocoa [1,11,12].

Pectinases used in the food industry are commercially produced by Aspergillus niger[13,14]. This fungal species produces various pectinases, including pectin methylesterase (PME), polygalacturonase (PG) and 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 regard to the production of pectinases, because they are unicellular, the growth is relatively simple, and the growth medium does not require an inducer. In addition, gene cloning and gene manipulation may improve enzyme production, thus suggesting that commercial enzyme 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 visitation 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, Lavras, 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 (Solanum pseudolulo), and umbu-cajá (Spondias tuberosa). Fresh fruit and fruit pulp from CEPLAC-BA, Fortaleza (EMBRAPA) and Corpoica-Rionegro (Colombia) 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 containing 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 μg of tetracycline or chloramphenicol 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 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 equivalent 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 continuously 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.

2.4.1 Polygalacturonase (PG)

Two methods were used to measure polygalacturonase activity in culture filtrates, namely by analyzing the release of reducing groups and the decrease in viscosity of the substrate. Polygalacturonase activity was assayed by measuring the increase in reducing groups derived from polygalacturonate using the method described by Miller [30]. One unit of enzyme activity was expressed as μmol of galacturonic acid released min⁻¹μg 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 recorded at short intervals measured in seconds at 25 ± 0.01 °C using a Technico (Ekkattuthangal, Chennai, 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 defined 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 μmol 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 methods. 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 filtered through a cellulose nitrate membrane of 0.45 μm, and afterwards 1 ml of internal standard (202 mg of toluene/100 ml ethanol) was added to 0.5 ml of sample. A 1-μl 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.

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 incubation times (15, 30, 45 and 60 min), and different pH of the substrate (3.5, 4.5, 5.5 and 6.5) and incubation temperatures (30, 35, 40, 45 and 50 °C). The results obtained were extrapolated using a standard curve for galacturonic 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 pectinase in, either, solid medium containing polygalacturonic acid and glucose and/or galactose as carbon source for polygalacturonase (PG) activity, solid medium 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 positive for pectin lyase activity (Table 1). PME was not detected in any of these yeast cultures filtrates. The isolates 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 carbon 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 better source of carbon than glucose using strains of Saccharomyces cerevisiae which were genetically modified to produce polygalacturonase [13]. The results obtained in this study indicate that the yeast Debaryomyces hansenii (SL-140) also secreted β-galactosidase (data not shown).

1

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

Strain	Yeast isolates	MP5-glucosea (PG)	MP5-galactoa (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 either polygalacturonic acid or pectin.

a

Schwan et al., 1997 [7].

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 associated with fruits [35,36] or fruit pulp [22]. It is likely that some strains developed pectinolytic activity to optimise 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 sugars occurring on the side chains of pectin such as rhamnose, galactose and arabinose. The utilisation of these sugars by the yeasts isolated was demonstrated in biochemical tests utilised in the identification at the species level. All 21 yeasts able to produce pectinase could utilise 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, comprising thirteen species of yeasts, were found capable to secrete 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. angusta (1), P. anomala (1), P. guilliermondii (2), Zygosacchoromyces 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).

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
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 μmol of galacturonic acid released min⁻¹μg 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%).

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 Tukey's test at the 5%-probability level. K. wickerhamii strain 185 showed the highest enzyme activity with the conditions tested (viz. 24.0 μmol galacturonic acid released min⁻¹μg protein⁻¹). K. marxianus and S. smithiae also showed high enzyme activities of 14.2 and 12.9 μmol of galacturonic acid released min⁻¹μg protein⁻¹, respectively. These values were, however, approximately 50% of the enzyme activity secreted by K. wickerhamii strain 185.

The assay to determine pectin lyase activity in the culture 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. wickerhamii 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 μmol of polygalacturonic acid min⁻¹μg protein⁻¹, occurred in K. wickerhamii strain 185 after 15 min incubation, at pH 4.5 and at a temperature 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].

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: (♦) 30 °C, (◻) 35 °C, (●) 40 °C, (*) 45 °C. (a) Kluyveromyces wickerhamii (strain185), (b) Kluyveromyces marxianus (strain 166). PG activity was defined as μmol of galacturonic acid released min⁻¹μg total protein⁻¹.

3.5 Mechanism of enzyme action

Determination of polygalacturonase activity by the level 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 substantial 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 linkage 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 viscosity 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% decrease 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 α-1,4-d-galacturonide glycanohydrolase (EC 3.21.15) or an endo-PG. The yeast S. smithiae strain 168 showed a relatively high polygalacturonase activity of 12.9 μmol of galacturonic acid min⁻¹μg of protein⁻¹ (Table 2) as measured by the release of reducing groups, but there was no decrease in viscosity of a 3.2% (w/v) polygalacturonic acid solution after 120 min of incubation at 25 °C. This behaviour strongly suggests that the PG secreted by this isolate acts by an exo-splitting mechanism [15]. The other yeasts studied showed low polygalacturonase activity as determined by both methods.

2

Decrease in viscosity produced by endo-PG secreted from (○) Kluyveromyces wickerhamii (strain185), (△) Debaryomyces hansenii (strain SL-140) and (.×.) Pichia anomala (strain SL-125).

It has already been demonstrated that the yeast K. marxianus has considerable economic advantages over Aspergillus as a source of endo PG even without genetic improvement of the strain [7,13,19,43]. The use of PG from yeasts could be highly advantageous for the wine, cider and fruit juice industries and in the softening of vegetables for the preparation of baby foods. Based on the data obtained in the present study, it can be concluded that yeasts from tropical fruits have a great potential for the production of pectinolytic enzymes, which could be used in the food industry. More detailed studies on the secretion of enzymes from yeasts occurring on tropical fruits are needed to further provide support for their potential utilisation in the food industry.

Acknowledgements

This work was supported by a European Union INCO-DC Project Grant (IC18 CT97 0182) and by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível 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.