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Keywords:

  • cold-active enzyme;
  • Mrakia frigida;
  • pectate lyase;
  • pectinase;
  • psychrophilic yeast

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  This study was conducted to determine optimal conditions for pectate lyase (PL) production by two psychrophilic yeast strains and to compare the properties of the cold-active enzymes using mesophilic PL as reference enzyme.

Methods and Results:  Two psychrophilic yeasts isolated from remote geographical locations (European Alps, north Siberia) produced extracellular cold-active PL. Both strains were identified as Mrakia frigida by analysis of ITS and large subunit (LSU) rRNA sequences. Maximum enzyme production occurred at a cultivation temperature of 1 or 5°C. The apparent optimum for enzyme activity was observed at 30°C and pH 8·5–9. The enzymes were thermolabile, but were resistant to repeated freezing and thawing.

Conclusion:  We describe for the first time alkaline PL-producing representatives of the yeast species M. frigida. The two strains produce cold-active PL with similar properties, but have a different enzyme production pattern.

Significance and Impact of the Study:  The enzymes described in this study could be useful for a wide range of applications, such as low-temperature pretreatment of wastewater containing pectic substances.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

During the past decade it has been recognized that cold-adapted micro-organisms provide a large biotechnological potential, offering numerous economical and ecological advantages over the use of organisms and their enzymes which operate at higher temperatures (Ohgiya et al. 1999; Margesin et al. 2002a). Cold-active enzymes are characterized by high catalytic efficiency at low and moderate temperatures at which homologous mesophilic enzymes are less or not active, and are thermolabile. These properties are of interest for both basic research and industrial application. A wide variety of cold-adapted bacteria producing cold-active enzymes have been described. Comparatively little is known about cold-adapted yeasts, although they represent a promising source for biodegradation processes and enzyme production (Birgisson et al. 2003; Margesin et al. 2003; Pazgier et al. 2003; Nakagawa et al. 2004).

Pectic substances are ubiquitous in the plant kingdom. Enzymes attacking these substances are broadly known as pectinases. Depending on their mode of action, they are classified into pectinesterases, which de-esterify pectin by removal of methoxy residues, and depolymerases, which cleave the main chain. The depolymerizing enzymes include polygalacturonases (hydrolases) and lyases, which cleave the glycosidic bonds by β-elimination (Alkorta et al. 1998). Alkaline pectinases, such as pectate lyases (PL), are among the most important industrial enzymes and are of great significance in biotechnology for various applications, such as textile processing, raw cotton bioscouring, degumming of plant bast fibres, and pretreatment of wastewater containing pectic substances from the fruit and vegetable processing industry (Hoondal et al. 2002). The use of bacterial pectinases, which selectively remove pectic substances from the wastewater and thus facilitate decomposition by activated sludge treatment, represents a cost-effective and environmentally friendly alternative treatment method. An extracellular alkaline PL from Bacillus sp. was used effectively to remove pectic substances from industrial wastewater (Tanabe et al. 1987). Enzymes that are active at low temperatures are especially desirable for such degradation processes and would enable low energy treatment. Cold-adapted PL producers described so far are predominantly bacterial representatives of a number of genera (Magro et al. 1994; Laurent et al. 2000; Truong et al. 2001), whereas yeasts are mainly known for their ability to produce acidic pectinases (Blanco et al. 1999; Birgisson et al. 2003; Nakagawa et al. 2004).

In this study, we compared two PL-producing psychrophilic yeast strains that originated from remote geographical locations. We determined the optimal conditions for growth and enzyme production and compared the properties of the enzymes using mesophilic PL as reference enzyme.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation and identification of strains

Two yeast strains were selected on the basis of their ability to produce PL at 10°C (Margesin et al. 2003). Strain A15 was isolated from Alpine glacier cryoconite as described (Margesin et al. 2002b). Strain AG25 originated from a sediment sample containing mud, spring water and moss, which was collected in the Gyda peninsula in north Siberia (Gounot 2001). Species identification was determined by sequencing of the internal transcribed spacer (ITS) region and the 5′-end of the large subunit (LSU) rRNA gene including the variable domain D1 and D2. For DNA isolation, cells were harvested from 5-day-old subcultures and lyophilized. DNA was isolated by the CTAB method (O'Donnell et al. 1997). Primers used for the two distinct PCRs and sequencing of the two fragments were ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′), IT2 (5′-CCTCCGCTTATTGATATGCTTAAG-3′), F63 (5′-GCATATCAATAAGCGGAGGAAAAG-3′) and LR3 (5′-TCCTCCGCTTATTGATATGC-3′).

Conditions for the two PCR reactions were identical. DNA was amplified through 35 cycles as follows: 30 s at 92°C, 30 s at 52°C, 1 min at 72°C. DNA sequencing was performed with primers ITS5, and IT2 using the Beckman-Coulter TM CEQ Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Inc., Fullerton, CA, USA). Identification was performed by comparison with sequences published before 1 November 2004. Sequences were deposited in the GenBank-EMBL with the accession numbers AJ866976 and AJ866979.

Effect of cultivation parameters on growth and enzyme production

Experiments were carried out in triplicate. Growth (OD 600 nm) and PL activity in the cell-free supernatant obtained by centrifugation were monitored at regular intervals over 10–14 d. To determine the effect of temperature on growth and PL production, the yeast strains were cultivated in R2A medium (pH 7) supplemented with 0·4% (w/v) polygalacturonic acid (PGA) at 180 rev min−1 and at temperatures ranging from 1 to 20°C. Bacillus subtilis was used as a PL-producing mesophilic reference strain and was cultivated in the same medium at 10–30°C. To determine the effect of pH, the yeast strains were cultivated at 5°C in R2A-PGA medium with the pH adjusted to 7 or 9 using 0·1 mol l−1 potassium phosphate or Tris-HCl buffer respectively. To evaluate the effect of aeration, cultivation was carried out at 5°C on a rotary shaker at 180 and 350 rev min−1, as well as in Erlenmeyer flasks with and without baffles at 180 rev min−1. To determine the effect of the media composition, cultivation occurred at 180 rev min−1 and 5°C in the following pH-neutral media supplemented with 0·4% (w/v) PGA: R2A, nutrient broth (NB), mineral salts medium (Margesin and Schinner 1998) containing yeast extract (10 mg l−1 and 1 g l−1).

Assays for PL activity

The screening for PL activity was performed on R2A agar plates containing PGA. After 4 d of growth at 10°C, the plates were flooded with 1 mol l−1 CaCl2. Distinct cloudy halos appeared around enzyme-producing colonies. PL activity was determined by monitoring the formation of C4 and C5 unsaturated reaction products spectrophotometrically at 235 nm using a modification of the methods described by Collmer et al. (1988) and Laurent et al. (2000). 800 μl of 60 mmol l−1 Tris-HCl buffer (pH 9) containing 0·6 mmol l−1 CaCl2 were mixed with 100 μl of 0·1% PGA (w/v, dissolved in buffer) and 100 μl of enzyme-containing supernatant. The reaction mixture was incubated for 20 min at 25°C. One unit was defined as the amount of enzyme which formed 1 μmol of 4,5-unsaturated product per min under the assay conditions. Relative activity was expressed in μmol of unsaturated product liberated per min per ml of supernatant.

Enzyme characterization

The culture supernatants from the two yeast strains and B. subtilis (grown in R2A-PGA medium, pH 7) were concentrated and desalted at 4°C by means of ultrafiltration using Centricon-10 (Amicon, Grace Co.; Danvers, MA, USA) with a 10 000-MW cutoff in order to remove medium components. These partially purifed enzyme solutions were stored at −20°C.

Characterization was performed with three replicates using the standard PL assay. To determine the optimum temperature for enzyme activity, the reaction was carried out at various temperatures (0–80°C) and at pH 9. For determination of the effect of temperature on enzyme stability, the enzymes were incubated for 15 min at pH 9 and 0–70°C. After cooling on ice, the residual activity was determined. The effect of freezing (−20°C) and thawing on enzyme stability was monitored after repeated freeze–thaw cycles. The effect of pH on enzyme activity was tested by using 60 mmol l−1 Tris-HCl (pH 7–10) or 60 mmol l−1 glycine-NaOH (pH 11) buffer. All buffers contained 0·6 mmol l−1 CaCl2. To test the effect of pH on enzyme stability, enzyme solutions were incubated with 60 mmol l−1 Tris-HCl buffer (pH 7–10) for 1 h at 20°C. Afterwards, the residual activity was determined. To check the effect of inhibitors and other reagents on enzyme activity, the enzymes were incubated with each of the tested reagents at 20°C for 30 min, and the residual activity was determined.

Electrophoresis

SDS-PAGE using 1 mm thick 12% Tris-HCl gels (Ready Gel; Bio-Rad, Munich, Germany) was carried out under constant voltage (200 V) in a Mini-Protean II electrophoresis cell (Bio-Rad), using 25 mmol l−1 Tris/192 mmol l−1 glycine/0·1% SDS (pH 8·3) as the running buffer. Enzyme solution was mixed with sample buffer, boiled for 5 min and cooled on ice, and applied to the gel. To determine molecular weight, SDS-PAGE molecular weight standards (Bio-Rad) were used. The gel was stained and destained as recommended by the manufacturer.

Isoelectric focusing (IEF) was performed as described (Margesin and Schinner 1991) on thin-layer polyacrylamide gel plates (pH 3·5–9·5; Amersham Biosciences Europe) at 8°C, 30 W, 1500 V, 50 mA with 585 Vh. A standard mixture of proteins (pH 3·5–9·3; Amersham Biosciences Europe) was used to indicate the pH gradient in the gel. Gels were fixed and stained as recommended by the manufacturer.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Identification of yeast strains

ITS sequences obtained from both strains were highly homologous (more than 99% identity for 612 positions aligned) to the sequences published for Mrakia frigida and M. nivalis (Diaz and Fell 2000). These two species names are considered synonyms and M. frigida takes precedence. Analysis of the partial LSU sequences confirmed this result with 99·7% identity for 601 positions aligned between the sequences of strain A15 and strain AG25 and more than 98% identity between these two sequences and the sequences published by Fell et al. (1999) for M. frigida. The psychrophilic nature of the two strains (growth at 1–20°C, no growth at 25°C) corresponds to the description of the species (Barnett et al. 2000).

Growth and PL production

Effect of temperature.  Temperature had a significant effect on growth and PL production of the strains tested (Fig. 1). Both M. frigida strains grew well at 1°C, had an optimum growth temperature on PGA (in terms of growth rate) of 15°C, and did not grow at 25°C. Notably, both strains showed comparable cell densities when cultivated at temperatures ranging from 1–15°C after 10 d of incubation. B. subtilis exhibited the properties of a mesophile showing maximum growth at 30°C and no growth at 55 and 10°C.

image

Figure 1. Effect of temperature on growth (top) and PL production (bottom) by the Alpine Mrakia frigida strain A15 (•), the Siberian M. frigida strain AG25 (bsl00066), and mesophilic Bacillus subtilis (bsl00000)

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PL was detected in the culture supernatants in the late logarithmic growth phase; enzyme production was highest in the early stationary growth phase. The two M. frigida strains showed a different enzyme production pattern. The Alpine strain A15 produced high amounts of PL only at a cultivation temperature of 1–10°C with a maximum of enzyme production at 5°C, despite high cell densities at a temperature range of 1–20°C. The Siberian counterpart AG25 produced PL over the whole growth temperature range (1–20°C); enzyme production was highest at 1°C and decreased with increasing cultivation temperatures. For both strains, enzyme production was more sensitive to increasing temperatures than growth. The mesophilic reference strain showed maximum PL production when grown at 25 and 30°C (Fig. 1) and produced a substantially (at least 10-fold) lower amount of PL than the yeasts. Enzyme production by the Siberian strain was approximately twice as high than production by the Alpine strain.

Effect of pH.  Both yeast strains preferred the pH-neutral over the alkaline R2A-PGA medium for growth as well as for production of the alkaline PL. Only 11% (Alpine M. frigida strain A15) or 12% (Siberian M. frigida strain AG25) of the cell yield produced in the pH neutral medium were detected when the alkaline medium was used. Similarly, the enzyme yield obtained in pH-neutral medium was reduced to 5% (Alpine yeast) or 24% (Siberian yeast) under alkaline conditions.

Effect of medium composition.  The strains were cultivated in various media at 5°C, and growth and enzyme yield were monitored over 10 d. The highest cell and enzyme yields were obtained using complex media (R2A-PGA and NB-PGA). Growth and enzyme production were significantly reduced in PGA-containing mineral medium. In the presence of a high amount of yeast extract (1 g l−1), growth was reduced by c. 25% compared with complex media, but PL production was reduced by two-thirds or more. A low amount of yeast extract (10 mg l−1) reduced growth and enzyme production of both strains substantially (Table 1).

Table 1.  Effect of media composition on relative growth (cell yield) and PL production (enzyme yield) by two psychrophilic Mrakia frigida strains after 10 d of cultivation at 5°C
MediumM. frigida A15M. frigida AG25
Cell yield (%)Enzyme yield (%)Cell yield (%)Enzyme yield (%)
  1. MM-PGA I, mineral salts medium with PGA and high amounts of yeast extract (1 g l−1); MM-PGA II, mineral salts medium with PGA and low amounts of yeast extract (10 mg l−1).

R2A-PGA100 ± 295 ± 14100 ± 278 ± 5
NB-PGA99 ± 1100 ± 992 ± 2100 ± 3
MM-PGA I78 ± 1023 ± 1174 ± 437 ± 5
MM-PGA II33 ± 22 ± 127 ± 312 ± 2

Effect of aeration.  Growth of both M. frigida strains was neither significantly affected by the agitation rate (180 and 350 rev min−1) nor by the type of cultivation flasks (with or without baffles). PL production, however, was highest when cultivation was done in flasks without baffles at 180 rev min−1. Agitation at 350 rev min−1 resulted in significantly reduced enzyme production. A similar result was obtained when using cultivation at 180 rev min−1 in baffled flasks (Fig. 2). This indicates the sensitivity of the PL towards high amounts of dissolved oxygen and shear stress.

image

Figure 2. Effect of agitation rate (bsl00001 180 rev min−1; • 350 rev min−1) and type of cultivation flasks (dashed line, with baffles; continuous line, without baffles) on PL production at 5°C by the Alpine Mrakia frigida strain A15

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Characterization of enzymes

Molecular weight and isoelectric point.  On SDS-PAGE, only protein bands with molecular masses of 37·5 ± 1·3 kDa (Alpine M. frigida), 38·0 ± 2·2 kDa (Siberian M. frigida) and 38·5 ± 0·5 kDa (B. subtilis) were detected and attributed to the partially purified PL. All three enzymes were characterized by several (at least five) closely focused bands of proteins within a pI range of 9·0 and >9·5, possibly revealing the presence of isoenzymes or different glycosylation levels of the enzymes. The pI was higher than that of the alkaline reference enzyme subtilisin Carlsberg (pI 8·6; Sigma).

Effect of temperature on activity and stability.  The apparent optimum temperature for activity of the two M. frigida PL was clearly at 30°C (Fig. 3). Both enzymes showed a similar activity pattern in the temperature range 0–30°C, being active at 0°C (16–21% of the maximum activity) and displaying already 76–68% of the maximum activity at 20°C. At temperatures above the apparent optimum for activity, the PL from the Siberian strain lost rapidly its activity. Total inactivation occurred at 50°C. The mesophilic PL had a significantly higher apparent optimum temperature for activity (60°C) and was active at temperatures at which the cold-active enzymes were inactivated (≥50°C). At 20 and 30°C, the cold-active enzymes showed a sevenfold and fourfold higher relative activity, respectively, than the mesophilic PL (Fig. 3). The activation energy of the enzyme-catalysed reactions was determined from the Arrhenius plot and revealed lower values for the cold-active yeast PL (28·7 and 35·6 kJ mol−1 for the Alpine and Siberian strain respectively) than for the mesophilic counterpart (46·0 kJ mol−1). The calculation of thermodynamic constants showed that the cold-active PL were also characterized by lower values of free energy (ΔG* = 55·6 and 54·3 kJ mol−1), enthalpy (ΔH* = 26·3 and 33·3 kJ mol−1), and entropy of activation (ΔS* = −100·0 and −72·2 J mol−1K−1) than the mesophilic enzyme (ΔG* = 60·3 kJ mol−1; ΔH* = 43·6 kJ mol−1; ΔS* = −57·1 J mol−1 K−1).

image

Figure 3. Effect of temperature (top) and pH (bottom) on activity of the PL produced by the Alpine Mrakia frigida strain A15 (•), the Siberian M. frigida strain AG25 (bsl00066), and mesophilic Bacillus subtilis (bsl00000)

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Concerning the effect of temperature on enzyme stability (Fig. 4), the cold-active enzymes lost already activity when incubated for 15 min at temperatures higher than 10°C. Only two-thirds of the initial activity were retained at 30°C. The mesophilic counterpart lost c. 50% of activity only after 15 min at 70°C. Activity of the cold-active PL was not affected after repeated (up to fourfold) freezing and thawing. Incubation for 24 h at 2°C did not affect enzyme activity, however, after 48 h at 2°C between 5% (Siberian M. frigida PL) and 21% (Alpine M. frigida PL) of the initial activity were lost.

image

Figure 4. Effect of temperature on stability of the PL produced by the Alpine Mrakia frigida strain A15 (•), the Siberian M. frigida strain AG25 (bsl00066), and mesophilic Bacillus subtilis (bsl00000)

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Effect of pH on activity and stability.  The mesophilic B. subtilis PL was active over a broad pH range with an apparent optimum at pH 7·5–8 and high activity in the alkaline pH range (Fig. 3). This enzyme retained full activity after an incubation of 1 h at pH 7–10. The cold-active enzymes showed a different pattern. Their maximum activity was distinctly at pH 9 (Alpine M. frigida) or 8·5 (Siberian M. frigida), and they were active only in a narrow pH range (Fig. 3). Both PL lost a considerable amount of activity (34–40%) when incubated for 1 h at pH 9–10.

Effect of inhibitors.  Susceptibility to the metal chelating agent EDTA indicated that the enzymes required metal ions for activity. 1 mmol l−1 EDTA affected the B. subtilis PL only to a small extent (12 ± 2% activity loss) but resulted in a strong inhibition of the M. frigida PL. These enzymes showed only 33 ± 5% (Alpine) or 15 ± 3% (Siberian) of residual activity. All three enzymes were almost completely inactived in the presence of 10 mmol l−1 EDTA. The absence of calcium ions resulted in an almost complete loss of activity of the two cold-active PL, whereas the B. subtilis enzyme still retained 71 ± 5% of its maximal activity. The anionic detergent SDS (1%) had a strong inhibiting effect on all three enzymes. Enzyme activity was also significantly affected in the presence of the oxidizing agent KMnO4 (10 mmol l−1) and the reducing agent l-cystein (10 mmol l−1).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this study, we characterized two psychrophilic yeast strains originating from remote geographical locations with regard to their taxonomy, growth, enzyme (PL) production and enzyme properties. The natural habitats of the two strains contain high numbers of psychrophilic yeasts (Gounot 2001; Margesin et al. 2002b). Psychrophiles predominate under permanently cold temperature conditions. In such a niche, they can compete successfully with cold-tolerant micro-organisms. Both yeast strains investigated in this study were assigned to the psychrophilic species M. frigida. Sexual reproduction occurs by teliospores, which may play a role in cold adaptation. M. frigida was previously isolated from deadwood or insect frass (Barnett et al. 2000), soil (Nakagawa et al. 2004) and frozen food (Moreira et al. 2001). Recently, cold-active acidic pectin-degrading enzymes produced by M. frigida and other psychrophilic yeasts were characterized (Nakagawa et al. 2004); however, PL activity was not detected. In this study, we thus report for the first time alkaline PL-producing representatives of this species.

In the second part of our study, we compared the optimal conditions for growth and PL production by the two M. frigida strains. An optimum growth temperature of 15°C on pectic substances was found in this study, and was also described by Birgisson et al. (2003) and Nakagawa et al. (2004). Interestingly, the two M. frigida strains investigated in our study had almost identical growth characteristics and requirements, but a completely different enzyme production pattern. This might be related to the natural temperature conditions, but could also be a strain-specific feature. While the Siberian M. frigida strain AG25 produced PL over the whole growth temperature range, enzyme production by the Alpine counterpart was optimal at 1–5°C, very low at 15°C, and absent at 20°C. This strain was isolated from glacier cryoconite where permanently cold conditions never exceeding +3°C prevail in summer, because the material is permanently saturated with cold meltwater from ablating snow patches. The Siberian strain originated from an Arctic sediment sample that might be subjected to more fluctuating conditions, which would explain enzyme production over the whole growth temperature range.

Thermal inhibition of extracellular enzyme production is a common feature of cold-adapted micro-organisms. The optimum temperature for substrate utilization is usually below the optimum growth temperature (Margesin et al. 2002a). However, optimal enzyme production at 1–5°C, such as observed with the M. frigida strains in this study, has been rarely described (Huston et al. 2000; Pazgier et al. 2003) and reflects the permanently cold natural habitat of the strains.

In the third part of our study, we compared the properties of the PL produced by the two psychrophilic M. frigida strains and the mesophilic reference enzyme. Both yeast enzymes displayed the typical features of cold-active enzymes, i.e. thermolability, maximum activity at 30°C (higher by 30°C than the mesophilic homologue) and high activity at 0–20°C, low values for activation energy and thermodynamic constants. Bacterial cold-active PL also displayed maximum activity at 30–35°C (Magro et al. 1994; Truong et al. 2001). Other properties of the PL investigated in this study, such as optimal activity in the alkaline pH range, the requirement of calcium ions for activity, molecular weight and isoelectric point, demonstrate that the cold-active enzymes produced by the two M. frigida strains had the typical characteristics of PL (Laurent et al. 2000; Soriano et al. 2000; Kobayashi et al. 2003). Considering the large biotechnological potential of cold-active enzymes, enzymes producers such as those described in this study represent a promising source, especially for the use in permanently cold areas.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The authors thank A.M. Gounot for kindly supplying strain AG25. P.A. Fonteyne is supported by the BCCM-IHEM, a programme of the Belgian Science Policy.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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