Purification and properties of an alpha-D-xylosidase from Aspergillus niger

Two components of alpha-D-xylosidase (alpha-D-xylosidase I and II) were detected in the culture filtrate of Aspergillus nigher grown in a medium containing Sanzyme 1000-treated Glyloid 2A. The major component (alpha-D-xylosidase I) was purified to an electrophoretically pure state. The purified enzyme showed approximately 540-fold increase in specific activity over the original culture filtrate. The purified enzyme was shown to be an oligomeric protein consisting of four subunits, each of which had a molecular weight of 123,000. The enzyme showed the highest activity at pH 2.5-3.0 and 45 degrees C, and was stable in the pH range from 3.0 to 7.0 and at the temperatures up to 60 degrees C. The isoelectric point of this enzyme was pH 5.6. The purified enzyme was highly specific for p-nitrophenyl alpha-D-xylopyranoside and isoprimeverose (6-O-alpha-D-xylopyranosyl-D-glucopyranose). The apparent Km and Vmax values of the enzyme for p-nitrophenyl alpha-D-xylopyranoside and isoprimeverose were 10.5 mM and 40.8 mumol/min/mg protein, and 2.2 mM and 30 mumol/min/mg protein, respectively. The purified enzyme could also split off the alpha-D-xylopyranosyl residue on the non-reducing terminal of the backbone of oligoxyloglucans such as alpha-D-xylopyranosyl-(1----6)-beta-D-glucopyranosyl- (1----4)-[(alpha-D-xylopyranosyl-(1----6)-]-beta-D-glucopyranosyl- (1----4)-] 2-D-glucopyranose.     

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.     

Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4

A thermotolerant yeast strain named Kluyveromyces marxianus IMB4 was used in a simultaneous saccharification and fermentation (SSF) process using Kanlow switchgrass as a feedstock. Switchgrass was pretreated using hydrothermolysis at 200 degrees C for 10 min. After pretreatment, insoluble solids were separated from the liquid prehydrolyzate by filtration and washed with deionized water to remove soluble sugars and inhibitors. Insoluble solids were then hydrolyzed using a commercial cellulase preparation and the released glucose was fermented to ethanol by K. marxianus IMB4 in an SSF process. SSF temperature was 37, 41, or 45 degrees C and pH was 4.8 or 5.5. SSF was conducted for 7 days. Results were compared with a control of Saccharomyces cerevisiae D(5)A at 37 degrees C and pH 4.8. Fermentation by IMB4 at 45 and 41 degrees C ceased after 3 and 4 days, respectively, when a pH 4.8 citrate buffer was used. Fermentation continued for all 7 days using IMB4 at 37 degrees C and the control. When pH 5.5 citrate buffer was used, fermentation ceased after 96 h using IMB4 at 45 degrees C, and ethanol yield was greater than when pH 4.8 citrate buffer was used (78% theoretical). Ethanol yield using IMB4 at 45 degrees C, pH 5.5 was greater than the control after 48, 72, and 96 h (P < 0.05).     

Physicochemical modification of the excretion product of Saccharomyces cerevisiae killer strains results in fungicidal activity against Candida albicans and Tricophyton mentagrophytes

It is known that certain yeast strains, so called 'killers', can produce and excrete proteinaceous toxins that can induce death of other sensitive strains. We obtained a stable fungicidal factor (SKF) through concentration and stabilization of the excretion product of certain killer strains of Saccharomyces cerevisiae (K1 and K2). The isolated proteinaceous complex exhibited activity at broad ranges of pH (4-7.5) and temperatures (20-37.5 degrees C). It was significantly lethal against Candida albicans and Tricophyton mentagrophytes. SKF showed stability and activity after storage, with a mean half-life of 6 months at 4 degrees C or at -20 degrees C.     

Production of feed enzymes (phytase and plant cell wall hydrolyzing enzymes) by<Emphasis Type="Italic">Mucor indicus</Emphasis> MTCC 6333: Purification and characterization of phytase

The production of phytase and associated feed enzymes (phosphatase, xylanase, CMCase, alpha-amylase and beta-glucosidase) was determined in a thermotolerant fungus Mucor indicus MTCC 6333, isolated from composting soil. Solid-substrate culturing on wheat bran and optimizing other culture conditions (C and N sources, level of N, temperature, pH, culture age, inoculum level), increased the yield of phytase from 266 +/- 0.2 to 513 +/- 0.4 nkat/g substrate dry mass. The culture extract also contained 112, 194, 171, 396, and 333 nkat/g substrate of phosphatase, xylanase, CMCase, beta-glucosidase and alpha-amylase activities, respectively. Simple 2-step purification employing anion exchange and gel filtration chromatography resulted in 21.9-fold purified phytase. The optimum pH and temperature were pH 6.0 and 70 degrees C, respectively. The phytase was thermostable under acidic conditions, showing 82% residual activity after exposure to 60 degrees C at pH 3.0 and 5.0 for 2 h, and displayed broad substrate specificity. The Km was 200 nmol/L and v(lim) of 113 nmol/s per mg protein with dodecasodium phytate as substrate. In vitro feed trial with feed enzyme resulted in the release of 1.68 g inorganic P/kg of feed after 6 h of incubation at 37 degrees C.     

Influence of culture conditions and virulence plasmids on expression of immunoglobulin-binding proteins of Yersinia pseudotuberculosis

The influence of culture conditions and plasmids on immunoglobulin (Ig)-binding activity of two isogenic strains of Yersinia pseudotuberculosis (plasmid-free strain 48(-)82(-) and strain 48(+)82(+) bearing plasmids pYV48 and pVM82) was studied. The highest activity was observed in the bacteria grown on glucose-containing liquid medium in the stationary growth phase. The Ig-binding activity of the bacteria cultured on the liquid medium at pH 6.0 was about 1.5-fold higher than that of the bacteria grown at pH 7.2. Expression of the Ig-binding proteins (IBPs) was most influenced by temperature of cultivation. The IBP biosynthesis was activated in the bacteria grown at 4 degrees C and markedly decreased in those grown at 37 degrees C. The Ig-binding activity of lysates from the bacteria was caused by proteins with molecular weights of 7-20 kD. The activities of the plasmid-free and plasmid-bearing Y. pseudotuberculosis strains (48(-)82(-) and 48(+)82(+), respectively) were analyzed, and the plasmids were shown to have no effect on the IBP expression and biosynthesis, which seemed to be determined by chromosomal genes.     

Assay for trans-p-coumaroyl esterase using a specific substrate from plant cell walls

Cell walls of Coastal Bermuda grass (Cynodon dactylon) were treated with polysaccharide hydrolases to release O-[5-O-(trans-p-coumaroyl)-alpha-L-arabinofuranosyl]-(1----3)-O-be ta-D- xylopyranosyl-(1----4)-D-xylopyranose (PAXX) which was isolated by liquid chromatography. The isolated PAXX was greater than 95% pure as determined by 1H NMR and was used as substrate for a sensitive assay of trans-p-coumaroyl esterase. PAXX was hydrolyzed by culture filtrates from the anaerobic fungus Neocallimastix MC-2. The trans-p-coumaric acid released by enzymatic hydrolysis was assayed by reverse-phase HPLC, and as little as 100 ng of acid could be determined. Steady-state velocities for the release of the acid obeyed Michaelis-Menten kinetics. Vmax was determined to be 1.17 mumol min-1 mg-1 and Km 13.2 microM at pH 7.5 and 30 degrees C.     

Exo-(1----3)-beta-glucanase, autolysin and trehalase activities during yeast growth and germ-tube formation in Candida albicans

Exo-(1----3)-beta-glucanase, beta-glucosidase, autolysin and trehalase were assayed in situ in Candida albicans during yeast growth, starvation and germ-tube formation. Cell viability, germ-tube formation, intracellular glucose-6-phosphate dehydrogenase and beta-glucosidase were unaffected in cells incubated in 0.1 M-HC1 for 15 min at 4 degrees C. However, in situ trehalase, (1----3)-beta-glucanase and autolysin activities in acid-treated cells decreased by 95, 50 and 35% respectively, indicating that these enzymes are, in part, associated with the cell envelope. Trehalase activity increased throughout yeast growth and remained elevated during the first hour of incubation for germ-tube formation. All of the in situ trehalase activity in starved yeast cells could be measured without the permeabilizing treatment. beta-Glucosidase activity declined throughout yeast growth and did not alter during germ-tube formation. Both the (1----3)-beta-glucanase and autolysin activities were optimal at pH 5 X 6, inhibited by gluconolactone and HgCl2, and maximal at 15-16 h during yeast growth. Although autolysin activity increased by 50-100% when starved yeast cells were incubated for germ-tube formation, the in situ (1----3)-beta-glucanase remained constant. When acid-treated starved yeast cells were similarly induced, in situ (1----3)-beta-glucanase increased 100% over 3 h of germ-tube formation. Yeast cells secreted (1----3)-beta-glucanase into the growth medium. This was highest in early exponential phase cultures (34% of the maximum in situ activity) and declined throughout growth. (1----3)-beta-Glucanase was also secreted into the medium during germ-tube formation and this represented 80-100% of the in situ activity in germ-tube forming cells. Both secretion of (1----3)-beta-glucanase and germ-tube formation were inhibited by 2-deoxyglucose, ethidium bromide, trichodermin and azaserine.     

Adaptation of the yeast Yarrowia lipolytica to heat shock

The adaptive response of the yeast Yarrowia lipolytica to heat shock has been studied. Experiments showed that, after 10 min of incubation at 45 degrees C, the survival rate of Yarrowia lipolytica cells was less than 0.1%. Stationary-phase yeast cells were found to be more thermotolerant than exponential-phase cells. The 60-min preincubation of cells at 37 degrees C or pretreatment with low concentrations of H2O2 (0.5 mM) and menadione (0.05 mM) made them more tolerant to heat and to oxidative stress (120 mM hydrogen peroxide). The pH dependence of yeast thermotolerance has also been studied. The adaptation of yeast cells to heat shock and oxidative stress was found to be associated with a decrease in the intracellular level of cAMP and an increase in the activity of antioxidant enzymes (catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and glutathione reductase).     

Isolation of thermotolerant mutants by using proofreading-deficient DNA polymerase delta as an effective mutator in Saccharomyces cerevisiae

Eukaryotic DNA polymerases delta and epsilon, both of which are required for chromosomal DNA replication, contain proofreading 3'-->5'exonuclease activity. DNA polymerases lacking proofreading activity act as strong mutators. Here we report isolation of thermotolerant mutants by using a proofreading-deficient DNA polymerase delta variant encoded by pol3-01 in the yeast Saccharomyces cerevisiae. The parental pol3-01 strain grew only poorly at temperatures higher than 38 degrees C. By stepwise elevation of the incubation temperature, thermotolerant mutants that could proliferate at 40 degrees C were successfully obtained; however, no such mutants were isolated with the isogenic POL3 strain. The recessive hot1-1 mutation was defined by genetic analysis of a weak thermotolerant mutant. Strong thermotolerance to 40 degrees C was attained by multiple mutations, at least one of which was recessive. These results indicate that a proofreading-deficient DNA delta polymerase variant is an effective mutator for obtaining yeast mutants that have gained useful characteristics, such as the ability to proliferate in harsh environments.     

Zygosaccharomyces lentus: a significant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature

Zygosaccharomyces lentus is a yeast species recently identified from its physiology and 18S ribosomal sequencing (Steels et al. 1999).The physiological characteristics of five strains of this new yeast so far isolated were investigated, particularly those of technical significance for a spoilage yeast, namely temperature range, pH range, osmotolerance, sugar fermentation, resistance to food preservatives such as sorbic acid, benzoic acid and dimethyldicarbonate (DMDC; Velcorin). Adaptation to benzoic acid, and growth in shaking and static culture were also investigated. Zygosaccharomyces lentus strains grew over a wide range of temperature (4-25 degrees C) and pH 2.2-7.0. Growth at 4 degrees C was significant. Zygosaccharomyces lentus strains grew at 25-26 degrees C in static culture but were unable to grow in aerobic culture close to their temperature maximum. All Z. lentus strains grew in 60% w/v sugar and consequently, are osmotolerant. Zygosaccharomyces lentus strains could utilize sucrose, glucose or fructose as a source of fermentable sugar, but not galactose. Zygosaccharomyces lentus strains were resistant to food preservatives, growing in sorbic acid up to 400 mg l-1 and benzoic acid to 900 mg l-1 at pH 4.0. Adaptation to higher preservative concentrations was demonstrated with benzoic acid. Resistance to DMDC was shown to be greater than that of Z. bailii and Saccharomyces cerevisiae. This study confirms that Z. lentus is an important food spoilage organism potentially capable of growth in a wide range of food products, particularly low pH, high sugar foods and drinks. It is likely to be more significant than Z. bailii in the spoilage of chilled products.     

Isolation of a bioemulsifier from Candida lipolytica.

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Isolation of a bioemulsifier from Candida lipolytica

The yeast Candida lipolytica produced an inducible extracellular emulsification activity when it was grown with a number of water-immiscible carbon substrates. Negligible emulsification activity was produced by this yeast when it was grown with glucose as the carbon substrate. In hexadecane-supplemented cultures, emulsification activity was first detected after 36 h of growth, with maximum production after 130 h. A water-soluble emulsification activity was partially purified by repeated solvent extractions of the culture filtrate. This emulsifier, which we named liposan, was primarily composed of carbohydrate. Maximum emulsification activity was obtained when the ratio of hexadecane to liposan was 50:1. Maximum emulsification activity was obtained from pH 2 to 5. Liposan was heat stable to temperatures up to 70 degrees C, with a 60% loss in activity after heating for 1 h at 100 degrees C. Liposan effected stable oil-in-water emulsions with a variety of hydrocarbons.     

Monoacylglycerols: glycolipid biosurfactants produced by a thermotolerant yeast, Candida ishiwadae

AIMS: To isolate and characterize biosurfactants produced by a thermotolerant yeast isolated in Thailand. MATERIALS AND RESULTS: Yeast strains isolated from plant material in Thailand were first screened for the ability to produce lipase and biosurfactant. A strain Y12, identified as Candida ishiwadae by physiological tests, survived at 45 degrees C and produced relatively large amounts of biosurfactants. From the culture filtrate of this strain, two glycolipid biosurfactants, a and b, were purified by solvent fractionation, silica gel and ODS column chromatographies. Compounds a and b were determined to be monoacylglycerols; 1-linoleylglycerol and 1-oleylglycerol, respectively. Both compounds exhibited higher surfactant activities tested by the drop collapse test than several artificial surfactants such as sodium dodecyl sulphate. CONCLUSIONS: Glycolipid biosurfactants produced by a thermotolerant yeast, C. ishiwadae were characterized to be monoacylglycerols which exhibited high surfactant activities. SIGNIFICANCE AND IMPACT OF THE STUDY: A thermotolerant yeast strain, C. ishiwadae, could be a potential candidate for producing monoacylglycerols which are useful in industrial applications.     

Growth of a methanol-utilizing yeast

A halophilic thermotolerant yeast species, identified as Hansenula polymorpha Morais et Maia, was isolated from a mixed culture obtained from sea-water from the Arabian Gulf. The species grew on methanol at 25–42°C, pH 3.5–6.7, and in a medium compounded with 75% sea-water. Either thiamin HCl and biotin or yeast extract proved essential for growth. In shake flask studies a depression of the yield was observed when methanol concentration increased; at concentrations in excess of 0.1%, v/v, inhibition of growth also occurred. In a batch culture grown in a 14 l fermenter, the values of T_d, μ and Y_s were found to be 3 h, 0.23 h⁻¹ and 0.38, respectively.     

Differences in thermotolerance induced by heat or sodium arsenite: cell killing and inhibition of protein synthesis

Chinese hamster ovary (CHO) cells became thermotolerant after treatment with either heat for 10 min at 45.5 degrees C or incubation in 100 microM sodium arsenite for 1 h at 37 degrees C. Thermotolerance was tested using heat treatment at 45 degrees C or 43 degrees C administered 6-12 h after the inducing agent. At 45 degrees C thermotolerance ratios at 10(-2) isosurvival levels were 4.2 and 3.8 for heat and sodium arsenite, respectively. Recovery from heat damage as measured by resumption of protein synthesis was more rapid in heat-induced thermotolerant cells than in either sodium arsenite-induced thermotolerant cells or nonthermotolerant cells. Differences in inhibition of protein synthesis between heat-induced thermotolerant cells and sodium arsenite-induced thermotolerant cells were also evident after test heating at 43 degrees C for 5 h. At this temperature heat-induced thermotolerant cells were protected immediately from inhibition of protein synthesis, whereas sodium arsenite-induced thermotolerant cells, while initially suppressed, gradually recovered within 24 h. Furthermore, adding cycloheximide during the thermotolerance development period greatly inhibited sodium arsenite-induced thermotolerance (SF less than 10(-6] but not heat-induced thermotolerance (SF = 1.7 X 10(-1] when tested with 43 degrees C for 5 h. Our results suggest that both the development of thermotolerance and the thermotolerant state for the two agents, while similar in terms of survival, differed significantly for several parameters associated with protein synthesis.     

Leptospirillum-like bacteria and their role in pyrite oxidation

Two strains of Leptospirillum-like bacteria isolated from dumps of Alaverdi and Akhtala sulfide ore deposits in Armenia were studied. The optimum and maximum temperatures for the growth of both strains were 37 and 40 degrees C, respectively. The pH optimum was 2.0-2.3. Bacterial growth and ferrous iron oxidation were inhibited by yeast extract. The pyrite-leaching activity of the Leptospirillum-like bacteria under mesophilic conditions was close to that of Acidithiobacillus ferroxidans and exceeded by 2.0-2.7 times the activity of these moderately thermophilic bacteria at 37 degrees C. The leaching of pyrite by Leptospirillum-like bacteria increased in the presence of sulfur-oxidizing bacteria, particularly, in their association with a thermotolerant sulfur-oxidizing bacterium.     

Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor-controlled bioreactor

AIMS: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor. METHODS AND RESULTS: The parental culture of Saccharomyces cerevisiae ATCC 26602 was mutated using UV treatment. A single thermotolerant mutant was isolated after extensive screening and optimization, and grown on molasses medium in liquid cultures. The mutant was 1.45-fold improved than its wild parent with respect to ethanol productivity (7.2 g l-1 h-1), product yield (0.44 g ethanol g-1 substrate utilized) and specific ethanol yield (19.0 g ethanol g-1 cells). The improved ethanol productivity was directly correlated with titres of intracellular and extracellular invertase activities. The mutant supported higher volumetric and product yield of ethanol, significantly (P相似文献   

Thermoresistance in Saccharomyces cerevisiae yeasts

Under natural conditions, yeast Saccharomyces cerevisiae reproduce, as a rule, on the surface of solid or liquid medium. Thus, life cycle of yeast populations is substantially influenced by diurnal changes in ambient temperature. The pattern in the response of unrestricted yeast S. cerevisiae culture to changes in the temperature of cultivation is revealed experimentally. Yeast population, in the absence of environmental constraints on the functioning of cell chemosmotic bioenergetic system, demonstrates the ability of thermoresistance when the temperature of cultivation switches from the range of 12-36 degrees C to 37.5-40 degrees C. During the transient period that is associated with the temperature switching and lasts from 1 to 4 turnover cycles, yeast reproduction rate remains 1.5-2 times higher than under stationary conditions. This is due to evolutionary acquired adaptive activity of cell chemosmotic system. After the adaptive resources exhausting, yeast thermoresistance fully recovers at the temperature range of 12-36 degrees C within one generation time under conditions of both restricted and unrestricted nourishment. Adaptive significance of such thermoresistance seems obvious enough--it allows maintaining high reproduction rate in yeast when ambient temperature is reaching a brief maximum shortly after noon.     

Effect of the medium composition and cultivation conditions on sporulation in chemolithotrophic bacteria

The possibility of controlling endospore formation by changing cultivation conditions was for the first time shown in acidophilic chemolithotrophic bacteria Sulfobacillus thermosulfidooxidans type strain 1269 and the thermotolerant strain K1 formerly described as "S. thermosulfidooxidans subsp. thermotolerans". Suppression of sporulation occurred when these strains were cultured in Manning's liquid medium with yeast extract. This medium was optimized by gradually reducing the concentrations of ferrous iron salts (the source of energy), phosphorous, nitrogen, and yeast extract and simultaneously increasing the concentrations of calcium, magnesium, and manganese (the elements important for sporogenesis) to attain higher yields of endospores by strains 1269 and K1. As a result, a new medium A was proposed, in which the life cycle of the strains studied culminated in sporulation at a level of 45 and 60%, respectively, of the total cell number. In a series of additional tests, the growth temperature and medium pH were adjusted to obtain the maximum yield of endospores. The optimal ranges found were 40-50 degrees C and pH 1.8-2.2 for strain 1269 and 35-40 degrees C and pH 2.5-2.7 for strain K1. An even higher yield of endospores, amounting to 55 and 75% for strains 1269 and K1, respectively, was obtained when the above growth conditions were combined (growth on medium A at optimal temperatures and pH). Our results suggest a new approach to optimizing sporulation by acidophilic chemolithotrophs, which consists in limiting the energy and nutrient sources and using temperature and pH values within the tolerance bounds of these cultures but outside their growth of optimum ranges.