Characterization of Schwanniomyces castellii mutants with increased productivity of amylases

Sills, A. M.; Zygora, P. S. J.; Stewart, Graham G.

doi:10.1007/bf00252589

Cited by 37 publications

(11 citation statements)

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“…Amylolytic activity in S. castellii is repressed at glucose concentrations greater than 3 mM (30). Expression of intact glucoamylase mRNA was repressed in strain 1402 cells when these cells were exposed to medium containing 56 mM glucose ( Fig.…”

Section: Resultsmentioning

confidence: 90%

“…2-Deoxyglucose represses expression of glucoamylase. The glucose analog 2-deoxyglucose is transported into but is believed not to be metabolized by yeast cells (1,30,35,44). This analog has been used with various regimens of mutagenesis to select carbon catabolite-derepressed mutants that produce amylolytic activity during growth on glucose (32).…”

Section: Resultsmentioning

confidence: 99%

“…This analog has been used with various regimens of mutagenesis to select carbon catabolite-derepressed mutants that produce amylolytic activity during growth on glucose (32). A few mutants of this type have been isolated from different Schwanniomyces species (6,20,30,39). To determine whether 2-deoxyglucose affects glucoamylase expression in the same way as glucose, cells adapted to minimal medium with 1% glucose were shifted to minimal medium containing 1% (wt/vol) 2-deoxyglucose, harvested at 30, 60, and 90 min, and analyzed for glucoamylase expression as described earlier (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…We and others are interested in developing the genetics of members of the budding yeast Schwanniomyces (7,24), particularly in view of their ability to hydrolyze starch (3,5,6,10,15,19,21,23,26,28,29,31,39,40). The 146-kilodalton (kDa) extracellular glucoamylase of Schwanniomyces castellii that we have characterized is of particular interest because it has both a-1,4 and ot-1,6 activity and is thermally labile (29,30). The general expression properties of this enzyme in relation to growth and carbon source were described by using enzymatic and immunological assays (7).…”

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confidence: 99%

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Expression and regulation of glucoamylase from the yeast Schwanniomyces castellii

Dowhanick¹,

Russell²,

Scherer³

et al. 1990

J Bacteriol

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Expression of the 146-kilodalton (kDa) extracellular glucoamylase by the budding yeast Schwanniomyces castellii is induced by maltose and starch. By use of antiglucoamylase antisera, we found that this expression was regulated at the level of the mRNA, taking place within 30 min after exposure of yeast cells to the respective sugars. Polyacrylamide gel electrophoresis analysis of the in vitro-translated products of total RNA from maltose-treated cells established that the glucoamylase precursor was approximately 120 kDa in size. Stable glucoamylase transcript was not produced in cells exposed to glucose, 2-deoxyglucose, and heat shock. Cells exposed to these two sugars also degraded intracellular and extracellular glucoamylase. In the presence of sugars such as cellobiose, galactose, lactose, and xylose or in the absence of any carbohydrate, a low-level, constitutive-like expression of this preglucoamylase occurred. The nascent glucoamylase underwent at least two posttranslational modifications, resulting in a 138-kDa cell-associated form and the 146-kDa active form that was found free in the medium. These results suggest that glucoamylase expression is tightly regulated similarly to expression of the enzymes responsible for maltose metabolism in Saccharomyces yeasts.

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Section: Resultsmentioning

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Expression and regulation of glucoamylase from the yeast Schwanniomyces castellii

Dowhanick¹,

Russell²,

Scherer³

et al. 1990

J Bacteriol

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“…High levels of hydrolytic enzymes would be advantageous at the beginning of fermentation to increase reducing sugars for fermentation. Sills et al (1984) and Wilson et al (1982) reported that insufficient glucoamylase activity was the main limiting factor for direct starch fermentation by Schwanniomyces spp.…”

Section: Resultsmentioning

confidence: 99%

Fermentation of starch byKlebsiella oxytoca p2, containing plasmids with ?-amylase and pullulanase genes

Santos

Araújo

Barros

et al. 1999

Biotechnol. Bioeng.

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Isolation and characterization of mutants of Trichosporon adeninovorans resistant to 2‐deoxy‐D‐glucose

Büttner

Scheit

Bode

et al. 1989

J. Basic Microbiol.

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We isolated stable mutants from Trichosporon adeninovorans resistant to 2-deoxy-~-glucose. All mutants were altered in the C-catabolite repression and produced different amounts of glucoamylase and 8-glucosidase when grown on several carbon sources. In addition to the C-catabolite derepression of these extracellular enzymes, a repression defect .of isocitrate lyase was found.Several yeast species have been investigated as potentially valuable candidates for the production of single cell protein or ethanol from starch. Therefore, in various laboratories extracellular amylolytic enzymes of yeasts have become subject of study (CLEMENTI et al. 1980, SA-CORREIA and VAN UDEN 1981, SPENCER-MARTINS 1982, TUCKER et al. 1984, SILLS et al. 1984a, MODENA et al. 1986, CLEMENTI and ROSS 1986.Mutants resistant to carbon catabolite repression by glucose have been investigated for the genetical characterization and regulation of production of extracellular enzymes. These mutants were isolated by selecting for 2-deoxy-~-glucose or glucoseamine resistance (ZIMMERMANN and SCHEEL 1977, VAN UDEN et al. 1980, CABECA-SILVA 1982, MCCANN and BARNETT 1984, SILLS et al. 1984b, MANCZINGER 1985, BROWN et al. 1987, DE MOT and VERACHTERT 1987.Recently we characterized an extracellular glucoamylase of a new yeast species, Trichosporon adeninovorans, described by MIDDELHOVEN et al. (1984) (BUTTNER et al. 1987). In this paper we report on the isolation and characterization of carbon catabolite derepression mutants from T. adeninovorans using 2-deoxy-~-glucose. Materials and methodsStrain and growth conditions: Trichosporon adeninovorans LS3 is a strain from our laboratory. Yeast cells were grown at 30 "C on a rotary shaker in 500-ml-flasks containing 200 ml of a minimal salt medium (TANAKA et al. 1967) supplemented with 1 mg biotin and 1 mg thiamine per litre. Ethanol, hexadecane, glucose, galactose, maltose or soluble starch were used as carbon source (1 %). The growth was followed by measuring the absorbance at 650 nm.Induction and isolation ofmutants: Cells ofa 12-h-culture werecentrifhgated and resuspended in 0.9 % NaCl solution. Mutants resistant to repression of starch hydrolysis were isolated after N-methyl-N -nitro-"-nitrosoguanidine mutagenesis. The concentration of the mutagen was 2.5 mM and a cell concentration of 10*/ml was used. After 2 h at 25 "C the cells were centrifugated and washed with 0.9 % NaCl solution. After resuspension the cells were plated on mineral salt medium containing 1 %corn starch and 0.1 % 2-deoxy-~-glucose, solidified with 2 % agar. Mutant colonies were selected after 72 h.Enzyme preparation: To determine some enzyme activities either culture supernatant, cell-free extract or permiabilized cells were used. To prepare cell-free extract yeast was resuspended in 50 mM Tris/HCl buffer (pH 8.0), disrupted by passing them twice through a X-pressure cell and centrifugated at 20,000 x g for 20 min. Permiabilized cells were produced by cooling of yeast cells overnight at -20 "C in 100 mM sodium acetate buffer (pH 5.0) ...

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Characterization of Schwanniomyces castellii mutants with increased productivity of amylases

Cited by 37 publications

References 14 publications

Expression and regulation of glucoamylase from the yeast Schwanniomyces castellii

Expression and regulation of glucoamylase from the yeast Schwanniomyces castellii

Fermentation of starch byKlebsiella oxytoca p2, containing plasmids with ?-amylase and pullulanase genes

Isolation and characterization of mutants of Trichosporon adeninovorans resistant to 2‐deoxy‐D‐glucose

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