Production of Rhodotorula glutinis: a yeast that secretes α-L-arabinofuranosidase
Financial support: This investigation
was financed by the grant Fondef D98I1037,
Keywords: α-L-arabinofuranosidase yeast, enzymes, wine.
Rhodotorula glutinis is a yeast that secretes the enzyme α-L-arabinofuranosidase (E.C. 18.104.22.168) into the culture medium and thus has an interesting biotechnological potential. To determine improved culture conditions of this organism, different factors of the culture media were evaluated such as the use of peptone as nitrogen source, salts composition, pH and growth temperature. Likewise, beet molasses and beet cosette were tested as industrial carbon sources to induce the production of the enzyme and how they influence the yeast growth. Based on these studies a culture medium is proposed for growth of this yeast in a continuous system. By assaying different dilution rates an average specific activity for the enzyme of 82.4 U/mg of protein was obtained.
The study of the aromas of fruits has revealed an important source of volatile compounds that may enrich the aromatic profile of juices and fermented drinks. The chemical composition of these potential aromas is formed by volatile compounds such as monoterpenes, derived from shikimate or C13 nonsoprenoids, which are linked to β-D-glucosides or diglicosides (Williams et al. 1982; Gunata et al. 1985). These compounds linked to sugars are released in two steps: initially, an α-arabinofuranosidase (Abf), an α-rhamnosidase or a β-apiosidase participate, followed closely by the action of a β-D-glucosidase (Gunata et al. 1988). Many of these aromatic compounds are naturally liberated during the fruit ripening (Cordonnier et al. 1989); however, the enzymatic activities of the plants are unable to liberate them completely leaving an important source of potential aromas in the juice (Gunata et al. 1985; Cordonnier et al. 1989). Therefore, some studies have been carried out to obtain endogenous enzymes, mainly from fungi and yeasts, which release these aromas. In general, these studies have been centred on identifying microorganisms with β-glucosidase activities and very few microorganisms have been considered that secrete the enzymes that participate in the first step of the reactions of the release of aromatic compounds (Gunata et al. 1990; Dupin et al. 1992; Gueguen et al. 1995; Lounteri et al. 1995; Charoenchai et al. 1997; Le Clinche et al. 1997, Spagna et al. 1998; Fernández et al. 2000; Manzanares et al. 2000; Spagna et al. 2000; Gallego et al. 2001; Strauss et al. 2001; Belancic et al. 2003). Generally, the glycosylated compounds are formed by a non-reductor α-L-arabinofuranoside (Gunata et al. 1990); therefore it would be interesting to use enzymes that hydrolyze this bond and allow the liberation of the volatile compound through the action of a β-glucosidase. This sequence of enzymatic activities would permit the enrichment of the aromatic profile of the product.
In grapes, the most abundant volatile compounds are monoterpenes, which can be free or linked to the disaccharides arabinofuranosilglucosides of geraniol, nerol and linalool (Williams et al. 1982; Gunata et al. 1988).
The α-L-arabinofuranosidase activity (E.C. 22.214.171.124) has been mainly described in fungus (Rombouts et al. 1988; Gunata et al. 1990; Le Clinche et al. 1997; De Ioannes et al. 2000). However, due to the physicochemical characteristics of some products, such as wine, it is difficult to maintain the enzymatic activities stable during the elaboration process. Therefore, some authors have searched for enzymes from microorganisms that participate in the must fermentation process like yeasts, and these studies have been mainly focused on the β-glucosidase activity more than on the activities responsible for hydrolysis of the disaccharide bond (Gueguen et al. 1995; Manzanares et al. 1999; Charoenchai et al. 1997; Fernández et al. 2000; Strauss et al. 2001; Belancic et al. 2003).
In light of the potential use that an Abf activity would have in the enological industry, in this work we studied the culture conditions of R. glutinis, a yeast that has been shown to produce Abf and are naturally present in fermentative processes. Furthermore, we evaluated the specific activity of this enzyme under continuous culture.
Rhodotorula glutinis strain L-1816 was isolated from Cabernet Sauvignon grapes and was maintained on YPD medium containing 2% (w/v) glucose, 2% (w/v) peptone and 1% (w/v) yeast extract.
The yeast inoculums for batch and continuous culture with 1 x 106 cells/mL were obtained by growth in YPD. Duplicate batch cultures were carried out in 250-mL flasks with 125 mL of assayed medium under mechanical shaking at 150 rpm. During the culture period growth was monitored by measuring absorbance at 475 nm every 0.5 and 1 hr, depending on the growth phase in which the culture was found. The dry cell mass concentration was determined from the optical density reading by using the following equation: dry cell mass = 0.31 OD475, 0.31 being the slope of a standard curve of dry cell mass (in g/L) versus OD475 (r2 = 0.9931). The specific growth rates (µ) were calculated by least-squares fitting to the linear part of the semilog growth plot. The basal culture media used contained 0.5% (w/v) yeast extract and a salts solution composed of 0.3% (w/v) (NH4)2SO4, 0.1% (w/v) KH2PO4 and 0.05% (w/v) MgSO4 x 7H2O. Modifications to the basal media are described below. Similarly, different pH and temperature conditions were evaluated.
a way of defining an improved culture media for the growth of R.
glutinis, several modifications to the basal media were evaluated.
Growth assays of the yeast were carried out in basal media with and
without peptone. Furthermore, assays were carried out with different
types and concentration of salts denominated II (0.3% (w/v) (NH4)
2SO4, 0.1% (w/v) KH2PO4, 0.05%
(w/v) MgSO4 x 7H2O and 0.005% (w/v) FeSO4
x 7H2O), III (0.3% (w/v) (NH4) 2SO4,
0.1% (w/v) KH2PO4, 0.05% (w/v) MgSO4
x 7H2O and 0.001% (w/v) MnCl2 x 4H2O)
and IV (0.3% (w/v) (NH4) 2SO4, 0.1%
(w/v) KH2PO4, 0.05% (w/v) MgSO4 x
7H2O, 0.005% (w/v) FeSO4 x 7H2O and
0.001% (w/v) MnCl2 x 4H2O). The salts composition
of the basal culture media was denominated as salt composition I.
Finally, as a means of determining the influence of the carbon source
on the yeast growth, 2% (w/v) beet cosette and 2% (w/v) beet molasses
were also evaluated. For this, the beet cosette was weighed, grinded
and mixed with 100 mL distilled water. This mix was vigorously agitated
and kept at
effect of the culture temperature on the growth of the yeast was also
studied. The temperatures assayed were 20, 25, 28 and
α-L-arabinofuranosidase activity was qualitatively determined
growth of R. glutinis was carried out in a laboratory constructed
fermentor under continuous operation. The fermentor was constructed
Statistical analyses were performed with Statgraphics Plus 4.0 software. Differences between treatment means were compared using the least significant differences (LSD) test with confidence level of 95%.
As a way of estimating how the constituents of the culture media influence the growth of R. glutinis and the production of Abf, different culture assays were carried out varying the composition of our base medium as indicated above.
yeast R. glutinis L-1816 was tested for its capacity to grow,
with or without 0.5% (w/v) peptone at
et al. (2001) have shown that the use of different nitrogen sources
in culture media affects yeast growth. Therefore, a study was carried
out to determine the most adequate salts composition in the culture
media for optimum growth of the yeast strain used in our investigation.
In these experiments, we evaluated the specific growth rates of R.
glutinis L-1816 using the basal media without peptone and varying
the type and concentration of salts as is described in Material and
Methods. As before, growth was followed for 72 hrs at
salts composition I (0.3% (w/v) of (NH4)2SO4;
0.1% (w/v) of KH2PO4 and 0.05% (w/v) of MgSO4
x 7H2O) is an adaptation of that described for Rhodotorula
flava by Uesaka et al. (1978) and our results
show that it also is enough to support the growth of R. glutinis
L-1816, being superior to the other salts compositions evaluated.
The addition of Fe or Mn in the salts composition II, III or IV seems
to not have important effects on yeast growth. The growth rate values
showed by R. glutinis L-
industrial carbon sources such as beet molasses and beet cosette have
been used in the production of this yeast (Roche et
al. 1994; Luonteri et al. 1995; De
Ioannes et al. 2000). Likewise, beet cosette has been studied
as a cheap substrate source in
Because the production of Abf in microorganisms is dependent on the substrate present in the culture media (De Ioannes et al. 2000) and considering the eventual applicability of beet molasses or beet cosette as industrial carbon sources for yeast growth, we study the effect of these two substrates on the production of Abf in qualitative assays. For this, R. glutinis L-1816 was grown in petri dishes with different concentrations of beet molasses or beet cosette (Table 2). Our results show that an increase in beet molasses concentration has a negative effect on the Abf activity. This observation could be a consequence of an increase in the sucrose content present in beet molasses whichcan have an inhibitory effect on the production of Abf. For practical considerations, the greatest enzyme secretion was obtained at a concentration of 0.2% (w/v) of beet molasses. Also, beet molasses has approximately 62% (w/v) of carbohydrates and other residues that could induce the production of Abf by the yeast.
On the other hand, the effect shown by the beet cosette on the production of Abf by R. glutinis L-1816 is independent of its concentration in the culture media. Illanes and Schaffeld (1983) by a hydrolysis experiment of this substrate indicated that its chemical composition includes 58.5% (w/v) of crude fiber. In our case, a hydrolysis was not carried out prior to its incorporation into the culture media, however, part of these components may have passed into solution and it is therefore possible that some induce the production of Abf. De Ioannes et al. (2000) showed that for the case of Penicillium purpurognum, beet cosette is a good inducer for the production of this enzyme.
growth temperature conditions described for R. glutinis have
the other hand, we have carried out assays in Petri dishes of several
native isolates to obtain yeasts with Abf activity (
study of pH evolution in unbuffered R. glutinis L-1816 72 hrs
cultures showed an increase of this parameter reaching a value of
7.0, when basal media without peptone at
the assays above, we proposed that the best growth conditions for
Abf production by R. glutinis L-1618 are 0.2% (w/v) beet molasses,
0.3% (w/v) (NH4)2SO4, 0.1% (w/v)
KH2PO4, 0.05% (w/v) MgSO4 x 7H2O,
0.5% (w/v) yeast extract, pH 5.2 and
Using the culture conditions described in the previous paragraphs, the growth of R. glutinis L-1816 was carried out in a continuous culture system for80 hrs to produce Abf. Two dilution rates were assayed as is shown in Table 4. Under these conditions it was possible to obtain a positive correlation between dilution rate and Abf activity, where the latter increased almost five times. Furthermore, an increase in the production of total proteins is also observed, which allowed the specific activity of the enzyme under study be maintained constant.
Uesaka et al. (1978) by growing R. flava in a medium with purified beet arabinan as carbon source obtained an α-arabinofuranosidase activity of 3.6 mU/mL, a lower value than that obtained in our study at the different flow velocities assayed. On the other hand, the specific activity described by these authors was 0.26 U/mg. In our case, the specific activity obtained for the R. glutinis strain L-1816, did not show significant differences between the flows velocities assayed, obtaining an average value of 82.4 U/mg, approximately 23 times greater than that described by Uesaka et al. (1978). This is mainly because of the low amount of proteins secreted by the yeast, which could correspond to the enzyme under study and therefore our growth conditions for this yeast permit a greater increase in the Abf production.
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