Enzyme formation by a yeast cell wall lytic Arthrobacter species: formation of amylase

The kinetics of amylolytic enzyme formation by a yeast cell wall lytic Arthrobacter species were studied. Cultivation on autoclaved cells of baker's yeast showed that amylase formation was closely related to trehalose and glycogen dissimilation. Growth on yeast glycogen (0.5%) proceeded quite rapidly ( = 0.31 h^–1) with extensive amylase formation during exponential cell multiplication and a further low increase in activity during the stationary phase. Beside amylolytic activity [450 units (U) l^–1] the formation of a relatively high level of -glucosidase (90 U l^–1) was detected, the latter almost exclusively bound to bacterial cells. Growth on 0.5% trehalose occurred at a reduced rate ( = 0.22 h^–1) with post-logarithmic enzyme synthesis in the stationary phase. Amylase activity attained a level of 1200 U l^–1, whereas -glucosidase was very low at 7.7 U l^–1. Continuous culture experiments in the chemostat showed maximal volumetric productivity of amylase (105 U l^–1 h^–1) at a dilution rate of 0.15 h^–1. Growth on various carbohydrates revealed low levels of amylolytic activity (<100 U l^–1), which were increased by a -1,4-glucans and oligosaccharides such as starch, dextrin, maltotriose and maltose. On 0.5% maltose, growth-associated enzyme synthesis (230 U l^–1) was detected at a reduced growth rate ( = 0.14 h^–1). Amylolytic enzyme preparations from the culture fluid showed an unusual cleavage pattern; acting on starch, the polymer was almost completely hydrolysed to maltotriose and maltose in a molar ratio of 3:1.Correspondence to: W. A. Hampel     

Ethanol production in multistage continuous, single stage continuous, Lactobacillus-contaminated continuous, and batch fermentations

Saccharomyces cerevisiae-based ethanol fermentations were conducted in batch culture, in a single stage continuous stirred tank reactor (CSTR), a multistage CSTR, and in a fermentor contaminated with Lactobacillus that corresponded to the first fermentor of the multistage CSTR system. Using a glucose concentration of 260 g l^–1 in the medium, the highest ethanol concentration reached was in batch (116gl^–1), followed by the multistage CSTR (106gl^–1), and the single stage CSTR continuous production system (60gl^–1). The highest ethanol productivity at this sugar concentration was achieved in the multistage CSTR system where a productivity of 12.7gl^–1h^–1 was seen. The other fermentation systems in comparison did not exceed an ethanol productivity of 3gl^–1h^–1. By performing a continuous ethanol fermentation in multiple stages (having a total equivalent working volume of the tested single stage), a 4-fold higher ethanol productivity was achieved as compared to either the single stage CSTR, or the batch fermentation.     

Batch cultivation and astaxanthin production by a mutant of the red yeast,Phaffia rhodozyma NCHU-FS501

Phaffia rhodozyma cells were treated with the mutagenic agent NTG several times and plated on yeast-malt agar containing -ionone as a selective medium. This mutagenesis of the yeast yielded a mutant (NCHU-FS501) with a total carotenoid content of 1454 g g^–1 dry biomass. Temperature and pH had only a slight effect on the volumetric pigment production by the red yeast, however astaxanthin yield and specific growth rate were influenced more significantly by temperature and pH. The optimum inoculum size, temperature and air flow rate for astaxanthin formation by the mutant in a bench-top fermentor were 7.5% (v/v), 22.5°C and 3.6 vvm, respectively. Glucose (1%, w/v) as carbon source yielded the highest volumetric astaxanthin production (6.72 g ml^–1). Peptone (15.8% total nitrogen) was the best nitrogen source for astaxanthin production (6.72 g ml^–1). Pigment formation by the mutant was further improved by increasing the glucose concentration to 3.5%, where the astaxanthin concentration was 16.33 m ml^–1. At 4.5% glucose or above astaxanthin formation was inhibited. Control of the pH of the fermentation broth did not improved pigment production.     

Chitinase production byTalaromyces emersonii

Summary Talaromyces emersonii, when grown on medium containing chitin, yielded extracellular chitinase and chitobiase activities of 0.45 mol.h^–1.ml^–1 culture fluid and 1.4 mol. min^–1.ml^–1, respectively, after 2–4 days of growth under pH-controlled conditions. The enzyme system was optimally active at pH 5.0–5.5, c. 65°C and the least stable components had half-lives of 20 min at 76°C, pH 5.0.     

Examining the relationship between bacteria and heterotrophic nanoflagellates in Funka Bay (Japan) using the size-fractionation method

The chemical and biological conditions, and the bacteria-heterotrophic nanoflagellate (HNF) relationship were investigated in the vicinity of Funka Bay, southwest of Hokkaido, Japan during early spring 1999. At the time of sampling, chlorophyll a concentration, bacteria, phycoerythrin rich-cyanobacteria, and HNF abundance were in the following ranges: 0.3–3.6 g l^–1, 2.5–5.6 × 10⁵ cells ml^–1, 0.6–1.2 × 10³ cells ml^–1, and 2.2–4.2 × 10³ cells ml^–1, respectively. Dissolved inorganic nitrogen, phosphate and silicate concentrations were in the ranges: 8.7–12.2 M, 0.9–2.0 M, and 21.6–25.5 M, respectively. Primary production ranged from 6.4 to 76.3 mg C m^–3 d^–1. Using water samples from regions of different productivity levels (in and outside bay), the bacteria - HNF relationship was uncoupled experimentally by the size-fractionation technique. Higher primary production (19.9 mg C m^–3 d^–1) in the bay supported higher bacterial growth rate (0.029 h^–1). However, outside the bay both primary production (6.4 mg C m^–3 d^–1) and bacterial growth rate (0.007 h^–1) were lower. The HNF growth rates and grazing rates were similar for both but by comparing both HNF grazing capacity and bacterial production, there was net decrease in bacterial abundance outside the bay and net increase inside the bay. The microbial parameters (rates and abundance) and the amount of carbon flow estimated through the phytoplankton – dissolved organic matter (DOM) – bacteria loop were different between the coastal station and the open ocean station. However HNF grazing and growth rates was similar for both stations.     

Thermostable cellulases from <Emphasis Type="Italic">Streptomyces</Emphasis>sp.: scale-up production in a 50-l fermenter

Thermostable cellulase was produced by Streptomyces sp. T3-1 grown in a 50-l fermenter. Maximum cellulase activity was attained on the fourth day when agitation speeds and aeration rates were controlled at 300 rpm and 0.75 vvm, respectively. Maximum enzyme activities were: 148 IU CMCase ml^–1, 45 IU Avicelase ml^–1, and 137 IU -glucosidase ml^–1 with productivity of 326 IU l^–1 h^–1, which were 10--32% higher than the values obtained in shake-flask culturesRevisions requested 12 October 2004/1 November 2004; Received received 1 November 2004/14 December 2004     

Production of β-1.4-xylanase in continuous culture byAureobasidium Pullulans CBS 58475

Summary The fungusAureobasidium pullulans CBS 58475 can be fermented easily and economically in its yeast-like form. The enzyme production is strictly connected to biomass production. In continuous culture, at a dilution rate of D=0.085 h^–1, the fungus shows a volumetric -xylanase productivity of about 300 U/h·1.     

Enzyme formation by a yeast cell wall lytic Arthrobacter species: formation of α-mannanase

Summary The kinetics of -mannanase (EC 3.2.1.77) and -mannosidase (EC 3.2.1.24) formation by a yeast cell wall lytic Arthrobacter species were studied. Growth () on yeast mannan was multiphasic and caused by mannose (=0.29 h^–1) liberated by enzyme action from mannan. Early enzyme formation was soon repressed by mannose and depressed by its restricted availability during late exponential and stationary growth. Synthesis of -mannosidase occurred predominantly at the late stage of substrate utilization. Fructose was detected as an equally potent inducer for -mannanase formation as yeast mannan, being a simple and cheap substrate for large-scale cultivation. Growth on fructose was reduced (=0.20 h^–1), enzyme synthesis being growth associated; nevertheless, comparable -mannanase levels [180 (U) units l^–1] were formed. -Mannosidase activity was only detectable in small amounts. Continuous culture experiments gave values for maximal productivity of mannanase of 18 U h^–1 g^–1 and enzyme yield per biomass (Y _EA/X) of 100 U g^–1. Moreover, the substrate saturation constant (Monod constant) and maintenance coefficient were estimated for fructose as 115 mg l^–1 and 4 mg h^–1 g^–1, respectively.     

Selection of a support for immobilization of a microbial lipase for the hydrolysis of triglycerides

Baterial lipase from Staphylococcus carnosus (pLipMut2) has been immobilized on various supports in order to determine a suitable immobilization technique in terms of activity and stability, when utilized for the hydrolysis of tributyrin. The hydrophobic materials PBA Eupergit and PBA Eupergit 250L prooved to be appropriate supports, when the enzyme was crosslinked with glutaraldehyde after adsorption. No desorption of the immobilized enzyme occured during operation. The pore size of the support has a strong effect on the activity but does not influence stability.The initial activity for immobilized and soluble lipase is found to follow the Arrhenius equation at low temperature, where mass transfer does not affect reaction kinetics. Activation energies for soluble and immobilized lipase were evaluated to be 21.7 kJ mol^–1 and 60.8 kJ mol^–1, respectively.Operational stability was studied in a packed bed recirculation reactor. Thermal desactivation followed first order kinetics with a half-life of 1340 h at 10°C. Model calculations for productivity showed, that optimal temperatures for high productivity are well below the temperature of maximal activity.List of Symbols E _a [kJ mol^–1] activation energy - E _d [kJ mol^–1] activation energy of desactivation - H [–] half-number - k _d [h^–1] desactivation constant - k _d, [h^–1] constant - k _N [–] desactivation constant (number) - N [–] number of runs - p [mol dm^–3] productivity - t [h] time - t _0.5 [h] half-life - T [K] absolute temperature - V [U ml^–1] activity - V(N) [Uml^–1] activity exhibited in the n-th run - V _s,O [U ml^–1] initial activity of supernatant - V _s, [U ml^–1] activity of supernatant after immobilization - V _O [U ml^–1] initial activity - V [U ml^–1] constant - _imm [–] activity yield - [ml ml^–1] ratio of volume of support to volume of supernatant Financial support of this work by the Deutsche Forschungsgemeinschaft (SFB 145, A15) is gratefully acknowledged.     

Kinetics of beta-glucosidase production by Saccharomyces cerevisiae recombinants harboring heterologous bgl genes

The maximum productivity of -glucosidase by Saccharomyces cerevisiaerecombinants under the control of GALI promoter was 100 IU l^–1 h^–1. The highest productivity of -glucosidase by a S. cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea. The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1 g ethanol g^–1 cellobiose and g ethanol l^–1 h^–1, respectively.     

Optimization of cell density and dilution rate in <Emphasis Type="Italic">Pichia pastoris</Emphasis> continuous fermentations for production of recombinant proteins

This paper provides an approach for optimizing the cell density (X_c) and dilution rate (D) in a chemostat for a Pichia pastoris continuous fermentation for the extracellular production of a recombinant protein, interferon (INF-). The objective was to maximize the volumetric productivity (Q, mg INF- l^–1 h^–1), which was accomplished using response surface methodology (RSM) to model the response of Q as a function of X_c and D within the ranges 150 X_c 450 g cells (wet weight) l^–1 and 0.1 _mD0.9 _m (_m=0.0678 h^–1, the maximum specific growth rate obtained from a fed-batch phase controlled with a methanol sensor). The methanol and medium feed rates that resulted in the desired X_c and D were determined based on the mass balance. From the RSM model, the optimal X_c and D were 328.9 g l^–1 and 0.0333 h^–1 for a maximum Q of 2.73 mg l^–1 h^–1. The model of specific production rate (, mg INF- g^–1 cells h^–1) was also established and showed the optimal X_c=287.7 g l^–1 and D=0.0361 h^–1 for the maximum (predicted to be 8.92×10^–3 mg^–1 g^–1 h^–1). The methanol specific consumption rate (, g methanol g^–1 cells h^–1) was calculated and shown to be independent of the cell density. The relationship between and (specific growth rate) was the same as that discovered from fed-batch fermentations of the same strain. The approach developed in this study is expected to be applicable to the optimization of continuous fermentations by other microorganisms.     

Feeding rate inhibition in crowded Daphnia pulex

Feeding rates of Daphnia pulex fed a range of levels of the alga Chlamydomonas reinhardi of 15 °C are strongly density-dependent. At lower densities, Daphnia (30 1^–1) fed at higher rates than crowded (270 1^–1) Daphnia which manifest a relatively depressed saturation feeding response. At 30 individuals/liter, Daphnia consumed 8.5 – 15.7 × 10⁴ cells d^–1h^–1 (on a volume basis, 12.1 – 22.2 × 10⁶ m³), at 270 L^–1 3.7 – 3.9 × 10⁴ (5.2 – 5.5 = 10⁶ m³ cells d^–1h^–1 when feeding on algae at 80 000 cells ml^–1 (11.3 × 10⁶ m³ ml^–1). The feeding rate data best fit an Ivlev feeding function. An autoallelopath might be causing the repression. Water preconditioned with crowded Daphnia completely repressed feeding in uncrowded Daphnia after six hours.     

Improvement of the ethanol productivity in a high gravity brewing at pilot plant scale

A 2³ full factorial design was used to study the influence of different experimental variables, namely wort gravity, fermentation temperature and nutrient supplementation, on ethanol productivity from high gravity wort fermentation by Saccharomyces cerevisiae (lager strain), under pilot plant conditions. The highest ethanol productivity (0.69 g l^–1 h^–1) was obtained at 20°P [°P is the weight of extract (sugar) equivalent to the weight of sucrose in a 100 g solution at 20°C], 15°C, with the addition of 0.8% (w/v) yeast extract, 24 mg l^–1 ergosterol and 0.24% (v/v) Tween 80.     

Effect of the support matrix on biotransformation of benzaldehyde to benzyl alcohol by yeast cells in aqueous and aqueous-organic two phase systems

Summary Biotransformation of benzaldehyde to benzyl alcohol bySaccharomyces cerevisiae immobilized in different support matrices was investigated. Polymers with intrinsic hydrophobic and/or hydrophilic nature as well as mixed hydrophobic and hydrophilic supports were examined both in aqueous and bisphasic aqueous-organic systems. The hydrophobic support material ENTP-2000 or mixed silicone:alginate (50-2550-75) proved to be most suitable not only for nonconventional media but also for conventional aqueous media for production of benzyl alcohol. With ENTP-2000, catalytic activity and maximum yield were 159 mol h^–1 g^–1 dry weight catalyst and 0.89 mM, respectively, in hexane containing 2% moisture. Corresponding values in aqueous media were 246 mol h^–1 g^–1 dry weight catalyst and 1.53 mM. With 5050 silicone:alginate, catalytic activity and maximum yield were 177 mol h^–1 g^–1 dry weight catalyst and 1.18 mM, respectively, in hexane containing 2% moisture. Corresponding values in aqueous media were 192 mol h^–1 g^–1 dry weight catalyst and 0.8 mM.     

Soret spectroscopic and molecular graphic analysis of human semi-beta-hemoglobin formation

The interaction of heme-free (^o) and heme-containing (^h) chains of human hemoglobin has been monitored in 0.1 M potassium phosphate buffer, pH 7 or 8, at [5°C. Soret zero and first-derivative spectra were consistent with a uniform association reaction. Stopped-flow investigations demonstrated association rates on the order of 10⁷ M^–1 s^–1. This was 100-fold more rapid than the reported rate of combination of ^h and ^h proteins. This encounter-like rate of semi--hemoglobin (^oh) formation was increased by raising the pH from 7 to 8. pH change is known to affect the spatial arrangement of AB—GH helical entities. Molecular graphic analysis of modeled ^o protein superimposed over native ^h protein revealed an apo Mb-like structure with well-defined AB—GH segments. Repositioning of these core helical segments, resulting in increased conformational freedom of the ₁₁ interface, was apparently responsible for the enhanced association properties of the ^o protein.     

Thermal stability and thermodynamic analysis of native and methoxypolyethylene glycol modified trypsin

Four methoxypolyethylene glycols (MPEG, molecular masses 350, 750, 2000 and 5000 Da), each activated by nitrophenyl chloroformate, were used to modify trypsin. Compared with the native trypsin, the MPEG-modified trypsin was more stable against temperature between 30°C and 70°C, longer chain of MPEG moiety corresponding to higher thermal stability. The T for the native and the modified trypsin (0.4 mg ml^–1) was increased from 47°C to 66°C. The stabilization effect caused by MPEG modification was the result of decreasing in both the autolysis rate and the thermal denaturation rate. The thermodynamic analysis of the thermal denaturation process showed that the activation free energy (G^*) of the native and the modified trypsin at 60°C was increased from 102.9 to 109.3 kJ mol^–1; the activation enthalpy (H^*) was increased from 57.4 to 86.9 kJ mol^–1; the activation entropy (S^*) was increased from –136 to –67 J molK^–1. A possible explanation for the decreased thermal denaturation rate caused by MPEG modification was also discussed.     

Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis

Fed-batch cultures of Bacillus licheniformis produced poly--glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l^–1 with a productivity of 1 g l^–1 h^–1 by pulsed-feeding of citric acid (1.44 g h^–1) and l-glutamic acid (2.4 g h^–1) when citric acid was depleted from the culture medium.     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

Product inhibition during the feeding phasein fed-batch ethanol fermentation of sugar-cane blackstrap molasses

Summary The following equations represent the influence of the ethanol concentration (E) on the specific growth rate of the yeast cells () and on the specific production rate of ethanol () during the reactor filling phase in fed-batch fermentation of sugar-cane blackstrap molasses: = ₀ - k · E and v = v ₀ · K/(K +E) Nomenclature E ethanol concentration in the aqueous phase of the fermenting medium (g.L^–1) - E_m value of E when = 0 or = 0 (g.L^–1) - F medium feeding rate (L.h^–1) - k empirical constant (L.g^–1.h^–1) - K empirical constant (g.L^–1) - M_as mass of TRS added to the, reactor (g) - M_cs mass of consumed TRS (g) - M_e mass of ethanol in the aqueous phase of the fermenting medium (g) - M_s mass of TRS in the aqueous phase of the fermenting medium (g) - M_x mass of yeast cells (dry matter) in the fermenting medium (g) - r correlation coefficient - S TRS concentration in the aqueous phase of the fermenting medium (g.L^–1) - S_m TRS concentration of the feeding medium (g.L^–1) - t time (h) - T temperature (° C) - TRS total reducing sugars calculated as glucose - V volume of the fermenting medium (L) - V₀ volume of the inoculum (L) - X yeast cells concentration (dry matter) in the fermenting medium (g.L^–1) - filling-up time (h) - specific growth rate of the yeast cells (h^–1) - ₀ value of when E=0 - specific production rate of ethanol (h^–1) - ₀ value of when E=0 - density of the yeast cells (g.L^–1) - dry matter content of the yeast cells     

Lactulose production by β-galactosidase in permeabilized cells of<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis>

Lactulose production from lactose and fructose was investigated with several commercial -galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the -galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l^–1; concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60^°C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l^–1 lactulose in 3 h with a lactulose productivity of 6.8 g l^–1 h^–1. These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.