Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2006
Volume 9
Issue 4
Topic:
Biotechnology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Witkowska D. , Piegza M. 2006. CAPABILITY OF GEOTRICHUM CANDIDUM YEASTS FOR CELLULASES AND XYLANASES BIOSYNTHESIS, EJPAU 9(4), #41.
Available Online: http://www.ejpau.media.pl/volume9/issue4/art-41.html

CAPABILITY OF GEOTRICHUM CANDIDUM YEASTS FOR CELLULASES AND XYLANASES BIOSYNTHESIS

Danuta Witkowska, Micha³ Piegza
Department of Biotechnology and Food Microbiology, Wroc³aw University of Environmental and Life Sciences, Poland

 

ABSTRACT

In this paper the capability of eight strains of yeasts Geotrichum candidum for cellulases and xylanases production in two mineral media with addition of various components was examined. Mineral media were supplemented with four different sources of carbon, such as sugar beet pulp, wheat grain, CMC or glucose. Geotrichum candidum strains were characterized very different level of cellulase and xylanase biosynthesis. The highest cellulase activities for 6 strains from 8 ascertained in Saunders medium with sugar beep pulp and in MGP medium with a CMC. However highest xylanase activities observed in both media with CMC. Some strains produced cellulases and xylanases in mineral medium when glucose used as carbon sources.

Key words: Geotrichum candidum, cellulase, xylanase.

INTRODUCTION

Cellulolytic and xylanolytic capability in starter culture, proposal to use in malting, are important qualification. These enzymes could be helpful for own grain enzymes during the malting. Research in this direction is very advanced [1]. But there is not too much information about Geotrichum candidum cellulases and xylanases. The occurance of these enzymes among eight strains grown on milled malt and barley was already reported [7]. It was shown earlier by Rodionowa at al. that G. candidum strain produced beta-glucosidase able to degrade celobiose and laminaribiose and also endo-1,4-beta-xylanase [9, 10]. Both enzymes showed optimum activity at 50°C and pH=4.0-4.5. We also found information about two endocellulases produced by a Geotrichum strain isolated from soy bean [4]. These enzymes varied in molecular size but they acted in very similar catalytic way, showing optimum temperature at 50°C and pH stability in wide range. These endocellulases degraded CMC, but only one: endocellulase I utilized cellulose. Yaoi and Misthuishi [12, 13] characterized two other enzymes from Geotrichum sp. – xyloglucan endocellulase and oligoglucan glucosidase. These enzymes were not able to degrade neither CMC, cellulose not even barley glucan. The one of them utilized xyloglucans, but it demonstrated the lowest activity on barley’s xyloglucan.

The aim of this paper was to estimate usefulness of Geotrichum candidum strains, as a starter culture in malting, due to cellulase and xylanase production.

MATERIALS AND METHODS

Strains

Eight strains of Geotrichum candidum yeasts isolated from 5 sources: G.candidum 1 (OL) – collection/milk, G.candidum KB5 (X5) and G.candidum KB6 (X6) from Brie cheese, G.candidum Sc12 (Sc12) from Camembert cheese, G.candidum MSK311 (3–11) and G.candidum SS32B1 (B1) and G.candidum SS47D2 (D2) from malt, and also G.candidum PH1 (PH) from chicken feathers was stored on YM slants at 4°C.

Growth media

Two mineral media were used for cellulases and xylanases production: Saunders and MGP complemented by sugar beet pulp, wheat bran, CMC or glucose (Tab.1). Dynamics of xylanases biosynthesis was examined in SZW medium (Tab.1).

The cultivation of microorganism was conducted in 250 ml flasks (50 ml of medium) at 28°C for 7 days, at 168 rpm. Arthrospore suspension (1x107 cells in 0.5 ml of 1% Tween 80 water soluble) was used as an inoculum.

Table 1. Growth media (g/l)

Saunders

MGP

SZW

KH2PO4           0.20

   

K2HPO4           0.15

   

NaH2PO4         2.00

KH2PO4           2.00

 

Na2HPO4         1.50

MgSO4            1.00

 

NH4NO3           0.60

CaCl2              1.00

malt                    10.00

NaNO3             3.80

(NH4)2SO4       3.00

barley grain           5.00

MgSO4             0.30

pepton             2.00

sugar beet pulp       10.00

pepton              5.00

glucose            5.00

 

glucose             5.00
+microelements

   

complementation:
2% sugar beet pulp (S+sbp)
2% wheat bran (S+wb)
2% CMC (S+CMC)
1% glucose (S+glu)

complementation:
2% sugar beet pulp (MGP+sbp)
1.5% CMC (MGP+CMC)
1.5% wheat bran (MGP+wb)
1% glucose (MGP+glu)

 

Determination of enzymatic activity

The activities of cellulase and xylanase were determineted in 0.05 M acetic buffer pH=4.8 using carboxymethylcellulose-Na (1.0%) and xylan from birchwood (1.0%) (Sigma) as a substrates, respectively. The reducing substances – product of enzymatic hydrolysis (30 min, 50°C) were determined colorimetrically using dinitrosalicylic acid as a reagent (Sigma) [3]. The activities of enzymes were expressed in nKat/ml.

RESULTS AND DISCUSSION

The activity of cellulase and xylanase was examined in two mineral media with four different sources of carbon (sugar beet pulps, wheat bran, CMC and glucose).

Activity of cellulases (Fig. 1 and Fig. 2) for all 8 strains growth on both mineral media (S and MGP) complemented with various sources of carbon, reached level from 0 to 2.25 nKat/ml. Smaller differences in enzymatic activity were observed in MGP medium for G.candidum 1 (OL), G.candidum KB5 (X5), G.candidum KB6 (X6) and G.candidum MSK311 (3-11) (Fig.2). The remaining strains G.candidum PH1 (PH), G.candidum Sc12 (Sc12), G.candidum SS32B1 (B1) and G.candidum SS47D2 (D2) showed larger differentiation in the level of expression of enzymes depending on the source of carbon in both media.

Generally, the highest value of cellulase activity in MGP with addition of CMC was ascertained (Fig. 2). Among eight tested strains (except of D2 strain), the highest cellulase activity in Saunders medium was observed when sugar beet pulp was used as a carbon source (Fig. 1).However in the presence of wheat brain (wb) in Saunders medium biosynthesis of cellulase was generally poor. Only for strain PH and Sc12 such tendency was not observed.

The glucose, as a carbon source, in mineral medium generally did not favoured cellulase biosythesis. Only strain X5, Sc12, D2 produced effectively cellulase in present of glucose in medium than rest of using carbon source.

Fig. 1. Effect of the carbon source on cellulases production of Geotrichum candidum strain cultivated on Saunders medium

Fig. 2. Effect of the carbon source on cellulases production of Geotrichum candidum strain cultivated on MGP medium

The production of xylanases by Geotrichum candidum dependent on kind of mineral medium and carbon source used. The level of activity did not exceed 1 nKat/ml in Saunders medium and 0.6 nKat/ml in MGP medium (Fig. 3 and Fig. 4). Wheat brain was found as the worst carbon source, while the highest activities were observed in both media supplemented with CMC. Strains OL and X5 were not able to produce xylanases in MGP medium at all (Fig. 4).

The level of xylanase activity for strain OL, X5, 3-11, and D2 in MGP medium with glucose, as a carbon source, was highest, than with addition of the of rest of carbon source. This problem wasn’t observed in the richest Saunders medium (Fig. 3 and Fig.4).

Fig. 3. Effect of the carbon source on xylanases production of Geotrichum candidum strain cultivated on Saunders medium

Fig. 4. Effect of the carbon source on xylanases production of Geotrichum candidum strain cultivated on MGP medium

The dynamics of xylanases expression was studied using SZW medium, as more natural medium (Fig. 5). Generally on 7th day of culture, the activity of xylanases was higher that the ones achieved on mineral media (Fig 3, Fig. 4, Fig 5). Except of the strain OL, which after 3rd of day cultivation reached maximal level of their biosynthesis, the activities of the remaining 7 strains after second day of cultivation were on equal level.

Fig. 5. Dynamics of Geotrichum candidum xylanases biosynthesis in SZW medium

Moulds or yeasts cellulases (unlike bacterial) are not synthesized constitutively [11], but they are induced in the presence of cellulose’s substrates. Cellulose can not be the direct inductor of these enzymes, in view of its insolubility. The cellulase can be produced constitutively on low level and releasing cellooligosacharides, can to be inducer of enzymes apparatus of microorganism [11].

Mo and Hayashida studied two endocellulase in yeasts Geotrichum candidum (productive strain was isolated out from soya bean), which maximum activity level was achieved in the sixth’s day of growth [4]. Both enzymes were able to degraded carboxymethylocellulase, but only endocelulase I degraded cellulose. They were stable in a very wide range of pH and they showed maximum activity of 2-5 U/mg. Ours strains of Geotrichum candidum in the presence of malt and barley grains [7], achieved higher activity values were then in the presence of glucose or sugar beet pulp, but lower expression that cited strain from soya bean [4].

Cellulase and xylanase co-operates in the degradation of plant cellular walls, [5]. In the previously paper the yeasts were characterized by higher level of xylanase biosynthesis than cellulase. The level of their activity clearly grew up in the presence of carbon source of higher cellulose quantities. The presence of milled grain in a medium improved level of production of this enzymes imperceptibly, this decidedly higher activity including milled malt [7].

There research of Perez-Avalos et all. about Cellulomonas fravigena xylanase showed their induced character. Cellulose had a positive effect on the process of biosynthesis in contrast to xylan [6].

Lin at all. [2] studied xylanase from of Trichosporon cutanum, and suggested smaller efficiency in the its synthesis, when xylose or xylan used as carbon source, and practically completely slowed down when glucose or sucrose were used as a carbon source. However, hemicellulose appeared be a perfect inductor for their biosynthesis [2].

Purkarthofer and Steiner suggested, that xylanase from Thermomyces launginosus was produced constitutively on the low level (3.5 nKat/ml) in the presence of glucose, but the addition of xylan to a growth medium resulted in raising the level of activity to 7000 nKat/ml [8].

There is also known strain Geotrichum candidum 3C produced endo-1,4-xylanase with activity at 32.6 U/mg after 3rd day of growth. This enzyme characterized optimum activity in temperature 50°C and pH=4.0 [10]. In our studies optimum temperature for yeasts cellulases carried out 50°C, however for xylanases 50 – 60°C, meanwhile the stability was shaped in the range of 20 – 65°C in both cases. Optimum pH of cellulases and xylanases for most of tested strains equaled 6.0; except OL strain for which for cellulase was measured on the level 4.8. Stability of cellulase of G. candidum was measured in the pH range of 3.0 – 7.0 (non public data).

Because Geotrichum candidum strains shown in presented paper capability to cellulase and xylanase biosynthesis they can makes proposed strains suitable as a starter culture in malting.

CONCLUSION

  1. Geotrichum candidum yeasts show ability to cellulase and xylanase biosynthesis.

  2. The influence of kind of medium and carbon source on hydrolase biosynthesis was observed.

  3. Sugar beep pulp (in Saunders medium) and CMC (in MGP medium) specially was conductive to cellulase biosynthesis.

  4. CMC, as a carbon source in both mineral media was the best for increase xylanase biosynthesis.


REFERENCES

  1. Dziuba E., Foszczyńska B., 2001. Biological protection of barley grain and its influence on selected features of malt, Polish Journal of Food and Nutrition Sciences, vol 10, no 4, 49–53.

  2. Lin W., Zhu W., Lu Y., Kong J., Ma G., 1999. Production, partial purification and characterization of xylanase from Trichosporon cutaneum SZ 409, Process Biochemistry, 33, 3, 331-336.

  3. Miller G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem., 31, 426-428.

  4. Mo K., Hayashida S., 1988. Production and characteristics of two types of endocellulase from Geotrichum candidum, Agric. Biol. Chem. 52, 7, 1675-1682.

  5. Murashima K., Kosugi A., Doi R.H., 2003. Synergistic effect of cellulosomal xylanase and cellulase from Clostridium celllovovans on plant cell wall degradation, Journal of Bacteriology, 1518-1524.

  6. Perez-Avalos O., Ponce-Noyola T., Magana-Plaza I., de la Torre M., 1996. Induction of xylanase and β-xylosidase in Cellulomonas flavigena growing on different carbon source, Appl. Microbiol Biotechnol, 46, 405-409.

  7. Piegza M., Witkowska D., Stempniewicz R., Rywińska A., 2005. Geotrichum hydrolytic activity in milled malt and barley, EJPAU, Biotechnology, vol 8, issue 1.

  8. Purkarthofer H., Steiner W., 1995. Induction of endo-β-xylanase in the fungus Thermomyces lanuginous, Ezyme and Microbial Technology, 17, 114-118.

  9. Rodionova NA., Tavobilov IM., Martinovich LI., Buachidze TS., Kvesitadze GI., Bezborodov AM., 1987. Beta-glucosidases from cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum as typical glycosidases, Biotechnology and Applied Biochemistry, 9, 239-250.

  10. Rodionowa N.A., Dubovaya N.V., Eneiskaya E.V., Martinowicz L.I., Grachewa I.M., Bezborodov A.M., 2000. Purification and characterization of endo-(1-4)-β-xylanase from Geotrichum candidum 3C, Appl Biochemistry and Microbiology, vol 36, no 5, 406.

  11. Suto M., Tomita F., 2001. Induction and catabolite repression mechanisms of cellulase in fungi, Journal of Bioscience and Bioengineering, 92, 4, 305-311.

  12. Yaoi K., Mitsubishy Y., 2002. Purification, characterization, cloning and expression of a novel xyloglucan-specyfic glucosidase, oligoxyloglucan reducing end-specyfic cellobiohydrolase, The Journal of Biological Chemistry, vol 227, no 50, 48276-48281.

  13. Yaoi K., Mitsuishi Y., 2004. Purification, characterization, cDNA clonning and expression of xyloglucan endoglucanase from Geotrichum sp. M128, FEBS Letters, 560, 45-50.

 

Accepted for print: 4.12.2006


Danuta Witkowska
Department of Biotechnology and Food Microbiology,
Wroc³aw University of Environmental and Life Sciences, Poland
Che³mońskiego 37/41
51-630 Wroc³aw
Poland
email: wit@ozi.ar.wroc.pl

Micha³ Piegza
Department of Biotechnology and Food Microbiology,
Wroc³aw University of Environmental and Life Sciences, Poland
Che³mońskiego 37/41
51-630 Wroc³aw
Poland
email: mikey@wnoz.ar.wroc.pl

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