Purification and properties of an extracellular esterase from a cold-adapted <Emphasis Type="Italic">Pseudomonas mandelii</Emphasis>

An extracellular esterase, EstK, was purified from the psychrotrophic bacterium Pseudomonas mandelii grown at 25°C. Prior to harvest, cells were treated with 0.2 M MgCl₂ to precipitate lipopolysaccharides in the outer membranes, which otherwise form aggregates with the secreted enzymes. EstK was purified to homogeneity using standard procedures. It had substrate specificity towards esters of short-chain fatty acids, particularly, p-nitrophenyl acetate. Optimum activity of EstK was at 40°C; at 4°C the activity was ~50% of its maximum. EstK has a unique substrate preference for p-nitrophenyl acetate and remains active at low temperatures.     

First description of the environmental niche of the epibenthic dinoflagellate species Coolia palmyrensis,C. malayensis,and C. tropicalis (Dinophyceae) from Eastern Australia

Environmental variables such as temperature, salinity, and irradiance are significant drivers of microalgal growth and distribution. Therefore, understanding how these variables influence fitness of potentially toxic microalgal species is particularly important. In this study, strains of the potentially harmful epibenthic dinoflagellate species Coolia palmyrensis, C. malayensis, and C. tropicalis were isolated from coastal shallow water habitats on the east coast of Australia and identified using the D1‐D3 region of the large subunit (LSU) ribosomal DNA (rDNA). To determine the environmental niche of each taxon, growth was measured across a gradient of temperature (15–30°C), salinity (20–38), and irradiance (10–200 μmol photons · m^?2 · s^?1). Specific growth rates of Coolia tropicalis were highest under warm temperatures (27°C), low salinities (ca. 23), and intermediate irradiance levels (150 μmol photons · m^?2 · s^?1), while C. malayensis showed the highest growth at moderate temperatures (24°C) and irradiance levels (150 μmol photons · m^?2 · s^?1) and growth rates were consistent across the range of salinity levels tested (20–38). Coolia palmyrensis had the highest growth rate of all species tested and favored moderate temperatures (24°C), oceanic salinity (35), and high irradiance (>200 μmol photons · m^?2 · s^?1). This is the first study to characterize the environmental niche of species from the benthic harmful algal bloom genus Coolia and provides important information to help define species distributions and inform risk management.     

Novel Use for the Osmolyte Trimethylamine N-oxide: Retaining the Psychrophilic Characters of Cold-Adapted Protease Deseasin MCP-01 and Simultaneously Improving its Thermostability

The low thermostability of cold-adapted enzymes is a main barrier for their application. A simple and reliable method to improve both the stability and the activity of cold-adapted enzymes is still rare. As a protein stabilizer, the effect of trimethylamine N-oxide (TMAO) on a cold-adapted enzyme or protein has not been reported. In this study, effects of TMAO on the structure, activity, and stability of a cold-adapted protease, deseasin MCP-01, were studied. Deseasin MCP-01 is a new type of subtilase from deep-sea psychrotolerant bacterium Pseudoalteromonas sp. SM9913. Fluorescence and CD spectra showed that TMAO did not perturb the structure of MCP-01 and therefore kept the conformational flexibility of MCP-01. One molar TMAO improved the activity of MCP-01 by 174% and its catalytic efficiency (k _cat /K _m) by 290% at 0°C. In the presence of 1 M TMAO, the thermostability (t _1/2) of MCP-01 increased by two- to fivefold at 60∼40°C. Structural analysis with CD showed that 1 M TMAO could keep the structural thermostability of MCP-01 close to that of its mesophilic counterpart subtilisin Carlsberg when incubated at 40°C for 1 h. Moreover, 1 M TMAO increased the melting temperature (T _m) of MCP-01 by 10.5°C. These results suggest that TMAO can be used as a perfect stabilizing agent to retain the psychrophilic characters of a cold-adapted enzyme and simultaneously improve its thermostability.     

The upper thermal tolerance for a Texas population of the hairy maggot blow fly Chrysomya rufifacies Macquart (Diptera: Calliphoridae)

1. The hairy maggot blow fly (Chrysomya rufifacies: Macquart) is an invasive necrophagous fly found throughout the continental United States. Chrysomya rufifacies is of medical/veterinary, forensic, and ecological importance due to its ability to cause myiasis, colonise human remains, and displace native Diptera. However, little is known about their upper thermal tolerance, which could be used to better predict their invasion potential.
2. We investigated the upper thermal tolerance of C. rufifacies exposed to different temperatures (20–45 °C), times (1–6 h), and nutrients (no food or water, water only, or a food-water mixture) for both sexes and two age ranges (young = 6–8 days post pupal emergence; old = 9–11 days post pupal emergence).
3. As temperature or duration increased, the probability of knockdown increased (0–100% at 20 and 45 °C and from 41 to 75% at 1 and 6 h), while the probability of survival decreased (99–2% at 20 and 45 °C and from 75 to 28% at 1 and 6 h). The availability of nutrients increased thermal tolerance at moderate temperatures (40 and 42 °C). Female flies were more thermally tolerant than males (probability of knockdown = 49% vs 58%; probability of survival = 58% vs 46%). Thermal tolerance did not differ by age.
4. These data reveal details about the upper thermal tolerance for a single population of C. rufifacies, and suggest that environmental and organismal factors ought to be considered in order to make meaningful predictions about the invasion potential of C. rufifacies in North America.

     

Temperature‐dependent development of the double‐spined spruce bark beetle Ips duplicatus (Sahlberg, 1836) (Coleoptera; Curculionidae)

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A cold-adapted endoglucanase from camel rumen with high catalytic activity at moderate and low temperatures: an anomaly of truly cold-adapted evolution in a mesophilic environment

Endoglucanases are important enzymes in plant biomass degradation. They have current and potential applications in various industrial sectors including human and animal food processing, textile, paper, and renewable biofuel production. It is assumed that the cold-active endoglucanases, with high catalytic rates in moderate and cold temperatures, can improve the cost-effectiveness of industrial processes by lowering the need for heating and, thus, energy consumption. In this study, the endoglucanase CelCM3 was procured from a camel rumen metagenome via gene cloning and expression in Escherichia coli BL21 (DE3). The maximum activity of the enzyme on carboxymethyl cellulose (CMC) was obtained at pH 5 and 30 °C with a V_max and K_m of 339 U/mg and 2.57 mg/ml, respectively. The enzyme with an estimated low melting temperature of 45 °C and about 50% activity at 4 °C was identified to be cold-adapted. A thermodynamic analysis corroborated that CelCM3 with an activation energy (E_a), enthalpy of activation (ΔH), and Gibb’s free energy (ΔG) of, respectively, 18.47 kJ mol⁻¹, 16.12 kJ mol⁻¹, and 56.09 kJ mol⁻¹ is a cold-active endoglucanase. In addition, CelCM3 was tolerant of metal ions, non-ionic detergents, urea, and organic solvents. Given these interesting characteristics, CelCM3 shows promise to meet the requirements of industrial applications.

     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Effects of temperature on the immature development and emergence of five species of <Emphasis Type="Italic">Trichogramma</Emphasis>

Five constant temperatures between 14 and 30°C were used to evaluate their effect on the development time and adult emergence of five Trichogramma species found parasitizing eggs of the velvetbean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) on soybeans in subtropical Southern Brazil. Host eggs were parasitized at 20°C and then transferred to the study temperatures to follow development and emergence of parasitoids. All five species were able to develop and emerge within the range of temperatures evaluated, and the effect of temperature on development rates could be described by linear regression. Trichogramma acacioi Brun, Moraes & Soares and T. rojasi Nagaraja & Nagarkatti were the most cold-tolerant species, with lower threshold temperatures of 8.1 ± 0.16°C and 9.2 ± 0.16°C, respectively. Trichogramma atopovirilia Oatman & Platner was the least cold-adapted species, with a lower threshold of 10.2 ± 0.13°C. Degree-day accumulation ranged from 153.8 DD for T. atopovirilia to 190.7 DD for T. acacioi. Adult emergence was higher than 90% for T. atopovirilia and T. pretiosum at all temperatures, whereas T. lasallei Pinto emergence dropped to 71.3% at 14°C and to 58.3% at 26°C, both significantly lower than the emergence of T. pretiosum and T. atopovirilia. Significantly less T. acacioi adults emerged at 30°C than either T. pretiosum or T. atopovirilia. The sex-ratio was not affected within the range of temperatures studied, and varied from 0.65 to 0.88 (female/(male + female)). Differences among Trichogramma spp. densities in the field can be attributed to slower development rates and/or reduced emergence of adults, both at low and high temperatures.

Temperature × light interaction and tolerance of high water temperature in the planktonic freshwater flagellates Cryptomonas (Cryptophyceae) and Dinobryon (Chrysophyceae)

Using microcosm experiments, we investigated the interactive effects of temperature and light on specific growth rates of three species each of the phytoplanktonic genera Cryptomonas and Dinobryon. Several species of these genera play important roles in the food web of lakes and seem to be sensitive to high water temperature. We measured growth rates at three to four photon flux densities ranging from 10 to 240 μmol photon · m^?2 · s^?1 and at 4–5 temperatures ranging from 10°C to 28°C. The temperature × light interaction was generally strong, species specific, and also genus specific. Five of the six species studied tolerated 25°C when light availability was high; however, low light reduced tolerance of high temperatures. Growth rates of all six species were unaffected by temperature in the 10°C–15°C range at light levels ≤50 μmol photon · m^?2 · s^?1. At high light, growth rates of Cryptomonas spp. increased with temperature until the temperature optimum was reached and then declined. The Dinobryon species were less sensitive than Cryptomonas spp. to photon flux densities of 40 μmol photon · m^?2 · s^?1 and 200 μmol photon · m^?2 · s^?1 over the entire temperature range but did not grow under a combination of very low light (10 μmol photon · m^?2 · s^?1) and high temperature (≥20°C). Among the three Cryptomonas species, cell volume declined with temperature and the maximum temperature tolerated was negatively related to cell size. Since Cryptomonas is important food for microzooplankton, these trends may affect the pelagic carbon flow if lake warming continues.     

A temperate pollinator with high thermal tolerance is still susceptible to heat events predicted under future climate change

1. Mutualisms may be particularly vulnerable to climate change as interacting species are likely to respond differently, which could destabilise interactions. 2. Temperate zone insects typically experience mean temperatures below their thermal optima, making them less vulnerable than tropical insects to small increases in mean temperature. However, they are likely to experience a higher frequency of extreme heat events, putting mutualism persistence in jeopardy. 3. This study investigated the potential impacts of climate change on Pleistodontes imperialis, a temperate Australian fig wasp that pollinates Port Jackson figs (Ficus rubiginosa). Wasp emergence and longevity were measured at temperatures ranging from those commonly experienced in nature (25 °C) to high values (> 40 °C) that are currently infrequent, but which are becoming more common with climate change. 4. Wasp emergence was unaffected by temperatures up to 39 °C, but it declined drastically above 39 °C. Adult longevity was unaffected by temperatures up to 30 °C, but decreased at 35 °C and above. Low humidity reduced wasp longevity across all temperatures. 5. Fitness reductions were observed at temperatures ～5 °C above the summer daily mean maximum, suggesting that P. imperialis has a high thermal tolerance, but is vulnerable to extreme heat. Figs located in the shade may provide protected microhabitats under hot conditions. 6. Tropical pollinators may be threatened by small increases in mean temperature. In contrast, it is shown here that temperate pollinators may face a different primary threat from climate change – the increasing frequency of extreme heat events – despite their higher thermal tolerances.     

Effect of temperature on immature development and longevity of two introduced opiine parasitoids on Bactrocera invadens

Temperature‐dependent development, parasitism and longevity of the braconid parasitoids, Fopius arisanus Sonan and Diachasmimorpha longicaudata Ashmed on Bactorcera invadens Drew Tsuruta & White, was evaluated across five constant temperatures (15, 20, 25, 30 and 35°C). Developmental rate decreased linearly with increasing temperature for both the parasitoid species. Linear and Brière‐2 nonlinear models were used to determine the lower temperature threshold at which the developmental rate (1/D) approached zero. For F. arisanus, lower thresholds to complete development estimated with the linear and nonlinear models were 10.1 and 6.9°C, respectively. The total degree‐days (DD) required to complete the development estimated by the linear model for F. arisanus was 360. In D. longicaudata, the linear and nonlinear models estimated lower thresholds of 10.4 and 7.3°C, respectively, and the total DD estimated was 282. In F. arisanus, percentage parasitism differed significantly across all temperatures tested and was highest at 25°C (71.1 ± 2.5) and lowest at 15°C (46.4 ± 1.4). Parasitoid progeny sex ratio was female biased at all temperatures except at 20°C. In D. longicaudata, percentage parasitism was highest at 20°C (52.2 ± 4.0) and lowest at 15°C (27.7 ± 2.5). Parasitoid progeny sex ratio was female biased and similar for all temperatures. Adult longevity of both parasitoids was shortest at 35°C and longest at 15°C, and females lived significantly longer than males at all temperatures tested. Our findings provide some guidance for future mass rearing and field releases of the two parasitoids for the management of B. invadens in Africa.     

Morphological and functional seed traits of the wild medicinal plant Dioscorea strydomiana,the most threatened yam in the world

• Morphological and functional seed traits have important roles in characterising the species regeneration niche and help to understand the reproductive biology of rare and threatened plants, which can thus support appropriate plant conservation measures.
• Seed morphometric and dispersal kinetics of the critically endangered Dioscorea strydomiana were measured and compared with those of four other Dioscorea species, and seed germination response under constant temperatures (5–35 °C) was compared with that of the congeneric and widespread D. sylvatica.
• Seed mass of D. strydomiana (ca. 14 mg) was twice that of D. sylvatica, but similar to or smaller than the other species examined. Seeds of D. strydomiana have the lowest speed of descent and lowest variability in most of the morphological traits considered, suggesting lower phenotypic plasticity but higher variance in the wing‐loading value. Seeds of D. strydomiana reached maximum germination at 15 °C (ca. 47%), which decreased slightly to ca. 37% at 25 °C and was completely inhibited at 35 °C. D. sylvatica seeds started to germinate at 10 °C (ca. 3%), reached 75–80% germination at 15–20 °C and maximum (ca. 90%) at 25–30 °C. Base temperatures for germination (T_b) were 9.3 and 5.7 °C, for D. strydomiana and D. sylvatica, respectively. Due to the higher germination percentages of D. sylvatica, ceiling and optimum temperatures could also be modelled for this species, suggesting higher sensitivity to high temperature for seeds of D. strydomiana.
• The detected poor seed lot quality of D. strydomiana suggests difficulties in reproduction from seed, highlighting the need for further investigation and conservation actions for this threatened yam species.

     

The green alga Dictyosphaerium chlorelloides biomass and polysaccharides production determined using cultivation in crossed gradients of temperature and light

The green microalga Dictyosphaerium chlorelloides was identified as promising microorganism for biotechnological production of exopolysaccharides (EPS). In stationary phase the culture suspension solidifies to thick gel, with very high viscosity and high content of EPS which may be interesting for many biotechnological applications. To develop cultivation protocol for maximum biomass/polysaccharide production, the optimum conditions for growth and polysaccharides production were determined in this study using the crossed gradient cultivation method. Temperature and irradiance requirements of Dictyosphaerium chlorelloides were evaluated by statistical analyses for growth rate/biomass, extracellular (EPS) and intracellular (IPS) polysaccharides contents in crossed gradients of temperature (4–45°C) and irradiance (2–18 W/m², 9.1 – 82.3 μmol/(m² s)). The maximum relative growth rate was observed at temperatures around 19.2°C and relatively low irradiances in range 2.6–11 W/m² (11.9–50.3 μmol/(m² s)). The maximum IPS production was observed at temperatures around 19.2°C and irradiance around 11 W/m² (50.3 μmol/(m² s)). The maximum production of EPS was observed at temperatures around 25.7°C and similar irradiances as IPS production. Due to temperature separation of growth and EPS production, development of cultivation protocol based controlled temperature manipulation is possible.     

Characterization of a recombinant cold-adapted purine nucleoside phosphorylase and its application in ribavirin bioconversion

Ribavirin is a broad-spectrum antiviral drug and can be produced by enzymatic synthesis by purine nucleoside phosphorylase (PNP). In this study, we describe the application of such a cold-adapted XmPNP in ribavirin bioconversion which showed approximately 15°C lower optimum temperature and 1.80-fold higher catalytic efficiency (k_cat/K_m) at 37°C within substrate inosine than homolog in E. coli. By contrast, E. coli (XmPNP) took only 12 h to reach maximum substrate conversion rate (70%) under its optimum temperature (50°C) by using recombinant strain cell as enzyme source, but E. coli (EcPNP) did at 24 h. These results suggest cold-adapted PNP is one attractive candidate for ribavirin bioconversion and other nucleoside medications to improve the catalytic efficiency.     

Temperature‐dependent developmental model of Neoseiulus californicus (McGregor) (Acari,Phytoseiidae)

The developmental time and survival of immature stages of Neoseiulus californicus were studied at nine constant temperatures (12, 16, 24, 24, 28 32, 36, 38 and 40°C), 60–70% RH, and a photoperiod of 16 : 8 (L : D) h. The total mortality of immature N. californicus was lowest at 24°C (4.5%) and highest at 38°C (15.2%). The total developmental time decreased with increasing temperature between 12°C (18.38 days) and 32°C (2.98 days), and increased beyond 32°C. The relationship between the developmental rate and temperature was fitted by five nonlinear developmental rate models (Logan 6, Lactin 1, 2 and Briere 1, 2). The nonlinear shape of temperature development was best described by the Lactin 1 model (r² = 0.98). The developmental variation of each stage was well described by the three‐parameter Weibull distribution model (r² = 0.91–0.93). The temperature‐dependent developmental models of N. californicus developed in this study could be used to determine optimal temperature conditions for its mass rearing, to predict its seasonal population dynamics in fruit tree orchards or greenhouse crops, or to develop a population dynamics model of N. californicus.     

Effects of temperature on life histories of three endangered Japanese diving beetle species

To elucidate population-increasing factors in the diving beetle Cybister tripunctatus lateralis (Fabricius) (Coleoptera: Dytiscidae) in Japan in recent years, life histories and oviposition patterns were compared among three endangered diving beetle species, Cybister brevis Aubé (qualified by the Japanese Red Data List as ‘near threatened’), Cybister chinensis Motschulsky (vulnerable), and C. tripunctatus lateralis (vulnerable). Oviposition in C. brevis, C. chinensis, and C. tripunctatus lateralis was observed from late April to mid-June, from late April to early July, and from late May to mid-August, respectively, under semi-outdoor conditions. There were no interspecies differences in total hatchling production during the reproductive season. In rearing experiments at various temperatures (20, 23, 25, 28, and 30 °C), the mortality of C. tripunctatus lateralis larvae was higher at 20 °C, and gradually lower with increasing temperature up to 30 °C. Adult body size of females in C. tripunctatus lateralis is larger than that of males but there were no significant differences among temperatures (25–30 °C). Cybister brevis had a higher emergence rate at 23–28 °C than at 20 and 30 °C. In C. brevis, the body size of adults reared at 25 or 28 °C was significantly larger than at other temperatures. Cybister chinensis did not differ in emergence rate and adult body size among the five temperature conditions. The developmental zero (i.e., the lower developmental threshold) from the first instar to adult emergence was 11.1 °C for C. brevis, 8.7 °C for C. chinensis, and 16.8 °C for C. tripunctatus lateralis. We speculate how the influence of global warming may have a positive impact on the growth and survival of C. tripunctatus lateralis.     

TEMPERATURE-GROWTH RESPONSES OF ALGAL ISOLATES FROM ANTARCTIC OASES1

Thirty-five taxa (128 clonal cultures) of Antarctic algae isolated from various habitats were assayed for growth over a range of 2–34°C. Isolates, all unialgal and two axenic, varied markedly in their temperature-growth responses. Only four taxa belonging to either the Chlamydomonadaceae or Ulotrichaceae were obligately cold-adapted and incapable of growth at ≥20°C. All isolates grew at temperatures ranging from 7.5 to 18°C, and a few were incapable of growth at ≤5°C. Over one-third of the isolates grew at 30°C, but none grew at 34°C. Percentages of cold-adapted clones correlated well with the more stable low temperature habitats. Four chlamydomonad isolates displayed optimum temperatures for growth near their maximum temperatures for growth, both temperatures being well above those of the native habitats. This temperature-growth response suggests a closer relationship to algae from more moderate thermal regions than one might have supposed. However, the ability to grow at low temperatures and the inability to grow at 34°C suggest that these Antarctic algae are cold temperature adapted. Growth capability at low in situ temperatures is considered more useful ecologically than physiologically-defined categories for algae based on their maximum temperature for growth.     

Biology of Doryctobracon brasiliensis at different temperatures: development of life table and determining thermal requirements

Doryctobracon brasiliensis (Szépligeti) is a parasitoid larval–pupal of fruit flies and has great potential to be used in biological control programmes as it feeds on other Anastrepha species in addition to Anastrepha fraterculus (Wiedemann). This study investigated the biology of D. brasiliensis at different temperatures to design a life fertility table and determine thermal requirements. The parasitoids were multiplied in larvae of A. fraterculus in air‐conditioned chambers at 15, 18, 20, 22, 25, 28 and 30°C, 70 ± 20% RH and photophase of 12 h. We determined the number of offspring, sex ratio, longevity of males and females and duration of egg–adult period. The temperature range 18–22°C ensures higher fecundity and at 20°C, and the average number of offspring per female was 152.77 parasitoids. The sex ratio of offspring produced was reduced with increasing temperatures. Longevity of males and females of D. brasiliensis was reduced by increasing temperatures. At 15, 28 and 30°C, there was no development of immature stages. For the temperature range 18–25°C, the duration of egg–adult period of D. brasiliensis was inversely proportional to temperature. At 20 and 22°C, we observed the highest values of net reproduction rate (Ro) and finite reason of increase (λ), meaning that at the estimated optimum temperature (21°C), the population of D. brasiliensis increased 47 times each generation. The lower temperature threshold for development was 10.01°C and the thermal constant (K) 303.21 degree/days. This information confirms that D. brasiliensis is better suited to temperate environments, which implies a significant potential for the use of D. brasiliensis in the control of A. fraterculus, because most areas occupied by this pest are in temperate regions. In addition, D. brasiliensis is useful in mass rearing systems in laboratory.     

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.