Catabolite Inactivation in the Methylotrophic Yeast Pichia pastoris

Inactivation of the alcohol oxidase enzyme system of Pichia pastoris, during the whole-cell bioconversion of ethanol to acetaldehyde, was due to catabolite inactivation. Electron microscopy showed that methanol-grown cells contained peroxisomes but were devoid of these microbodies after the bioconversion. Acetaldehyde in the presence of O₂ was the effector of catabolite inactivation. The process was initiated by the appearance of free acetaldehyde, and was characterized by an increase in the level of cyclic AMP, that coincided with a rapid 55% drop in alcohol oxidase activity. Further enzyme inactivation, believed to be due to proteolytic degradation, then proceeded at a constant but slower rate and was complete 21 h after acetaldehyde appearance. The rate of catabolite inactivation was dependent on acetaldehyde concentration up to 0.14 mM. It was temperature dependent and occurred within 24 h at 37°C and by 6 days at 15°C but not at 3°C. Alcohol oxidase activity was psychrotolerant, with only a 17% decrease in initial specific activity over a temperature drop from 37 to 3°C. In contrast, protease activity was inhibited at temperatures below 15°C. When the bioconversion was run at 3°C, catabolite inactivation was prevented. In the presence of 3 M Tris hydrochloride buffer, 123 g of acetaldehyde per liter was produced at 3°C, compared with 58 g/liter at 30°C. By using 0.5 M Tris in a cyclic-batch procedure, 140.6 g of acetaldehyde was produced.     

Der Einfluß des Thioharnstoffs auf den Sauerstoffverbrauch der Fische in hypothermischen Bedingungen

Zusammenfassung Die Wirkung des in Wasser gegebenen Thioharnstoffs auf den Sauerstoffverbrauch der Fische (Carassius auratus gibelio Bloch.) wird untersucht. Die Fische waren an niedrige Temperaturen angepaßt.Der Sauerstoffverbrauch der Fische bei niedrigen Temperaturen (7–9° C) während einer 30tägigen Behandlung mit Thioharnstoff (1 g/l) erreicht eine durchschnittliche Steigerung von 61,23%.In ähnlichen Temperaturbedingungen (5–7° C) und während der gleichen Behandlungsperiode verursacht eine doppelte Thioharnstoffdosis (2 g/l) eine Steigerung des Sauerstoffverbrauchs der Fische.Der hypermetabolische Effekt des Thioharnstoffs ist schon nach den ersten drei Tagen der Behandlung deutlich; er zeigt eine beträchtliche Verminderung in den folgenden 24 Std nach dem Einstellen der Behandlung.

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The action of thiourea administered in water on the oxygen consumption of fishes in hypothermic conditions

Summary The action of thiourea administered in water on the oxygen consumption of fishes (Carassius auratus gibelio Bloch.) acclimatized to low temperatures (7–9° C) was investigated.The results of the performed experiments led to the following conclusions.At 7 to 9° C, during a thiourea treatment period of 30 days (1 g/l of water), the oxygen consumption of fishes recorded an average increase by 61.23 per cent.Under similar conditions (5–7° C) and doubling the thiourea dosis (2 g/l of water) an increase of the oxygen consumption by 158.92 per cent was stated.The hypermetabolic effect of thiourea is onsetting after the first three days of treatment and decreases significantly only after 24 hours from stopping the latter.

     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Microbial production of xylitol from D-xylose using Candida tropicalis

Candida tropicalis DSM 7524 was used to produce xylitol from d-xylose. The fermentation conditions were optimized during continuous cultivation. The strain employed showed no great dependence upon temperature in a range between 30° C and 37° C. It achieved its best yield of xylitol from d-xylose at a pH value of 2.5. Such low pH values allow non sterile cultivation, which is a major economic factor. With an oxygen uptake rate of 0.8–1 ml oxygen per litre culture medium, the C. tropicalis produce xylitol at a yield of between 77% and 80% of the theoretical value. Higher yeast extract concentrations prevent the conversion of d-xylose into xylitol. d-xylose acts as a growth inhibitor in higher concentrations. The maximum xylitol yield was reached at a d-xylose concentration of around 100 g/l. In a non sterile batch culture with substrate shift 220 g/l xylitol were produced from 300 g/l d-xylose at a xylitol productivity rate of 0.37 g/(lh). In order to increase the specific yield, C. tropicalis was immobilised on porous glass and cultivated in a fluidized bed reactor. In a continuous non sterile cultivation with immobilised cells 155 g/l d-xylose produced 90–95% g/l xylitol with a productivity of 1.35 g/(lh).Mr. S. S. da Silva was a visiting scientist to the GBF. He was supported by a scholarship from the National Council of Scientific and Technological Development, Brasilia, Brazil (CNPq).We also would like to gratefully acknowledge the support of Prof. Dr. Michele Vitolo of the University of Sao Paulo, and the Centre for Biotechnology and Chemistry, Lorena, S. P. Brazil, in particular the Department of Fermentative Process.We are grateful to Prof. Rainer Jonas, head of the International Cooperation between Germany/Brazil for the helpful discussions and Dr. Heinrich Lönsdorf (GBF) for the Scanning electron micrographs.Dedicated to the 65th birthday of Prof. Dr. Fritz Wagner.     

Influence of temperature,pH and dissolved oxygen on growth of Brevibacterium linens in a fermentor

Summary The effects of temperature (20°, 25°, 30° C), pH (7.5, 8.0, 8.5) and dissolved oxygen (40%, 45%, 50%, 55%, 60% of concentration in air-saturated medium) on the growth of Brevibacterium linens were studied with cultures on sodium l-lactate and casamino acids in a regulated fermentor, using a factorial design methodology and an analysis of variance programme. The effect on growth rates was very striking: an extremely significant quadratic effect of oxygen, a marked negative effect of pH and a great negative interaction between pH and temperature were observed. Growth, transition from cocci to rods and pigmentation were inhibited by dissolved oxygen levels of 40% and 60%, while the level 50% produced higher absorbance values and growth rates.     

Isolation of mutants of Penicillium purpurogenum resistant to catabolite repression

 The strain Penicillium purpurogenum P-26 was subjected to UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine treatment and mutants were isolated capable of synthesizing cellulase under the conditions of a high concentration of glucose. Initially mutants resistant to catabolite repression by 2-deoxy-D-glucose were isolated on Walseth’s cellulose/agar plates containing 15–45 mM 2-deoxy-D-glucose. These mutants were again screened for resistance to catabolite repression by glycerol or glucose on Walseth’s cellulose/agar plates containing 50 g/l glycerol or 50 g/l glucose respectively. Four mutants with different sizes of clearing zone on Walseth’s cellulose/agar plates containing 50 g/l glucose were selected for flask culture. Among them, the mutant NTUV-45-4 showed better carboxymethylcellulase activity in flask culture containing 1% Avicel plus 3% glucose than did the parental strain. Received: 9 October 1995/Received revision: 27 November 1995/Accepted: 8 January 1996     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

Stability studies and effect of the initial oleic acid concentration on lipase production by Candida rugosa

The production of lipase by Candida rugosa in batch cultures was studied. The initial concentration of the carbon source employed, oleic acid, had an important effect on the final lipolytic activity levels. The maximum lipase/substrate yield and specific productivity obtained correspond to an initial oleic acid concentration of 2 g/l. At higher concentrations, up to 8 g/l oleic acid, specific productivity decreased. Lipase production was not observed below 1 g/l oleic acid. Lipase inactivation in culture broth due to surface forces and shear stress at the gas/liquid interface was not observed. There was no shear stress denaturation at stirring rates of 250, 500 and 750 rpm. No temperature inactivation was detected up to 50° C. Two different lipases with a similar molecular weight of 60kDa were purified from culture broth.     

Some factors affecting the respiration of some aquatic plants

Summary The oxygen consumption of four aquatic plants has been determined at various concentrations of dissolved oxygen and at three different temperatures.Oxygen consumption rates (mg oxygen/g dry weight per hr) at 20°C in air-saturated water were Berula erecta, 1.25; Callitriche obtusangula, 2.8; Hippuris vulgaris, 1.96; and Ranunculus pseudofluitans, 1.90.Oxygen consumption rates increased with increase in dissolved-oxygen concentration within the experimental limits of 1.2–17 p.p.m. dissolved oxygen. The relation of oxygen consumption to this range of oxygen concentrations can be described by the empirical equation R = aC ^b. Increase of temperature has been shown to increase the rates of oxygen consumption. Q₁₀ values ranging from 1.32 to 3.48 have been obtained.     

Continuous 2-keto-<Emphasis Type="SmallCaps">l</Emphasis>-gulonic acid fermentation from <Emphasis Type="SmallCaps">l</Emphasis>-sorbose by <Emphasis Type="Italic">Ketogulonigenium vulgare</Emphasis> DSM 4025

A single-stage continuous fermentation process for the production of 2-keto-l-gulonic acid (2KGA) from l-sorbose using Ketogulonigenium vulgare DSM 4025 was developed. The chemostat culture with the dilution rate that was calculated based on the relationship between the 2KGA production rate and the 2KGA concentration was feasible for production with high concentration of 2KGA. In this system, 112.2 g/L of 2KGA on the average was continuously produced from 114 g/L of l-sorbose. A steady state of the fermentation was maintained for the duration of more than 110 h. The dilution rate was kept in the range of 0.035 and 0.043 h⁻¹, and the 2KGA productivity was 3.90 to 4.80 g/L/h. The average molar conversion yield of 2KGA from l-sorbose was 91.3%. Under the optimal conditions, l-sorbose concentration was kept at 0 g/L. Meanwhile, the dissolved oxygen level was changing in response to the dilution rate and 2KGA concentration. In the dissolved oxygen (DO) range of 16% to 58%, it was revealed that the relationship between DO and D possessed high degree of positive correlation under the l-sorbose limiting condition (complete consumption of l-sorbose). Increasing D closer to the critical value for washing out point of the continuous fermentation, DO value tended to be gradually increased up to 58%. In conclusion, an efficient and reproducible continuous fermentation process for 2KGA production by K. vulgare DSM 4025 could be developed using a medium containing baker’s yeast without using a second helper microorganism.     

Histidine decarboxylase production by Lactobacillus hilgardii: Effect of organic acids

Histidine decarboxylase production from Lactobacillus hilgardii 5w, isolated from wine, was inhibited by the presence of l-malic acid in the basal culture medium. The inhibition was related to l-malic acid concentration. The maximal production of the enzyme at 12 h of culture incubated at 30°C was inhibited 71% by 2 g/L l-malic acid and 47% by 0.5 g/L. In these conditions l-malic acid consumption was 16% and 20% respectively. The addition of 300 mg/L citric acid to the basal medium stimulated the enzyme production from 9 to 45 nmoles/min/mg dry weight, and the increase was correlated with citric acid concentration. When different concentrations of l-malic acid were added to the basal medium plus 200 mg/L citric acid, reversion of stimulation was observed, achieving the maximum at a concentration of 2 g/L. In this case, citric acid comsumption was not modified, whereas L-malic acid utilization was higher.     

Production of rosmarinic acid by<Emphasis Type="Italic">Lavandula vera</Emphasis> MM cell suspension in bioreactor: effect of dissolved oxygen concentration and agitation

The relationship between dissolved oxygen (DO) concentration, agitation rate and growth of Lavandula vera MM and rosmarinic acid biosynthesis was investigated in 3 l laboratory bioreactor. Lavandula vera MM cell suspension accumulated the highest amounts of biomass (34.8 g/l) and rosmarinic acid (1870.6 mg/l) on day 12 of cultivation at 50% dissolved oxygen and agitation speed 100 rpm and at 30% dissolved oxygen and agitation speed 300 rpm, respectively.     

The effect of pH,temperature and sucrose concentration on high productivity continuous ethanol fermentation using Zymomonas mobilis

Summary A flocculent strain of Zymomonas mobilis was used for ethanol production from sucrose. Using a fermentor with cell recycle (internal and external settler) high sugar conversion and ethanol productivity were obtained. At a dilution rate of 0.5 h^-1 (giving 96% sugar conversion) the ethanol productivity, yield and concentrations respectively were 20 g/l/h, 0.45 g/g and 40 g/l using a medium containing 100 g/l sucrose. At a sucrose concentration of 150 g/l, the ethanol concentration reached 60 g/l. The ethanol yield was 80% theoretical due to levan and fructo-oligomer formation. No sorbitol was detected. This fermentation was conducted at a range of conditions from 30 to 36°C and from pH 4.0 to 5.5.     

Biotransformation of benzeldehyde to l-phenylacetylcarbinol,an intermediate in l-ephedrine production,by immobilized Candida utilis

Biotransformation of benzaldehyde to l-phenylacetylcarbinol (l-PAC) as a key intermediate for l-ephedrine synthesis has been evaluated using immobilized Candida utilis. During biotransformation, the benzaldehyde level and respiratory quotient significantly affected both l-PAC and by-product benzyl alcohol formation. By controlling the benzaldehyde level at 2 g/l, maintaining a respiratory quotient of 5–7 and pulse feeding glucose, a final concentration of 15.2 g/l l-PAC was achieved in a fed-batch process. This compares with previous published results of 10–12 g/l in batch culture and 10 g/l l-PAC in a semicontinuous process with immobilized Saccharomyces cerevisiae. In a single stage continuous process with immobilized C. utilis, the steady state l-PAC concentration was significantly reduced because of the sustained toxic effects of benzaldehyde.     

Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

Ethanol and glycerol production under aerobic conditions by wild-type,respiratory-deficient mutants and a fusion product of Saccharomyces cerevisiae

Summary Experiments were performed to investigate growth, ethanol and glycerol production by wild-type strains (RHO) and respiratory-deficient (rho) mutants of Saccharomyces cerevisiae. Furthermore protoplasts were fused in order to enhance the fermentation capacity of a flocculent strain. At high substrate conditions, 150 g/l of saccharose, there is no difference in cell growth. However, at a glucose concentration of 10–20 g/l the mutants grow much slower. After 3 days of incubation at 28° C in a complete medium the viability of the two strains is the same. In minimal medium on the other hand the number of viable cells of the mutant is 100-fold reduced. All mutants tested showed a higher specific activity of alcohol dehydrogenase (ADH I) and an enhanced production of glycerol compared with the wild-type strain. By protoplast fusion a modified flocculent strain was obtained with higher specific activity of ADH I and a reduced biosynthesis of glycerol. However, the yields of ethanol (75–78%) are about the same for the wild-type strain and the rho mutants under aerobic conditions in absence of catabolite repression.     

Glucose overflow metabolism and mixed-acid fermentation in aerobic large-scale fed-batch processes with Escherichia coli

Industrial 20-m³-scale and laboratory-scale aerobic fed-batch processes with Escherichia coli were compared. In the large-scale process the observed overall biomass yield was reduced by 12% at a cell density of 33 g/l and formate accumulated to 50 mg/l during the later constant-feeding stage of the process. Though the dissolved oxygen signal did not show any oxygen limitation, it is proposed that the lowered yield and the formate accumulation are caused by mixed-acid fermentation in local zones where a high glucose concentration induced oxygen limitation. The hypothesis was further investigated in a scale-down reactor with a controlled oxygen-limitation compartment. In this scale-down reactor similar results were obtained: i.e. an observed yield lowered by 12% and formate accumulation to 238 mg/l. The dynamics of glucose uptake and mixed-acid product formation (acetate, formate, d-lactate, succinate and ethanol) were investigated within the 54 s of passage time through the oxygen-limited compartment. Of these, all except succinate and ethanol were formed; however, the products were re-assimilated in the oxygen-sufficient reactor compartment. Formate was less readily assimilated, which accounts for its accumulation. The total volume of the induced-oxygen-limited zones was estimated to be 10% of the whole liquid volume in the large bioreactor. It is also suggested that repeated excretion and re-assimilation of mixed-acid products contribute to the reduced yield during scale-up and that formate analysis is useful for detecting local oxygen deficiency in large-scale E. coli processes. Received: 7 November 1998 / Received revision: 4 February 1999 / Accepted: 5 February 1999     

Synthesis of l-tyrosine by immobilized Escherichia intermedia cells

Summary Cells of Escherichia intermedia were immobilized by entrapment in a polyacrylamide gel and used for the enzymatic production of l-tyrosine from phenol, pyruvate, and ammonia. A preparation containing 50 mg of cells/g of gel retained 60% of its original activity. The effect of temperature, pH and substrate concentration on the activity of free cells was almost identical with the effect on immobilized cells. Phenol showed inhibition and inactivation of the catalyst at high concentration. Synthesis of l-tyrosine (up to 10 g/l) was demonstrated in batch reactors with high conversion yields (95–100%) and a maximal productivity of 2 g/l/h. In continuous reactor the catalyst showed a very high operational stability (more than 54 days without losses).     

Identification and physiological characterization of the nitrogen fixing bacterium Corynebacterium autotrophicum GZ 29

The coryneform hydrogen bacterium strain GZ 29, assigned to Corynebacterium autotrophicum fixed molecular nitrogen under autotrophic (H₂, CO₂) as well as under heterotrophic (sucrose) conditions. Physiological parameters of nitrogen fixation were measured under heterotrophic conditions. The optimal dissolved oxygen concentration for cells grown in a fermenter with N₂ was rather low (0.14 mg O₂/l) compared with cells grown in the presence of NH ₄ ⁺ (4.45 mg O₂/l). C. autotrophicum GZ 29 had a doubling time of 3.7 h at 30°C with N₂ as N-source and sucrose as carbon source and at optimal pO₂. Acetylene reduction reached values of 12 nmoles of ethylene produced/minxmg protein. Although the oxygen concentration in the growing culture was kept constant, the optimal dissolved oxygen tension for the acetylene reduction assay shifted to higher pO₂-values. The overall efficiency of nitrogen fixation amounted to 22 mg N fixed/g sucrose consumed; it reached a maximal value of 65 mg N fixed/g sucrose consumed at the beginning of the exponential growth phase. Intact cells reduced acetylene even under anaerobic test conditions; further anaerobic metabolic activity could not be ascertained so far.     

Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.