Pectinolytic enzymes secreted by yeasts from tropical fruits

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.     

Extracellular enzymatic activities of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina)

As part of a project aimed at the selection of cold-adapted yeasts expressing biotechnologically interesting features, the extracellular enzymatic activity (EEA) of basidiomycetous yeasts isolated from glacial and subglacial waters of northwest Patagonia (Argentina) was investigated. Ninety-one basidiomycetous yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Dioszegia, Mrakia, Rhodotorula, Rhodosporidium, Sporobolomyces, Sporidiobolus, Cystofilobasidium, and Udeniomyces) were screened for extracellular amylolytic, proteolytic, lipolytic, esterasic, pectinolytic, chitinolytic, and cellulolytic activities. Over 15% of the strains exhibited three or more different EEAs at 4 degrees C and more than 63% had at least two EEAs at the same temperature. No chitinolytic or cellulolytic activities were detected at 4 and 20 degrees C. Cell-free supernatants exhibited significantly higher (P < 0.01) protease and lipase activities at < or = 10 degrees C, or even at 4 degrees C. In light of these findings, cold environments of Patagonia (Argentina) may be considered a potential source of cold-adapted yeasts producing industrially relevant cold-active enzymes.     

Pectinolytic yeasts from cold environments: novel findings of Guehomyces pullulans,Cystofilobasidium infirmominiatum and Cryptococcus adeliensis producing pectinases

One hundred and three yeasts isolated from soil samples from King George Island and Tierra del Fuego province were screened in relation with their capability to produce pectinolytic enzymes. Although all the yeasts showed well-developed colonies at 20 °C, only eight showed a clear halo around the colony, indicative of pectin degradation. A secondary screening demonstrated that only four yeasts were capable to produce pectinases at low temperatures (8 °C). It could be seen that the selected yeasts were able to grow and produce high levels of polygalacturonase activity when submerged fermentations were performed using pectin-containing fruit wastes as substrates. None of the strains produced neither lyase nor rhamnogalacturonan hydrolase activities. Regarding pectin esterase activity, it was only produced in lower amounts by G. pullulans 8E (0.022 U ml⁻¹). A TLC analysis of the substrate cleavage pattern of the pectinolytic systems was consistent with an endo-type activity. The clarification of apple juice was only accomplished by G. pullulans pectinolytic system, with a clarification of 80% (%T₆₅₀) using 4 U/ml of enzyme at 20 °C. As far as we concern this work describes for the first time the production of pectinases by the cold-adapted yeasts species Cystofilobasidium infirmominiatum, Cryptococcus adeliensis and G. pullulans.

     

Pectinolytic activity secreted by yeasts isolated from fermented citrus molasses

AIMS: The aim of this study was to obtain improved strains of pectinolytic yeasts adapted to the conditions of an industrial fermentation process, which was continuously operated to convert citrus molasses into ethanol. METHODS AND RESULTS: The starter yeast of the industrial fermentation process was a commercial baker's yeast, which was capable of growing without forming any secretion halo of pectinase activity on solid medium. Nevertheless, isolates showing secretion of pectinolytic activity on plates were obtained from the fermentation process. The secretion of pectin-degrading activity by isolates on plates was repressed by galactose and improved as the result of colony aging on polygalacturonic acid plates at 30 degrees C. Liquefaction of polygalacturonate gels as well as the splitting of the pectin-degrading activity into a wall-linked and a supernatant fraction were also observed when the starter yeast was propagated under agitation in liquid medium containing pectin. CONCLUSIONS: Isolates capable of secreting pectinolytic activity on plates were predominant at the end of the citrus molasses fermentation. Nevertheless, the sizes of the secretion haloes on plates were not necessarily an indication of the levels of pectinolytic activity secreted in the liquid medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Improved pectinolytic strains of Saccharomyces can be used as a source of pectinases for a variety of applications. This organism also participates in plant deterioration processes.     

Yeasts from sub-Antarctic region: biodiversity,enzymatic activities and their potential as oleaginous microorganisms

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. Sixty-one yeasts strains were isolated from King George Island, Antarctica which were characterized physiologically and identified at the molecular level using the D1/D2 region of rDNA. Fifty-eight yeasts (belonging to the genera Cryptococcus, Leucosporidiella, Rhodotorula, Guehomyces, Candida, Metschnikowia and Debaryomyces) were screened for extracellular amylolytic, proteolytic, esterasic, pectinolytic, inulolytic xylanolytic and cellulolytic activities at low and moderate temperatures. Esterase activity was the most common enzymatic activity expressed by the yeast isolates regardless the assay temperature and inulinase was the second most common enzymatic activity. No cellulolytic activity was detected. One yeast identified as Guehomyces pullulans (8E) showed significant activity across six of seven enzymes types tested. Twenty-eight yeast isolates were classified as oleaginous, being the isolate 8E the strain that accumulated the highest levels of saponifiable lipids (42 %).     

Isolation and characterization of psychrophilic yeasts producing cold-adapted pectinolytic enzymes

AIMS: The present study was conducted to screen for psychrophilic yeasts that are able to degrade pectin compounds at low temperature, and to examine the cold-active pectinolytic enzymes produced by the isolated psychrophilic yeasts. METHODS AND RESULTS: Psychrophilic yeasts, which grow on pectin as a sole carbon source, pectinolytic-psychrophilic yeast (PPY) strains PPY-3, 4, 5 and 6, were isolated from soil from Abashiri (Hokkaido, Japan). The sequences of 28S rDNA D1/D2 of strains PPY-3 and 4 indicated a taxonomic affiliation to Cryptococcus cylindricus and Mrakia frigida, respectively, strains PPY-5 and 6 belonged to Cystofilobasidium capitatum. The isolated strains were able to grow on pectin at below 5 degrees C, and showed the activities of several cold-active pectinolytic enzymes. CONCLUSION: The findings of this study indicate the possibility that the isolated strains produce novel pectinolytic enzymes that are able to degrade pectin compounds at low temperature. Significance and Impact of the Study: It is possible that the cold-active pectinolytic enzymes from the isolated strains can be applied to the food industry, e.g. the clarification of fruit juice below 5 degrees C.     

Molds in Brined Cucumbers: Cause of Softening During Air-Purging of Fermentations

Softening of cucumbers in fermentations purged at high air-flow rates was caused by molds growing in the brined cucumbers, not in the brine. This conclusion is based on the following results: (i) no microorganisms were isolated in significant numbers from brines that caused softening of pasteurized brined cucumbers, (ii) no pectinolytic enzyme activities were produced in cucumber brines in the absence of cucumbers, (iii) the pickles in some air-purged fermentations became very soft without the appearance of any pectinolytic enzyme activity in the brine, (iv) mold hyphae were consistently observed in tissues of soft pickles, (v) molds consistently developed in cultures of slices of surface sterilized cucumbers taken from fermentations in which soft pickles were subsequently found, and (vi) molds belonging to the genera Alternaria, Fusarium, and Mucor isolated from slices all softened pasteurized brined cucumbers.     

Minimum inhibitory concentrations of various antifungal agents against Basidiomycetes clinical isolates]

The basidiomycete yeasts are often isolated from clinical samples. A minimal inhibiting concentrations (MIC) of ten antifungals of different groups--azols, allilamines, polyens etc.--against isolates of Rhodotorula, Cryptococcus [symbol: see text] Trichosporon yeasts genera were estimated. No one of these cultures was sensitive to azoles at concentrations 0-256 mcg/ml. A vitality of cultures after incubation during 10 days with antifungals was investigated. Miramistin was the most potent fungicidal agent against all cultures.     

Identification and characterization of antimicrobial activity in two yeast genera

A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera. Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue. Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited. No antibacterial activity was evident against four gram-negative bacteria used in this study. Optimal activities were found to be expressed after yeasts were grown at pH 6. A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated.     

Identification and characterization of antimicrobial activity in two yeast genera.

A general screening test for the expression of antibacterial activity was performed on over 400 cultures belonging to 31 yeast genera. Of these cultures, only two, Kluyveromyces thermotolerans and Kloeckera apiculata, were found to produce zones of inhibition of bacterial growth on Diagnostic Sensitivity Test Agar medium supplemented with 0.002% methylene blue. Of nine bacteria used as test organisms, only Lactobacillus plantarum and Bacillus megaterium were inhibited. No antibacterial activity was evident against four gram-negative bacteria used in this study. Optimal activities were found to be expressed after yeasts were grown at pH 6. A requirement for cultivation in the presence of methylene blue added to culture media for the expression of apparent antibacterial activity was demonstrated.     

A study of the polygalacturonase activity of several yeast strains isolated from cocoa

Summary During the traditional fermentation of cocoa, yeasts with pectinolytic activity are involved in the degradation of the pulp. Saccharomyces chevalieri, Torulopsis candila, and Candida norvegensis, as well as Kluyveromyces fragilis included in this study as a control strain all have a pectinolytic activity (endopolygalacturonase E.C. 3.2.1.15). The enzymes studied have the same optimal pH of activity [5] but are different from each other in their optimal temperature and their thermal stability. The enzymes of Torulopsis candida and Kluyveromyces fragilis have the highest optimal temperature (60° C). Among the strains studied, Candida norvegensis produced the greatest amount of exocellular enzyme.     

Stimulation of the production of extracellular pectinolytic activities of Aspergillus sp. by galacturonic acid and glucose addition

We studied the effect of the addition of galacturonic acid and of glucose on the production of the pectinolytic activities by Aspergillus sp. The results showed that a differential response in the production of the activities was produced. The production of the pectinolytic activity as measured by reducing groups was not negatively affected when low concentrations of eithr glucose or galacturonic acid were added to growing pectin containing cultures of Aspergillus sp. regardless of the time of addition. The production of the activity, measured by viscosimetry, was stimulated by addition of galacturonic acid at 24 h, suggesting that galacturonic acid participates in the induction of pectinolytic activities of Aspergillus sp. When the addition was made at t = 0, the increase in pH seems to affect the production of this activity. The addition of glucose at 24 h had no effect on the production of the viscosimetric activity; however, catabolic repression was observed upon the addition at t = 0. The production of both pectinolytic activities was strongly repressed when glucose was added at high concentration and was affected less when galacturonic acid was added at the same high concentration.     

Storage of Stock Cultures of Filamentous Fungi, Yeasts, and Some Aerobic Actinomycetes in Sterile Distilled Water

Castellani's procedure for maintaining cultures of filamentous fungi and yeasts in sterile distilled water was evaluated. Four hundred and seventeen isolates of 147 species belonging to 66 genera of filamentous fungi, yeasts, and aerobic actinomycetes were maintained in sterile distilled water at room temperature over periods ranging from 12 to 60 months in four independent experiments. Of the 417 cultures, 389 (93%) survived storage in sterile distilled water. The selection of good sporulating cultures and sufficient inoculum consisting of spores and hyphae suspended in sterile distilled water were the most important factors influencing survival in water over a longer period of time. The technique was found to be simple, inexpensive, and reliable.     

Yeasts associated with fresh and frozen pulps of Brazilian tropical fruits

The occurrence of yeasts on ripe fruits and frozen pulps of pitanga (Eugenia uniflora L), mangaba (Hancornia speciosa Gom.), umbu (Spondias tuberosa Avr. Cam.), and acerola (Malpighia glaba L) was verified. The incidence of proteolytic, pectinolytic, and mycocinogenic yeasts on these communities was also determined. A total of 480 colonies was isolated and grouped in 405 different strains. These corresponded to 42 ascomycetous and 28 basidiomycetous species. Candida sorbosivorans, Pseudozyma antarctica, C. spandovensis-like, C. spandovensis, Kloeckera apis, C. parapsilosis, Rhodotorula graminis, Kluyveromyces marxianus, Cryptococcus laurentii, Metchnikowia sp (isolated only from pitanga ripe fruits), Issatchenkia occidentalis and C. krusei (isolated only from mangaba frozen pulps), were the most frequent species. The yeast communities from pitanga ripe fruits exhibited the highest frequency of species, followed by communities from acerola ripe fruits and mangaba frozen pulps. Yeast communities from frozen pulp and ripe fruits of umbu had the lowest number of species. Except the yeasts from pitanga, yeast communities from frozen pulp exhibited higher number of yeasts than ripe fruit communities. Mycocinogenic yeasts were found in all of the substrates studied except in communities from umbu ripe fruits and pitanga frozen pulps. Most of the yeasts found to produce mycocins were basidiomycetes and included P. antarctica, Cryptococcus albidus, C. bhutanensis-like, R. graminis and R. mucilaginosa-like from pitanga ripe fruits as well as black yeasts from pitanga and acerola ripe fruits. The umbu frozen pulps community had the highest frequency of proteolytic species. Yeasts able to hydrolyse casein at pH 5.0 represented 38.5% of the species isolated. Thirty-seven percent of yeast isolates were able to hydrolyse casein at pH 7.0. Pectinolytic yeasts were found in all of the communities studied, excepted for those of umbu frozen pulps. The highest frequency of pectinolytic activity was found in mangaba frozen pulp communities. Around 30% of all isolates produced pectinases. The ability to split arbutin was observed in all communities ranging from 8% in yeasts from pitanga frozen pulps to 40.6% in acerola ripe fruit communities. Among 432 species tested, 125 were active for beta-glucosidase production, and Kloeckera apis, P. antarctica, C. sorbosivorans, and C. spandovensis-like were the most active species.     

Utilisation of differential killer toxin sensitivity patterns for fingerprinting and clustering yeast strains belonging to different genera

The differential killer sensitivity of 103 yeast cultures belonging to 12 species (genera Debaryomyces, Kluyveromyces, Saccharomyces, and Zygosaccharomyces), all previously taxonomically certified by nDNA-nDNA reassociation, against a given panel of 39 killer yeasts was used as a fingerprinting tool. All strains, with the only exception of eight cultures belonging to the species Zygosaccharomyces bailii, were characterised by a specific, individual sensitivity pattern (killer formula). Cluster analysis of binary sequences based on killer sensitivity of strains belonging to different genera is presented and discussed.     

Control of benzylamine oxidase activity in Kluyveromyces fragilis grown on primary amines as nitrogen source

Abstract After growth on a mixture of ammonium and either methylamine or n -butylamine as nitrogen sources, benzylamine oxidase activity in yeasts from a number of different genera was found to be repressed to a lesser extent by ammonium than was methylamine oxidase. Catalase activity was better repressed by ammonium with methylamine as the nitrogen source than with n -butylamine. During growth of Kluyveromyces fragilis on equimolar mixtures of ammonium and an amine as nitrogen sources, benzylamine oxidase synthesis began during the period of exclusive growth on ammonium, and a period of simultaneous use of both nitrogen sources was observed just before the ammonium was exhausted. Addition of ammonium to cultures growing on n -butylamine as nitrogen source had no immediate repressive effect on benzylamine oxidase or catalase synthesis. However, growth on limiting ammonium in the absence of amines did give rise to low levels of amine oxidase and derepression of catalase activity. It is concluded that benzylamine oxidase in yeasts is induced strongly by amines as well as being less strongly repressed by ammonium than methylamine oxidase.     

Distribution of aldoxime dehydratase in microorganisms

The distribution of phenylacetaldoxime-degrading and pyridine-3-aldoxime-degrading ability was examined with intact cells of 975 microorganisms, including 45 genera of bacteria, 11 genera of actinomyces, 22 genera of yeasts, and 37 genera of fungi, by monitoring the decrease of the aldoximes by high-pressure liquid chromatography. The abilities were found to be widely distributed in bacteria, actinomyces, fungi, and some yeasts: 98 and 107 strains degraded phenylacetaldoxime and pyridine-3-aldoxime, respectively. All of the active strains exhibited not only the aldoxime-dehydration activity to form nitrile but also nitrile-hydrolyzing activity. On the other hand, all of 19 nitrile-degrading microorganisms (13 species, 7 genera) were found to exhibit aldoxime dehydration activity. It is shown that aldoxime dehydratase and nitrile-hydrolyzing activities are widely distributed among 188 aldoxime and 19 nitrile degraders and that the enzymes were induced by aldoximes or nitriles.     

Distribution of Aldoxime Dehydratase in Microorganisms

The distribution of phenylacetaldoxime-degrading and pyridine-3-aldoxime-degrading ability was examined with intact cells of 975 microorganisms, including 45 genera of bacteria, 11 genera of actinomyces, 22 genera of yeasts, and 37 genera of fungi, by monitoring the decrease of the aldoximes by high-pressure liquid chromatography. The abilities were found to be widely distributed in bacteria, actinomyces, fungi, and some yeasts: 98 and 107 strains degraded phenylacetaldoxime and pyridine-3-aldoxime, respectively. All of the active strains exhibited not only the aldoxime-dehydration activity to form nitrile but also nitrile-hydrolyzing activity. On the other hand, all of 19 nitrile-degrading microorganisms (13 species, 7 genera) were found to exhibit aldoxime dehydration activity. It is shown that aldoxime dehydratase and nitrile-hydrolyzing activities are widely distributed among 188 aldoxime and 19 nitrile degraders and that the enzymes were induced by aldoximes or nitriles.     

pH dependence and thermostability of lipases from cultures from the ARS culture collection

Summary Previously we used a simple, sensitive agar plate method to screen lipase activity from 1229 selected cultures including 508 bacteria, 479 yeasts, 230 actinomycetes and 12 fungi covering many genera and species. About 25% of the cultures tested were lipase-positive. These lipase-positive strains were further classified as good, moderate or weak enzyme producers. We have expanded our screening method to focus specifically on the pH dependence and thermostability of these lipase activities. The lipases exhibited various pH sensitivities and were divided into three groups: (i) lipases which are active at pH 5.5 but not at pH 7.5—produced by 36 bacteria, 23 yeasts and four actinomycetes; (ii) lipases which are active at pH 7.5 but not at pH 5.5—produced by 17 bacteria, four yeasts, two actinomycetes and one fungus; and (iii) lipases which are active at both pH 5.5 and pH 7.5—produced by 112 bacteria, 90 yeasts, 15 actinomycetes and five fungi. By screening at 60°C and pH 9.0, we further identified 50 bacteria and 26 yeasts that produce thermostable alkali-tolerant lipases. Product analyses confirmed our screening results. Lipases with specific pH dependency and thermostability have potential to be developed into industrial enzymes.     

Psychrophilic yeasts in glacial environments of Alpine glaciers

The presence of psychrophilic yeasts in supra- and subglacial sediments, ice and meltwater collected from two glaciers of the Italian Alps (Forni and Sforzellina-Ortles-Cevedale group) was investigated. After incubation at 4 degrees C, subglacial sediments contained from 1.3 x 10(3) to 9.6 x 10(3) CFU of yeasts g(-1). The number of yeast cells in supraglacial sediments was c. 10-100-fold lower. A significant proportion of isolated yeasts exhibited one or more extracellular enzymatic activities (starch-degrading, lipolytic, esterolytic, proteolytic and pectinolytic activity) at 4 degrees C. Selected isolates were able to grow at 2 degrees C under laboratory-simulated in situ conditions. In all, 106 isolated yeasts were identified by MSP-PCR fingerprinting and 26S rRNA gene sequencing of the D1/D2 region as belonging to 10 species: Aureobasidium pullulans, Cryptococcus gilvescens (over 50% of the total), Cryptococcus terricolus, Mrakia gelida, Naganishia globosa, Rhodotorula glacialis, Rhodotorula psychrophenolica, Rhodotorula bacarum, Rhodotorula creatinivora and Rhodotorula laryngis. Four strains, all belonging to a new yeast species, yet to be described, were also isolated.