HYDROLASES OF GEOTRICHUM CANDIDUM YEASTS IN BARLEY BETA-GLUCAN DEGRADATION

Michał Piegza, Danuta Witkowska, Małgorzata Robak
Department of Biotechnology and Food Microbiology, Wrocław University of Environmental and Life Sciences, Poland

ABSTRACT

The research was focused on the capability of eight Geotrichum candidum strains to produce hydrolases, which can be useful for beta-glucan degradation. The evaluation of synthesis character, in synthetic media with sugar beet pulps and with malt, barley and sugar beet pulp, was checked. The degradation of barley glucan and its monitoring by HPLC of the reaction products was performed.

The yeasts were able to the secrete of enzymes degradation laminarin, lichen and beta-glucan, and the activity of those enzymes reached satisfactory level. The products size of beta-glucan hydrolyze showed the that enzymes of Geotrichum candidum mainly attack the endogenous bonds in glucan structure.

Key words: Geotrichum candidum, laminarinase, lichenase, beta-glucan, HPLC.

INTRODUCTION

Brewing barley contained up to 68% of starch, and up to 9% pentozanes – with a considerable beta glucan part [9]. The level of glucan degradation is being considered, as one of the most important indexes of malt quality [2]. Than the capability of Geotrichum candidum to secrete enzymes which can degrade this non-starch polysaccharide becomes purposeful. This analysis was directly involved with using discuss yeasts, as a starter cultures in malting process. Previous research confirmed usefulness of this microorganism to suppress the growth limit of pathogenic moulds and also to enhance domestic barley’s enzymes in correcting technological malt quality [3, 4, 5, 6, 7, 10, 14]. Confirmation the ability of Geotrichum candidum to produce beta-1,3-glucanase (laminarinase) and beta-1,3(1,4)-glucanase (lichenase) in medium contain milled malt and barley was observed [11]. The main purpose of presented paper was estimated the Geotrichum candidum yeasts to synthesis of extracellular lichenase and laminarinase and also checked the influence of microorganism’s enzymes on degradation of barley beta-glucan.

MATERIAL AND METHODS

The full characteristic of all 8 tested Geotrichum candidum strains was presented earlier [12].

Two mineral growth media were used for laminarinases biosynthesis: Saunders and MGP (Mineral Glucose Peptone) (Tab.1) complemented by sugar beep pulp and glucose. In the remaining cases enzymes were obtained in SZW medium (Table 1).

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

Activities of beta-1,3-glucanase, beta-1,3(1,4)-glucanase and beta-glucanase were determined with laminarin (0.25%), lichen (0.25%) and beta-glucan (0.5%) (Sigma) as a substrate in 0.05 M acetic buffer pH=4.8 respectively. The reducing sugars – products of enzymatic reaction (30 min, 50°C) – were determined colorimetrically, using dinitrosalicylic acid (Sigma) [9]. Activities of enzymes were expressed in nKat/ml.

Influence of temperature and pH on stability and activity of examinated enzymes was tested in 20-80°C range, and 3.0-8.0 range, using McIlvain’s buffer (Na₂HPO₄+citric acid) respectively.

Detection of products after beta-glucan hydrolysis using HPLC:

The reaction was conducted in eppendorf tubes using 0.5% barley glucan in acetic buffer (0.05M, pH=4.8). Enzymatic reaction with 0.25 ml enzyme and 0.5 ml substrate was performed. The reaction was stopped with 0.75 ml 96% ethanol. The mixtures were filtered on Millipore 0.45 micro-m Nylon Mebrane (Millex-HN) and applied on chromatographic column. The separation of the products of hydrolysis was performed in HPLC – Beckman 110b, on Aminex HPX 87H Ion Exclusion Column 300 mm x 7.8 mm; detected on Beckman 156 refractometr. The products of separation (0.3 m/s, 30 min) were analyzed according to the following standards: glucose, cellobiose, cellotriose, laminaritiose, laminaritetraose and cellopentaose. The largeness of obtained peaks and also retention time were settled on the basis of Beckman GOLD program.

RESULTS AND DISCUSSION

All tested strains, suggested in earlier works for malting, as a starter cultures, characterized by the capability of synthesis beta-glucanase. The activity in both minerals medium with sugar beet pulp was on the level between 1.3 – 8 nKat/ml.

G. candidum 1 (OL), G. candidum SS47D₂ (D2), G. candidum SS32B₁ (B1), G. candidum KB6 (X6), and G. candidum KB5 (X5) were much more successful in synthesis laminarinase in Saunders than MGP medium (Fig. 1). But other strains (Sc12, 3-11, PH) characterized higher activity in MGP than Saunders medium. When there was only glucose, as the source of carbon, the level of enzymes activity was 2-3 times lower. These results suggested, that these enzymes could be synthesized in the presence of glucose, so no catabolytic repression was observed.

Using SZW medium, the dynamics of biosynthesis of enzymes which could degradated lichen, laminarin and beta-glucan was checked (Fig. 2). The tested strains started to secret the enzymes at the second day of the culture. The intensification of enzymes biosynthesis was observed at the 4th day. Strain 3-11 reached maximum level of laminarinase after 4 days, strain X5 after 5 days, but strain OL after 7 days. Strain X6 after 5 days of culture reached high (proportionally to the rest of strains) lichenase activity which was on stabile level till the end of the culture.

The difference of strains capability to degrade beta-glucan, was observed and the OL strain reached maximum level in the 6th day of culture.

The optimal condition for activity and stability of lichenase and laminarinase was examined. Optimal lichenase activity was observed at 50°C and pH=6.0, but laminarinase at 40-50°C and pH=6.0 too. Laminarinase and lichenase exhibited the highest stability in the range 20-60°C and at pH 3.4-7.4.

The Geotrichum candidum 1 and Geotrichum candidum PH1 hydrolases was used in enzymatic degradation of beta-glucan. The commercial lichenase (Megazyne) and laminarinase (Sigma) were used as the control of beta-glucan degradation process (activity was approximated to activity of yeasts enzymes).

Generally in every case after enzymatic hydrolysis (2h) of beta-glucan was displayed on chromatographs the two peaks with large surfaces and the retention below 12 minutes
(Fig. 3 and Fig. 4). There was indicate the few smaller peaks (above 100 times) with retention time above 13 minutes (retention time of glucose – 16.872 min.). On the basis of obtained results it was possible to suppose the endogenic effect of enzymes hydrolyse. The beta-glucan chain was cut inside and accumulating oneself large – probably over 10G and more glucose fragments. Differences, which were possible to observe on glucan chromatogram, were showed onto diverse way of hydrolysis. It was considerable the similarity among the effects of working of yeasts enzymes and commercial preparation – lichenase. The disclosed oneself fractions with retention time above 13 minutes was identified as a fragments composing from 2, 3 of glucose molecule. The process of hydrolysis beta-glucan results in fast degrading of structure of polysaccharide chain, but slow releasing of reducing sugars.

In literature, data’s of relationship between direct capable of yeasts Geotrichum candidum with the production of barley beta-glucan degrading enzymes, were not found. Early investigations confirmed possibility of studied yeasts to hydrolysis of lichen and laminarin only [11]. It was found, that beta-glucosidase, from yaests and moulds, disclosed capable to slow hydrolysis laminarin [1]. The different beta-glucosidase and their hydrolysis products were examined (cellosaccharides and laminarinsaccharides) [13]. It should be underlined, that in spite of the fact that usually laminarin and lichen are degradated by β-1,3- and β-1,3(1,4)-glucanases, and yet it is possible for other enzymes to participate in this process. It was proven, that beta-1,4-glucanase (cellulase) may participate indirectly in the degradation of glucan [14]. However, the slow increase of reducing sugars does not surprise. Commercial enzymes degradated of polysaccharide chain rapidly, but simultaneously glucose - as the final effect of hydrolysis – appear very late [8].

CONCLUSIONS

The Geotrichum candidum hydrolases could be useful in degradate the barley glucan as activity supporting barley own enzymes, specially supporting the increasing the viscosity of worth. This also confirms that presented strains would be perfect as a starter culture in malting.

ACKNOWLEDGEMENTS

This research was supported by a grant from State Committee for Scientific Research within the project no. 2 P06T 001 26.

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Accepted for print: 9.11.2006

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