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

Authors


Tomoyuki Nakagawa, Department of Food Science and Technology, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan (e-mail: t-nakaga@bioindustry.nodai.ac.jp).

Abstract

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°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°C.

Introduction

There is an industrial tendency to treat foodstuffs under mild conditions in order to avoid spoilage, and changes in taste and nutritional value at an ambient temperature. Therefore, cold-active enzymes are used for processing foods (Margesin and Schinner 1994; Russell 1998; Gerday et al. 2000). Psychrophiles are micro-organisms exhibiting optimal growth at 15°C or lower temperatures (Morita 1975), have attracted attention as sources of enzymes with potential for low-temperature catalysis. Indeed, a variety of cold-active enzymes have been found in a psychrophile (Feller et al. 1996; Marshall 1997; Gerday et al. 2000).

However, pectin is one of the main constituents of primary cell walls, and the middle lamellae of higher plant cells together with cellulose and xyloglucan. It is a complex heteropolysaccharide, composed mainly of d-galacturonic acid residues joined through α-1,4-linkages that form homogalacturonan chains, and the d-galacturonic acid residues may be methylated (Sakai et al. 1993). In the food industry, pectin compounds may be the cause of problems arising during the extraction, filtration, concentration and clarification of fruit juice (Pilnik and Rombouts 1985). Therefore, pectinolytic enzymes are also widely used for the degradation of pectin compounds in the fruit and vegetable processing industries (Sakai et al. 1993; Alkorta et al. 1998).

Pectinolytic enzymes can be classified into two main groups, i.e. pectin methylesterase (PME), which removes methoxyl group from pectin, and depolymerizing enzymes, which cleave the bonds between galacturonate units. Pectin-depolymerizing enzymes include pectin lyase (PNL), pectate lyase (PAL) and polygalacturonase (PG).

In the previous paper, we reported a pectinolytic–psychrophilic yeast (PPY) Cystofilobasidium capitatum strain PPY-1 (Nakagawa et al. 2002). The present study was designed to isolate psychrophilic yeasts that are able to degrade pectin compounds at low temperature, and to determine the characteristics of the yeasts isolated from soil from Abashiri (Hokkaido, Japan), in order to have several more types of PPY strains. Moreover, we investigated the pectinolytic enzyme activities in the extracellular fractions of these PPY strains.

Materials and methods

Screening and cultivation conditions

The yeast strains used in this study were isolated from soil from forest in Abashiri (Hokkaido, Japan). For the screening of psychrophilic micro-organisms utilizing pectin, the mineral synthetic medium (MI medium) (Sakai et al. 1998;Nakagawa et al. 2003) containing 1% (w/v) pectic compounds as a sole carbon source was used. The pectic compounds used were citrus pectin, whose degree of esterification was ca 90% (Sigma Chemicals, St Louis, MO, USA), and polygalacturonate (Sigma Chemicals). The pectic compounds were purified by washing with acidified 60% ethanol (5 ml concentrated HCl/100 ml of 60% ethanol), followed by washing with 60 and 90% neutral ethanol (Stratilova et al. 1998). The initial pH of the medium was adjusted to 5·0.

Phylogenetic analysis of isolated strains

For phylogenetic analysis, the partial 28S rDNA-D1/D2 gene was amplified with genomic DNA of the isolated strains as a template by the PCR method, as described previously (Nakagawa et al. 2002). The amplified 28S rDNA was used as the sequencing template. Multiple alignment of sequences, calculation of nucleotide substitution rates, and construction of a neighbour-joining phylogenetic tree were carried as given in Saitou and Nei (1987).

Enzyme assays

PME activity was measured by titrating with 0·01 n NaOH (Nakagawa et al. 2000). One unit was defined as the amount of enzyme that released 1 μmol of carboxy group per minute at 5°C. PG activity was assayed by measuring the increase in reducing groups derived from polygalacturonate (Nakagawa et al. 2000). One unit was defined as the amount of enzyme that released 1 μmol of reducing group per minute at 5°C. PNL and PAL activities were determined by monitoring the absorbance at 235 nm during incubation with 1% pectin or 1% polygalacturonate (Ishii and Yokotsuka 1972). One unit was defined as the increase in absorbance at 235 nm of 1·0 of the reaction mixture per minute at 5°C.

Zymogram analysis of pectin-degrading enzymes

Electrophoresis of the extracellular pectin-degrading enzymes was performed on a 10% (w/v) polyacrylamide gel. For detection of pectin-degrading enzyme activity, 0·1% pectin or 0·1% polygalacturonate was polymerized in the gel matrix. Staining for pectinolytic enzymes was performed with a 0·02% ruthenium red solution (Cruickshank and Wade 1980).

Determination of the N-terminal amino acid sequence

The extracellular proteins separated by SDS-PAGE were transferred to a polyvinylidene difluoride membrane (ProBlott, Applied Biosystems, Foster City, CA, USA), and then their N-terminal amino acid sequences were determined with an automated protein sequence analyzer (Model 477A/120A; Applied BioSystems, Foster City, CA, USA).

Results

Isolation and characterization of PPY strains

First, we isolated four micro-organisms, strains PPY-3, 4, 5 and 6, which can utilize pectin at 5°C, from soil from Abashiri (Hokkaido, Japan). The colonies of strains PPY-5 and 6 were pinkish in colour, and those of strains PPY-3 and 4 were white. These strains were the PPY, because they had yeast-like cells, and could grow at 5°C, but not at 25°C.

To confirm the phylogenetic relationship with other yeasts, partial 28S rDNA D1/D2 sequences of the isolated strains were determined and compared with available 28S rDNA D1/D2 sequences (Fig. 1). The results of sequencing revealed that the four isolates belong to three different species. The 28S rDNA D1/D2 sequences of strains PPY-5 and PPY-6 showed high isology with that of Cys. capitatum. However, the 28S rDNA D1/D2 sequences of strains PPY-3 and 4 showed high isology with those of Cryptococcus cylindricus and Mrakia frigida, respectively.

Figure 1.

Phylogenetic tree obtained on comparison of 28S rDNA D1/D2 sequences. The sequences were compared with 28S rDNA sequences obtained from the GenBank database. The accession numbers are shown in parentheses

Pectinolytic enzymes from PPY strains

The pectinolytic characteristics of the PPY strains were examined. All isolated strains were able to grow on pectic compounds, i.e. polygalacturonate and pectin. The optimum growth temperature on pectic compounds was 15°C, although all strains could grow below 5°C. Moreover, the amount of pectin in the medium decreased with the growth of all isolated strains at 5°C, although the PPY-3 strain exhibited very low ability (data not shown). These findings indicate the possibility that these strains produce pectinolytic enzymes, which function at low temperature.

Next, the activities of pectinolytic enzymes of the PPY strains were examined at 5°C. The PPY-4, 5 and 6 strains grown on pectin exhibited PME and PG activities, and the PPY-5 and 6 strains produced PNL activity in the extracellular fraction even at low temperature like the PPY-1 strain, although strain PPY-3 did not show all the enzyme activities and none of the isolated strains exhibited PAL activity (Table 1). Also, zymogram analyses of the pectinolytic enzymes in the extracellular fractions of the PPY strains were performed. The extracellular fraction of strain PPY-4 grown on pectin gave a single activity band for pectate-depolymerizing enzymes, which produce clear zones on a gel stained with ruthenium red, although strain PPY-3 did not show any activity bands (Fig. 2a). Strain PPY-5 gave three activity bands for pectate-depolymerizing enzymes, and PPY-6 showed five activity bands like strain PPY-1. However, on pectin gels, all PPY strains gave a single activity band for PME, which gives dark-stained band, at different positions (Fig. 2b).

Table 1.  Specific activities of pectinolytic enzymes on extracellular fractions from PPY strains
StrainSpecific activity (mU mg−1)
PMEPNLPALPG
  1. PME, pectin methylesterase; PNL, pectin lyase; PAL, pectate lyase; PG, polygalacturonase; n.d., not detected. The enzyme activities were measured at 5°C.

PPY-3n.d.n.d.n.d.n.d.
PPY-416·0 ± 2·45n.d.n.d.7·61 ± 0·80
PPY-52·68 ± 1·323·58 ± 0·47n.d.25·5 ± 2·96
PPY-61·33 ± 0·661·20 ± 0·38n.d.12·6 ± 0·49
Figure 2.

Zymogram analysis of pectinolytic enzymes in the extracellular fractions of the PPY strains. Twenty microlitres of extracellular fraction was applied to (a) a 0·1% polygalacturonate or (b) a 0·1% pectin-containing 10% polyacrylamide gel. The strains were: lane 1, PPY-1; lane 2, PPY-3; lane 3, PPY-4; lane 4, PPY-5; and lane 5, PPY-6 strain

Moreover, the components of the extracellular fraction of strain PPY-1 grown on pectin were analysed by SDS-PAGE. The extracellular fraction of strain PPY-1 contained at least seven types of protein, named A–G (Fig. 3). N-terminal amino acid sequences of the band A, B and C materials were not determined. However, the band D–F materials had the same sequence, WSATISSLNDVAAAKKCTSI, which shows high identity with that of PG from Saccharomyces cerevisiae (52·9% identity). This showed that PG from strain PPY-1 was processed at least three times through signal peptidases, proteases or other modifications. However, the N-terminal amino acid sequence of the band G material was YADPGVVSGNVNVHDPGLAK, which showed high identity with that of arabinase from Streptomyces coelicolor (55% identity), which is one of the protopectinases.

Figure 3.

SDS-PAGE of the extracellular fraction of strain PPY-1 grown on pectin. Twenty micrograms of extracellular fraction was applied to a 10% SDS-poyacrylamide gel

Discussion

In this study, we isolated PPY strains that could grow on pectin at 5°C, and strains PPY-3 and 4 were identified as Cry. cylindricus and M. frigida, respectively, and strains PPY-5 and 6 as Cys. capitatum, like strain PPY-1. Recently, we reported Cys. capitatum strain PPY-1, which produces some pectinolytic enzymes (Nakagawa et al. 2002). Moreover, Birgisson et al. (2003) reported about eight cold-adapted polygalacturonase-producing yeasts isolated from frozen environmental samples in Iceland, which belong to Cys. lari-marini, Cys. capitatum, Cry. macerans and Cry. aquaticus. From these facts, it seems that Cys. capitatum is one of the most dominant PPY strains in natural environments, and Cry. cylindricus and M. frigida isolated in this study are novel PPY species that have not been reported previously.

However, the isolated strains produced cold-active enzymes that degrade pectin, like strain PPY-1. Moreover, we showed that strain PPY-1 has arabinase, which is one of the protopectinases, in addition to pectinolytic enzymes. Based on these findings, it is possible that some pectinolytic enzymes and protopectinases from these strains will be applicable to the food industry and other fields.

Acknowledgements

We are grateful to Ms Takako Kanai and Ms Yoshiko Motohashi for their skilful assistance. We are also indebted to Professor Toshihiro Watanabe (Tokyo University of Agriculture), for his help in the amino acid sequence determination. Identification of the PPY strains was conducted by NCIMB Japan Co., Ltd (Shimizu, Japan).

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