Claudio E. Voget

Yeast growth and β-galactosidase production during aerobic batch cultures in lactose-limited synthetic medium

Abstract A lactose-limited synthetic medium was used to analyse the dynamics of batch growth and β-galactosidase production in the yeasts Kluyveromyces and Candida , under fully aerobic conditions. In shake flasks cultures, no significant differences were observed in the growth yields among 18 tested strains and most of them showed comparable enzyme formation. Five of these strains, K. lactis CBS 2360, CBS 683, NRRL 1118, K. marxianus NRRL 1109 and C. pseudotropicalis NCYC 744 were further studied in fermenter cultures. Metabolic behaviour was characteristic of the Crabtree-negative yeasts. Ethanol formation was absent or very low (less than 5% of the lactose metabolized). During the exponential growth phase typical fluxes for lactose and oxygen uptake were 3·0 and 13 15·5 mmol g −1 H −1 respectively. Enzyme levels were constant during the exponential phase and incresed 1·3–1·8-fold during the brief deceleration phase.

Some factors affecting pectinase production from apple pomace in solid-state cultures

Abstract Some factors affecting pectinase production from apple pomace in solid-state culture (medium composition, inoculum size, temperature and type of apple pomace used) were investigated. To obtain high enzymatic activities, apple pomace requires the addition of an adequate level of organic nitrogen components like those present in peptone, yeast extract or corn-steep. Inoculum size is not critical and optimum temperature for the fermentation process is 30°C. In connection with apple-pomace quality, the sugar concentration is the most important parameter which controls pectinase yields. By using Granny Smith pomace mixtures, containing 4% of sugars, production as high as 1300 U g−1 was attained in 36 h of culture. Pectinase levels ranging from 1000 to 2000 U litre−1 of juice for 1 to 3 h were necessary to achieve clarification in three different apple juices.

Butanol production from apple pomace

SummaryThe use of apple pomace for butanol production was studied by employing strains ofClostridium acetobutylicum andbutylicum. Yields of butanol between 1.9 and 2.2 % of fresh apple pomace are reported.

Aspergillus kawachii produces an acidic pectin releasing enzyme activity

A pectin-releasing (protopectinase, PPase) activity was found in a culture filtrate of Aspergillus kawachii IFO 4308. PPase activity was highest in the pH range of 2.0-2.5 and it was highly stable at 50 degrees C (85% of residual activity was found after a 10-h incubation in citrate-phosphate buffer, pH 3.0). Among other different enzyme activities, which are usually involved in plant cell-wall degradation, only polygalacturonase activity was detected. This result suggests that the PPase activity could correspond to a particular kind of polygalacturonase. Pectin extraction from lemon peels carried out at 50 degrees C for 2 h (pH 3.5) gave yields of ethanol-precipitated pectin equivalent to 17.4% of the initial total solids contained in the peels. Thus, this enzyme activity would allow carrying out a pectin extraction process at lower reaction pHs and higher temperatures in comparison with similar reports using other PPases. These properties seem to be very interesting from the practical point of view.

Apple pomace as raw material for pectinases production in solid state culture

Abstract Apple pomace was used for pectinases production on a small scale by solid state cultures. Viscosimetric enzyme activities as high as 1062 U g−1 were attained in 48 h. Characterization of the enzymatic pool showed that it is adequate for practical purposes.

The proteolytic system of the yeast Kluyveromyces lactis

Major proteolytic activities were characterized in the yeast K. lactis NRRL 1118, grown in chemostat cultures. This yeast expressed proteolytic activities similar to those found in S. cerevisiae. This fact was particularly evident in the case of proteases such as PrA, PrB and CpY with regard to substrate specificity, activation at pH 5·0 and inhibition patterns. The presence of a CpS activity could not be detected in either fresh or activated cell‐free extracts by using the dipeptide N‐Cbz‐Gly‐Leu, even in the presence of Zn+2. On the other hand, K. lactis exhibits at least two major intracellular Ap activities different from those reported in other yeasts, and these seem to be carried out by closely related proteins. These activities corresponded to molecular masses of about 60 kDa, close pI values, and a similar behaviour in non‐denaturing polyacrylamide electrophoresis. Both activities were enhanced by Co+2 and inhibited by EDTA. Among different aminoacyl‐p‐NAs, they preferentially hydrolysed Lys‐p‐NA. No increase of Ap activity was obtained by incubation of extracts at acid pH. The maximum PrA and PrB activities detected in N‐limited cultures were six‐fold higher than those expressed under C‐ or P‐limitation. The effect of culture conditions on the Cp and Ap expression was much less pronounced in comparison with PrA and PrB activities, Ap levels even being slightly higher in C‐limited cells. This fact suggests that hydrolysis of protein to peptides might be the limiting step in the pathway of general protein degradation in the vacuole. Copyright

Growth and β-galactosidase synthesis in aerobic chemostat cultures of Kluyveromyces lactis

Growth and β-galactosidase (β-gal) expression were characterized in the yeast Kluyveromyces lactis strain NRRL Y-1118 growing in aerobic chemostat cultures under carbon, nitrogen or phosphate limitation. In lactose or galactose-limited cultures, β-gal accumulated in amounts equivalent to 10–12% of the total cell protein. The induced β-gal expression was repressed when cells were grown under N- or P-limitation. In lactose medium, enzyme levels were 4–8 times lower than those expressed in C-limited cultures. A similar response was observed when galactose was the carbon source. These results suggest that a galactose-dependent signal (in addition to glucose) may have limited induction when cells were grown in carbon-sufficient cultures. Constitutive β-gal expression was highest in lactate-limited and lowest in glucose-limited media and was also repressed in glucose-sufficient cultures. Other K. lactis strains (NRRL Y-1140 and CBS 2360) also showed glucose repression (although with different sensitivity) under non-inducing conditions. We infer that these strains share a common mechanism of glucose repression independent of the induction pathway. The kinetics of β-gal induction observed in C-limited cultures confirms that β-gal induction is a short-term enzyme adaptation process. Applying a lactose pulse to a lactose-limited chemostat culture resulted in ‘substrate-accelerated death’. Immediately after the pulse, growth was arrested and β-gal was progressively inactivated. Yeast metabolism in C-limited cultures was typically oxidative with the substrate being metabolized solely to biomass and CO2. Cells grown under P- or N-limitation, either with glucose or lactose, exhibited higher rates of sugar consumption than C-limited cells, accumulated intracellular reserve carbohydrates and secreted metabolic products derived from the glycolytic pathway, mainly glycerol and ethanol.

Characterization of a glutaraldehyde stabilized yeast cell biocatalyst with β-galactosidase activity

A whole cell biocatalyst (Kluyveromyces lactis) consisting in permeabilized cells stabilized with glutaraldehyde under conditions that preserve the β-galactosidase activity was characterized in connection with autolytic processes, cell wall effects and sensitivity to shear stress and exogenous proteases. Permeabilized cells display a typical autolytic process that was characterized by the accumulation of extracellular amino-nitrogen due to proteolysis, carbohydrate solubilization and release of enzyme activities. The stabilization of the cell system was manifested by the inhibition of the autolytic process. This phenomenon was evident by the absence of proteolysis and enzyme solubilization. Cell wall autolysis appears not to be totally blocked by the glutaraldehyde treatment. The β-galactosidase contained in the stabilized cells was not solubilized when the cell wall was totally digested with exogenous lytic enzymes. Thus, a steric effect mediated by the cell wall was not involved in the insolubilization of the enzyme activity. Actually, the cross-linking reaction gave rise to the formation of an insoluble structure in which the intracellular, and partially the cell wall components, were immobilized. The property of this cluster of being insoluble is independent of the cell wall integrity. This structural feature were confirmed by electron microscopy. The formation of the insoluble structure requires the glutaraldehyde reaction to occur in the intracellular environment. When the cell components were dispersed in the reaction mixture, the cross-linking reaction produces soluble high molecular aggregates containing the β-galactosidase activity. The β-galactosidase was inactivated when the stabilized cells were agitated with glass beads, indicating that the whole cell biocatalyst was sensitive to shear stress. Treatment of the stabilized cells with exogenous proteases, such as trypsine, both solubilize and inactivate the β-galactosidase activity.

One-step Concentration and Partial Purification of Aspergillus kawachii Non-acidic Polygalacturonases by Adsorption to Glass Fiber Microfilters

The non-acidic polygalacturonases produced by Aspergillus kawachii in a glucose/tryptone medium were adsorbed to a glass fiber microfilter that was used to clarify the fermentation broth. Maximum adsorption occurred at pH 3 under low ionic strength conditions. The adsorbed activity could be readily released with a buffer solution at pH 5. Based upon these observations, a separation process was developed which enabled the broth to be clarified and, at the same time, the non-acidic polygalacturonases to be concentrated 20-fold and purified 100-fold in a unique filtration step. The practical advantage of recovering polygalacturonases by a filtration process lies in the simplicity and efficiency of the operation involved.

Solvents production from whey supplemented with corn steep and malt sprouts at 30°C and 37°C

SummaryCorn steep and malt-sprouts were used to replace yeast extract in solvents production from whey at 30°C and 37°C employing two clostridia producer strains. The results show different temperature and growth factors dependence in the two strains. Yields of solvents between 0.16 and 0.32 (g/g) were obtained.