Assimilation of Lemonade-Processing Wastewater by Yeasts

Of the five species of yeasts examined in shake-flask studies at 30°C, only Candida utilis and Saccharomyces fragilis gave higher cell yields (>2.5 g /liter) in 32 h and reduced approximately 87% of the biological oxygen demand in lemonade-processing wastewater fortified with 0.05% ammonium sulfate and 0.01% potassium phosphate. The latter, however, settled more rapidly than the former.     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.     

Aspects of inorganic nitrogen assimilation in yeasts

Cultures of Candida utilis utilise glutamate in preference to ammonia and ammonia in preference to nitrate. The nitrate reductase of this organism is induced by nitrate and repressed in cultures grown on glutamate or ammonia. Nitrate-grown cultures of C. utilis, irrespective of the medium nitrate concentration, behave as though nitrogen-limited. In contrast to C. utilis, Saccharomyces cerevisiae utilises ammonia in preference to glutamate. In eight yeasts studied the highest cellular contents of biosynthetic NADP-linked glutamate dehydrogenase were found in batch cultures containing low concentrations of ammonia or in nitrogen-limited chemostat cultures. NAD-linked glutamate dehydrogenase activity was detected in extracts of cells grown in the presence of glutamate but not in those grown in the presence of ammonia.     

Applicability of Yeast Extracellular Proteinases in Brewing: Physiological and Biochemical Aspects

A general screening survey for expression of extracellular acid proteinase production was performed on over 100 cultures belonging to the genus Saccharomyces. Although two strains of Saccharomyces cerevisiae showed positive extracellular proteinase phenotypes in plate tests, it was not possible to demonstrate proteolytic activities in cell-free culture supernatants in assays performed at beer pH values. Of several yeasts from other genera examined, Saccharomycopsis fibuligera and Torulopsis magnoliae produced extracellular proteinases with desirable properties. Proteolytic activities were detected in assays performed at beer pH values and at lower temperature. Brewer's wort served as a highly inducing medium for extracellular proteinase production, with T. magnoliae yielding enzyme of highest specific activity. In fact, commencement of enzyme production was detected shortly after the onset of exponential growth in brewer's wort. Inclusion of crude enzyme preparations in brewer's wort inoculated simultaneously with brewer's yeast reduced final ethanol yields slightly and was found to be effective in reducing chill haze formation in bottled beer.     

Molecular analysis of inorganic nitrogen assimilation in yeasts

Assimilation of nitrate and various other inorganic nitrogen compounds by different yeasts was investigated. Nitrate, nitrite, hydroxylamine, hydrazine, ammonium sulphate, urea and L-asparagine were tested as sole sources of nitrogen for the growth of Candida albicans, C. pelliculosa, Debaryomyces hansenii, Saccharomyces cerevisiae, C. tropicalis, and C. utilis. Ammonium sulphate and L-asparagine supported the growth of all the yeasts tested except D. hansenii while hydroxylamine and hydrazine failed to support the growth of any. Nitrate and nitrite were assimilated only by C. utilis. Nitrate utilization by C. utilis was also accompanied by the enzymatic activities of NAD(P)H: nitrate oxidoreductase (EC 1.6.6.2) and NAD(P)H: nitrite oxidoreductase (EC 1.6.6.4), but not reduced methyl viologen-or FAD-nitrate oxidoreductases (EC 1.7.99.4). It is demonstrated here that nitrate and nitrite reductase activities are responsible for the ability of C. utilis to assimilate primary nitrogen.     

Enzymatic Release of Invertase from Cell Ghosts of Candida utilis

1. The liberation of invertase (β-fructofuranosidase, EC 3.2.1.26) from Candida utilis at autolysis of the cells was found to begin after the autolysis was almost completed. The autolysis residue at this stage consisted mainly of cell walls (ghosts). A suspension of washed cell ghosts released invertase on further incubation and this liberation was stimulated by the addition of reducing agents such as mercaptoethanol, or proteolytic enzymes such as papain, as has been known in the release of the invertase of Saccharomyces cerevisiae.

2. The invertase activity of the cell ghosts was not lost when the suspension was heated at 60°C. However, the invertase of the heated cell ghosts was not liberated even if the above stimulative agents were added.

3. Several commercial enzymes were shown to stimulate the liberation of invertase from the heated cell ghosts and “Zymolyase,? one of the effective enzymes, was fractionated. One fraction isolated from the preparation showed a striking effect on the liberation of invertase but this fraction did not show lytic activity on brewer’s yeast cells.     

Production of intracellular enzymes by enzymatic treatment of yeast

Enzymatic extraction of intracellular enzymes from various yeasts by glucanase was investigated. Favourable conditions for lysis and release of intracellular enzymes were established. The effects of yeast concentration, growth phase of yeast, storage temperature and pretreatment of yeast were studied. The yeasts investigated can be divided into two groups. The first, Kluyveromyces lactis, Saccharomyces cerevisiae, Saccharomyces oviformis, Torulopsis glabrata, Hansenula polymorpha and local bakers' yeast, lysed relatively easily (70–80% of the cells), especially when cells from the logarithmic growth phase were treated. The second, Candida utilis and Candida vini, were more susceptible to lysis (40–50%) when cells were taken from the stationary phase. Release of two enzymes, glycerol kinase from Candida utilis grown on glycerol and formate dehydrogenase from Torulopsis glabrata grown on methanol was examined. The highest specific activities were obtained by incubating the cells with glucanase for 1.5 h at 37°C. Inactivation of the released enzyme was relatively low. After 12 h of enzymatic treatment at 28°C glycerol kinase maintained about 50%, and formate dehydrogenase over 80%, of the original activities.     

Accumulation and metabolism of selenium by yeast cells

This paper examines the process of selenium bioaccumulation and selenium metabolism in yeast cells. Yeast cells can bind elements in ionic from the environment and permanently integrate them into their cellular structure. Up to now, Saccharomyces cerevisiae, Candida utilis, and Yarrowia lipolytica yeasts have been used primarily in biotechnological studies to evaluate binding of minerals. Yeast cells are able to bind selenium in the form of both organic and inorganic compounds. The process of bioaccumulation of selenium by microorganisms occurs through two mechanisms: extracellular binding by ligands of membrane assembly and intracellular accumulation associated with the transport of ions across the cytoplasmic membrane into the cell interior. During intracellular metabolism of selenium, oxidation, reduction, methylation, and selenoprotein synthesis processes are involved, as exemplified by detoxification processes that allow yeasts to survive under culture conditions involving the elevated selenium concentrations which were observed. Selenium yeasts represent probably the best absorbed form of this element. In turn, in terms of wide application, the inclusion of yeast with accumulated selenium may aid in lessening selenium deficiency in a diet.     

Effect of growth rate on the glucose metabolism of yeast grown in continuous culture. Radiorespirometric studies

Specifically labelled¹⁴C-d-glucose was used to estimate the percentage participation of glycolysis and pentose phosphate cycle in the glucose catabolism ofCandida utilis andSaccharomyces cerevisiae. The two yeasts were cultivated at various growth rates (0.1 to 0.5 h^?1) in a chemostat on synthetic medium limited with glucose under aerobic conditions. The results show a considerable increase in the percentage participation of pentose phosphate cycle in the glucose catabolized bySaccharomyces cerevisiae with the increase in specific growth rate. However, inCandida utilis, the specific growth rate does not influence significantly the part of glucose catabolized via pentose phosphate cycle, but its absolute values are relatively higher than inSaccharomyces cerevisiae. A rough quantitative estimate indicates that a maximum of 60 to 72% of the assimilated glucose is catabolized through the pentose phosphate cycle while inSaccharomyces cerevisiae the percentage participation of the pentose phosphate cycle varies from 24 to 60% (maximum) and 9 to 34% (minimum).     

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.     

Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on apple processing wastes

Sequential cultures of the yeasts Saccharomycopsis fibuliger and Candida utilis were grown on selected wastes from the processing of apples. Effluent from cider manufacture supported the growth of 45.4 g cells/100 g substrate and C. utilis formed 96% of the viable cells in the harvested biomass. Whole, unripe apples yielded 44 g cells/100 g substrate with a reduction in the substrate viscosity of 84%. C. utilis formed 56% of the viable cells in the harvested biomass. Effluent from pectin manufacture contained a substantial proportion of reducing compounds and supported the growth of C. utilis without prehydrolysis by S. fibuliger, to yield 33 g cells/100 g substrate.     

Respiratory capacities of mitochondria of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 grown under glucose limitation

A comparative study was made of the in vitro respiratory capacity of mitochondria isolated from Saccharomyces cerevisiae and Candida utilis grown in glucose-limited chemostat cultures. An electron-microscopic analysis of whole cells revealed that the volume density of mitochondria was the same in both yeasts. Mitochondria from both organisms exhibited respiratory control with NADH, pyruvate + malate, 2-oxoglutarate + acetate or malate, and ethanol. The rate of oxidation of these compounds by isolated mitochondria was the same in both yeasts. The rate of oxidation of NADPH by mitochondria from S. cerevisiae was 10 times lower than by those from C. utilis. However, this low rate probably has no influence on the overall in vivo respiratory capacity of S. cerevisiae. The results are discussed in relation to the differences in metabolic behaviour between S. cerevisiae and C. utilis upon transition of cultures from glucose limitation to glucose excess. It is concluded that the occurrence of alcoholic fermentation in S. cerevisiae under these conditions does not result from a bottleneck in the respiratory capacity of the mitochondria.     

Production,characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications

In this review article, the extracellular enzymes production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. Several yeast strains which could produce different kinds of extracellular enzymes were selected from the culture collection of marine yeasts available in this laboratory. The strains selected belong to different genera such as Yarrowia, Aureobasidium, Pichia, Metschnikowia and Cryptococcus. The extracellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase and phytase, as well as killer toxin. The conditions and media for the enzyme production by the marine yeasts have been optimized and the enzymes have been purified and characterized. Some genes encoding the extracellular enzymes from the marine yeast strains have been cloned, sequenced and expressed. It was found that some properties of the enzymes from the marine yeasts are unique compared to those of the homologous enzymes from terrestrial yeasts and the genes encoding the enzymes in marine yeasts are different from those in terrestrial yeasts. Therefore, it is of very importance to further study the enzymes and their genes from the marine yeasts. This is the first review on the extracellular enzymes and their genes from the marine yeasts.     

Secreted xylanase XynA mediates utilization of xylan as sole carbon source in <Emphasis Type="Italic">Candida utilis</Emphasis>

The fodder yeast Candida utilis is able to use xylose mono- and oligomers as sources of carbon but not the abundant polymer xylan. C. utilis transformants producing the Penicillium simplicissimum xylanase XynA were constructed using expression vectors encoding fusions of the Saccharomyces cerevisiae Mfα1 pre-pro secretion leader to XynA. The Mfα1-XynA fusion was efficiently processed in transformants and XynA was secreted almost quantitatively into the culture medium. Secreted XynA was enzymatically active and allowed transformants to grow on xylan as the sole carbon source. Addition of a second expression unit for the heterologous green fluorescent protein (GFP) generated C. utilis transformants, which showed intracellular GFP fluorescence during growth on xylan. The results suggest that xylanase-producing C. utilis is suited as a cost-effective host organism for heterologous protein production and for other biotechnical applications.     

Biochemical characterization and evaluation of invertases produced from Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa for the production of fructooligosaccharides

Abstract

Invertases are used for several purposes; one among these is the production of fructooligosaccharides. The aim of this study was to biochemically characterize invertase from industrial Saccharomyces cerevisiae CAT-1 and Rhodotorula mucilaginosa isolated from Cerrado soil. The optimum pH and temperature were 4.0 and 70?°C for Rhodotorula mucilaginosa invertase and 4.5 and 50?°C for Saccharomyces cerevisiae invertase. The pH and thermal stability from 3.0 to 10.5 and 75?°C for R. mucilaginosa invertase, respectively. The pH and thermal stability for S. cerevisiae CAT-1 invertase from 3.0 to 7.0, and 50?°C, respectively. Both enzymes showed good catalytic activity with 10% of ethanol in reaction mixture. The hydrolysis by invertases occurs predominantly when sucrose concentrations are ≤5%. On the other hand, the increase in the concentration of sucrose to levels above 10% results in the highest transferase activity, reaching about 13.3?g/L of nystose by S. cerevisiae invertase and 12.6?g/L by R. mucilaginosa invertase. The results demonstrate the high structural stability of the enzyme produced by R. mucilaginosa, which is an extremely interesting feature that would enable the application of this enzyme in industrial processes.     

Screening and reaction engineering for the bioreduction of ethyl benzoylacetate and its analogues

Microbial reduction of benzoylacetates is already an established part of the synthetic toolbox to obtain chiral ethyl 3-hydroxy-3-phenylpropionate although bioreduction yields are low to moderate. A 30% increase in the enantioselectivity to 99% ee and a significant improvement in the yields to around 85% were achieved by combining simple screening procedures and a reaction engineering strategy. Three experimental parameters were selected for investigation: the influences of glucose, enzymatic inhibitor and biocatalyst immobilization. The screened yeasts Pichia kluyveri, Pichia stipitis and Candida utilis were found to give better yields and ee's for ethyl benzoylacetate 1a, p-nitrobenzoylacetate 1b and p-methoxybenzoylacetate 1c, respectively, with addition of glucose, α-chloroacetophenone as inhibitor and immobilization of the yeasts in alginate beads. Our results demonstrate that the optimized process can be implemented on a preparative scale without any loss in yield and ee.     

Production of yeast invertase from sauerkraut waste

Sauerkraut waste was found to be a favorable medium for the production of invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) by Candida utilis.     

Production of the Carotenoids Lycopene, β-Carotene, and Astaxanthin in the Food Yeast Candida utilis

The food-grade yeast Candida utilis has been engineered to confer a novel biosynthetic pathway for the production of carotenoids such as lycopene, β-carotene, and astaxanthin. The exogenous carotenoid biosynthesis genes were derived from the epiphytic bacterium Erwinia uredovora and the marine bacterium Agrobacterium aurantiacum. The carotenoid biosynthesis genes were individually modified based on the codon usage of the C. utilis glyceraldehyde 3-phosphate dehydrogenase gene and expressed in C. utilis under the control of the constitutive promoters and terminators derived from C. utilis. The resultant yeast strains accumulated lycopene, β-carotene, and astaxanthin in the cells at 1.1, 0.4, and 0.4 mg per g (dry weight) of cells, respectively. This was considered to be a result of the carbon flow into ergosterol biosynthesis being partially redirected to the nonendogenous pathway for carotenoid production.     

Growth of Saccharomycopsis fibuliger and Candida utilis in mixed culture on pectic materials

Hydrolysis of pectin by Saccharomycopsis fibuliger and cell growth on the products of hydrolysis by Candida utilis require different incubation conditions. A three-stage sequential culture is described in which S. fibuliger was first grown under aerobic conditions to generate cell mass. The concentration of dissolved oxygen was then reduced to promote pectolytic activity and reduce the number of viable cells in the culture. Finally culture conditions were adjusted to promote the growth of C. utilis in mixed culture with S. fibuliger. The presence of C. utilis increased the rate of pectolytic activity by S. fibuliger. A yeast product, containing 98% C. utilis cells, was obtained from the mixed culture grown on 10 g l⁻¹ pectin. Cell yields using starch or an equal mixture of starch and pectin were similar to those reported in the Symba process, although lower cell yields were recorded using pectin alone.