Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H₂. The enzyme showed a maximal activity of 120±40 mol H₂ oxidized · min^–1 · min^–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H₂ · min^–1 · mg^–1 with methyl viologen as electron donor, and an apparent K _m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S^2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F₄₂₀ or ferredoxin, nor with FAD, FMN, or NAD(P)⁺. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)     

Integral kinetics of the cleavage of maltose catalysed by α-glucosidase fromSaccharomyces carlsbergensis

Summary A new, sensitive and continuous assay for -glucosidase is described exploiting the different angles of rotation for the substrate maltose and the product glucose. Kinetic experiments revealed a very pronounced product inhibition of -glucosidase fromSaccharomyces carlsbergensis with a K_i of 4.85·10^–3 M for glucose.The K_M of maltose was found to be 37.8·10^–3 M. Taking these values, an integral kinetic curve for the enzymatic hydrolysis of maltose was calculated, which is shown to fit the experimental data.Symbols used k₁ (min^–1) pseudo first-order rate constant (for enzymatic cleavage) - k₂ (min^–1) rate constant (for mutarotation reaction) - I, P (mol/1) inhibitor (product) concentration - k_i (mmol/1) inhibitor constant - K_M (mmol/l) Michaelis constant - [M] ₅₈₉ ³⁰ (degree/m · l/mol) molecular rotation at 30°C and 589 nm - s (mmol/l) substrate concentration - R (mmol/mg · min) reaction rate - V_max (mmol/mg · min) maximal rate - U (mol/min) activity unit (here at 30°C and pH=6.8) Indices O initial value - max maximal value     

The substrate specificity of phosphoinositide phospholipase C in rat heart sarcolemma

In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [³H-]phosphatidylinositol 4,5-biphosphate ([³H-]PtdIns(4,5)P ₂) in an optimized assay mixture containing 15 leg SL protein, 100 mM NaCl, 1 mM free Ca²⁺,14 mM Na-cholate and 20 AM [³H-]PtdIns (4,5)P ₂ (400–500 dpm/gm-l) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14±0.55 nmol-mg^–1·2.5 min^–1. The addition of Mg²⁺ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na⁺ and at a low Ca²⁺ concentration (0.3 M), Mg²⁺ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC₅₀0.07 mM). PtdIns4P seemd to be a good substrate for the rat SL PLC (23.07 ± 1.57 nmol·mg^–1·2.5 min^–1) whereas PtdIns was hydrolysed at a very low rate (0.36 ± 0.08 nmol·mg^–1·2.5 min^–1). Unlike PtdIns(4,5)P ₂, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca²⁺ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed. (Mol Cell Biochem116: 27–31, 1992).Abbreviations DAG sn-1,2-diacylglycerol - EGTA ethyleneglycol-O,O-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - Ins(1,4,5)P ₃ inositol 1,4,5-trisphosphate - InsP inositol monophosphate (unidentified isomer) - InsP ₂ inositol bisphosphate (unidentified isomer) - InsP ₃ inositol trisphosphate (unidentified isomer) - InsP _x any inositol phosphate - PLC phospholipase C - PtdIns phosphatidylinositol - PtdIns(4,5)P ₂ phosphatidylinositol 4,5-bisphosphate - PtdIns4P phosphatidylinositol 4-monophosphate - SL sarcolemma     

Intraparticle mass-transfer resistance and apparent time stability of immobilized yeast alcohol dehydrogenase

The stability and, consequently, the lifetime of immobilized enzymes (IME) are important factors in practical applications of IME, especially so far as design and operation of the enzyme reactors are concerned. In this paper a model is presented which describes the effect of intraparticle diffusion on time stability behaviour of IME, and which has been verified experimentally by the two-substrate enzymic reaction. As a model reaction the ethanol oxidation catalysed by immobilized yeast alcohol dehydrogenase was chosen. The reaction was performed in the batch-recycle reactor at 303 K and pH-value 8.9, under the conditions of high ethanol concentration and low coenzyme (NAD⁺) concentration, so that NAD⁺ was the limiting substrate. The values of the apparent and intrinsic deactivation constant as well as the apparent relative lifetime of the enzyme were calculated.The results show that the diffusional resistance influences the time stability of the IME catalyst and that IME appears to be more stabilized under the larger diffusion resistance.List of Symbols C _A, C_B, C_E mol · m^–3 concentration of coenzyme NAD⁺, ethanol and enzyme, respectively - C _p mol · m³ concentration of reaction product NADH - d _p mm particle diameter - D _eff m² · s^–1 effective volume diffusivity of NAD⁺ within porous matrix - k _d s^–1 intrinsic deactivation constant - K _A, K_A, K_B mol · m^–3 kinetic constant defined by Eq. (1) - K _A ^x mol · m^–3 kinetic constant defined by Eq. (5) - r _A mol · m^–3 · s^–1 intrinsic reaction rate - R m particle radius - R _v mol · m^–3 · s^–1 observed reaction rate per unit volume of immobilized enzyme - t _E s enzyme deactivation time - t _r s reaction time - V mol · m^–3 · s^–1 maximum reaction rate in Eq. (1) - V ^x mol · m^–3 · s^–1 parameter defined by Eq. (4) - V _f m³ total volume of fluid in reactor - w _s kg mass of immobilized enzyme bed - factor defined by Eqs. (19) and (20) - kg · m^–3 density of immobilized enzyme bed - unstableness factor - effectiveness factor - Thiele modulus - relative half-lifetime of immobilized enzyme Index o values obtained with fresh immobilized enzyme     

Organic reduction of tapioca wastewaters using modified RBC

A modified Rotating Biological Contactor (RBC) was used for the treatability studies of synthetic tapioca wastewaters. The RBC used was a four stage laboratory model and the discs were modified by attaching porous nechlon sheets to enhance biofilm area. Synthetic tapioca wastewaters were prepared with influent concentrations from 927 to 3600 mg/l of COD. Three hydraulic loads were used in the range of 0.03 to 0.09 m³·m^–2·d^–1 and the organic loads used were in the range of 28 to 306 g COD· m^–2·d^–1. The percentage COD removal were in the range from 97.4 to 68. RBC was operated at a rotating speed of 18 rpm which was found to be the optimal rotating speed. Biokinetic coefficients based on Kornegay and Hudson models were obtained using linear analysis. Also, a mathematical model was proposed using regression analysis.List of Symbols A m² total surface area of discs - d m active depth of microbial film onany rotating disc - K _s mg ·l^–1 saturation constant - P mg·m^–2·^–1 area capacity - Q l·d^–1 hydraulic flow rate - q m³·m^–2·d^–1 hydraulic loading rate - S ₀ mg·l^–1 influent substrate concentration - S _e mg·l^–1 effluent substrate concentration - w rpm rotational speed - V m³ volume of the reactor - X _f mg·l^–1 active biomass per unit volume ofattached growth - X _s mg·l^–1 active biomass per unit volume ofsuspended growth - X mg·l^–1 active biomass per unit volume - Y _s yield coefficient for attachedgrowth - Y _A yield coefficient for suspendedgrowth - Y yield coefficient, mass of biomass/mass of substrate removed Greek Symbols hr mean hydraulic detention time - (_max)_A d^–1 maximum specific growth rate forattached growth - (_max)_s d^–1 maximum specific growth rate forsuspended growth - _max d^–1 maximum specific growth rate - d^–1 specific growth rate - _v mg·l^–1·hr^–1 maximum volumetric substrateutilization rate coefficient     

Factors which influence the sedimentation characteristics of Torulopsis glabrata after growth in a recirculation system

Summary Some environmental affects on cell aggregation described in the literature are briefly summarized. By means of a biomass recirculation culture (Contact system), using the yeast Torulopsis glabrata, the aggregation behavior of cells in static and in dynamic test systems is described. Sedimentation times required to obtain 50 g · l^–1 yeast dry matter in static systems were always higher than in dynamic ones.In addition to, influencing the biomass yield, the specific growth rate of the yeast also affected cell aggregation. The specific growth rate and therefore the aggregation could be regulated by the biomass recirculation rate as well as by the sedimenter volume.Abbreviations f_o Overflow flow rate (l·h^–1) - f_R Recycle flow rate (l·h^–1) - f_t0t Total flow rate through the fermenter (l·h^–1) - g Gram - h Hour - D_R Fermenter dilution rate due to recycle (h^–1) - D_S Fermeter dilution rate due to substrate (h^–1) - D_tot Total fermenter dilution rate (h^–1) - l Liter - Specific growth rate (h^–1) - P_F Fermenter productivity (g·l^–1·h^–1) - P_FS Overall productivity (g·l^–1·h^–1) - R_pM Rates per minute - RS Residual sugar content in the effluent with respect to the substrate concentration (%) - Y Yield of biomass with respect to sugar concentration (%) - Sed 50 Sedimentation time to reach a YDM of 50 g·l^–1 (min) - V Volume (l) - V_F Fermenter volume (l) - V_Sed Sedimenter volume (l) - VVM Volumes per volume and minute - X_F YDM in the fermenter (g·l^–1) - X_F YDM in the recycle (g·l^–1) - X_S Yeast dry matter due to substrate concentration (g·l^–1) - YDM Yeast dry matter (g·l^–1)     

Conversion of cellulose into fungal cell mass

Summary Chaetomium cellulolyticum (ATCC 32319) was cultivated on glucose, Avicel and/or Sigmacell in a 20-1 stirred tank batch reactor. The substrate (cellulose) concentration, the cell mass concentration (through protein and/or nitrogen content), reducing sugar concentration, the enzyme activity, the alkali consumption rate, the dissolved O₂ and CO₂ concentrations in the outlet gas were measured. The specific growth rate, the substrate yield coefficient, cell productivity, the oxygen consumption rate, the CO₂ production rate and the volumetric mass transfer coefficient were determined. At the beginning of the growth phase the oxygen utilization rate exhibits a sharp maximum. This maximum could be used to start process control. Because of the long lag phase periodic batch operation is recommended.Symbols CP cell protein concentration (g l^–1) - FPA FP enzyme activity (IU l^–1) - GP dissolved protein concentration (g l^–1) - IU international unit of enzyme activity - k_La volumetric mass tranfer coefficient (h^–1) - LG alkali (1 n NaOH) consumption (ml) - LGX specific alkali consumption rate per cell mass (ml g^–1 h^–1) - P cell mass productivity (g l^–1 h^–1) - specific oxygen consumption rate per cell mass (g g^–1 h^–1) - Q aeration rate (volumetric gas flow rate per volume of medium, vvm) (min^–1) - N impeller speed (revolution per minute, rpm) (min^–1) - S substrate concentration (g l^–1) - S⁰ S at t_F=0 (g l^–1) - S₀ S in feed (g l^–1) - SR acid consumption (ml) - TDW total dry weight (g l^–1) - T temperature (° C) - t_F cultivation time (h) - U substrate conversion - X cell mass concentration (g l^–1) - Y_X/S vield coefficient - specific growth rate (h^–1) - _m maximum specific growth rate (h^–1)     

Lactate content and lactate dehydrogenase activity inPalaemon serratus abdominal muscle during temperature changes

Summary Lactate concentration was measured in the abdominal muscle of the shrimpPalaemon serratus. Rapid and seasonal temperature changes result in an increase of the lactate content of approximately 3–4 fold.Lactate dehydrogenase from the abdominal muscle exhibits a temperature dependent pyruvate inhibition with pyruvate as substrate.The kinetic parameters of lactate dehydrogenase fromPalaemon serratus are found to vary during rapid temperature changes: V_max increases with temperature from 0.06 mol min^–1 (mg protein)^–1 at 10°C to 0.28 mol min^–1 (mg protein)^–1 at 30°C with lactate as substrate, and from 5.5 mol min^–1 (mg protein)^–1 at 10°C to 26.2 mol min^–1 (mg protein)^–1 at 30°C, with pyruvate (Table 1). The Hill coefficientn _H, decreases with temperature from 2.2 to 1.2 when the pyruvate reduction is examined, but remains near 1.2 when the activity is measured with lactate as substrate (Table 1). The S_0.5 values for lactate show a tendency to increase below 30 °C (18.9 mM l^–1 at 20 °C) whereas the S_0.5 for pyruvate is found to increase greatly with temperature (0.004 mM l^–1 at 10 °C and 0.06 mM l^–1 at 20 °C).Long term temperature changes involve variations of lactate dehydrogenase activity leading to inverse thermal compensation (Table 2).Activation energy (about 56 kJ both with pyruvate and lactate) does not vary during the year, suggesting that temperature adaptation does not induce important catalytic changes (Table 3).Abbreviation LDH lactate dehydrogenase     

Glutathione reductase activity in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum

Glutathione reductase activity was detected and characterized in heterocysts and vegetative cells of the cyanobacterium Nostoc muscorum. The activity of the enzyme varied between 50 and 150 nmol reduced glutathione· min^-1·mg protein^-1, and the apparent K_m for NADPH was 0.125 and 0.200 mM for heterocysts and vegetative cells, respectively. The enzyme was found to be sensitive to Zn⁺² ions, however, preincubation with oxidized glutathione rendered its resistance to Zn⁺² inhibition. Nostoc muscorum filaments were found to contain 0.6–0.7mM glutathione, and it is suggested that glutathione reductase can regenerate reduced glutathione in both cell types. The combined activity of glutathione reductase and isocitrate dehydrogenase in heterocysts was as high as 18 nmol reduced glutathione·min^-1·mg protein^-1. A relatively high superoxide dismutase activity was found in the two cell types; 34.2 and 64.3 enzyme units·min^-1·mg protein^-1 in heterocysts and vegetative cells, respectively.We suggest that glutathione reductase plays a role in the protection mechanism which removes oxygen radicals in the N₂-fixing cyanobacterium Nostoc muscorum.Abbreviations DTNB 5-5-dithiobis-(2-nitrobenzoic acid) - EDTA ethylenediaminetetra-acetic acid - GR glutathione reductase (EC1.6.4.2) - GSH reduced glutathione - GSSG oxidized glutathione - OPT O-phtaldialdehyde - SOD superoxide dismutase (EC 1.15.1.1)     

Enhancement of product selectivity via enzyme immobilization in sequential degradation reactions of polymeric substrates

The balance equations pertaining to the modelling of a slap-shaped bead containing immobilized enzyme uniformly distributed which catalyzes the sequential reactions of degradation of a polymeric substrate were written and analytically solved in dimensionless form. The effect of the Thiele modulus on the selectivity of consumption of each multimeric product was studied for a simple case. Whereas plain diffusional regime leads to lower selectivities than plain kinetic regime, improvements in selectivity of species A _i relative to species A_i+1 may be obtained at the expense of higher Thiele moduli within a limited range when the diffusivity of A _i is larger than that of A _i +1, or when the pseudo first order kinetic constant describing the rate of consumption of A _i is lower than that of A_i+1.List of Symbols A _i polymeric substrate containing i monomeric subunits - C _i mol·m^–3 normalized counterpart of C _i - C _i mol·m^–3 concentration of substrate A _i - C _i,0 mol·m^–3 initial concentration of substrate A _i - C _i,0 normalized counterpart of C _i,0 - D _ap,i m²·s^–1 apparent diffusivity of substrate A _i - k _i s^–1 pseudo-first order rate constant - K _m,i mol·m^–3 Michaelis-Menten constant associated with substrate A _i - L m half-thickness of the catalyst slab - N number of monomeric subunits of the largest substrate molecule - Th Thiele modulus - V _i mol·m^–3·s^–1 rate of rection of substrate A _i - V_max,i mol·m^–3·s^–1 maximum rate of reaction under saturating conditions of substrate A _i - x m longitudinal coordinate - S _i,i+1 selectivity of enzyme with respect to substrates with consecutive numbers of monomeric subunits Greek Symbols _i ratio of maximum rates of reaction - _i ratio of apparent diffusivities     

Simulation of a multicolumn recirculated packed bed reactor (MRPBR) for penicillin acylase

Enzyme reactors for the industrial hydrolysis of penicillin are analyzed in terms of biocatalyst stability to pH. A multicolumn system with packed beds placed in parallel and operating under recirculating conditions is proposed as an adequate reactor for this process. The system is studied both experimentally and with the aid of a simulation program.List of Symbols A transversal area (cm²) - C _A ammonia concentration in the reaction mixture (M) - C ₁ concentration of KH₂PO₄ in buffer (M) - C ₂ concentration of K₂HPO₄ in buffer (M) - d _p biocatalyst diameter (cm) - E enzyme or biocatalyst concentration (gcat l^–1) - K _APA APA non competitive inhibition constant (M) - K _IS excess substrate inhibition constant (M) - Km constant Michaelis-Menten (M) - K _PAA PAA competitive inhibition constant (M) - Q recirculation flow rate (cm³ min^–1) - Q _T recirculation flow rate per column (cm³ min^–1) - Re Reynolds number - S _E substrate concentration entering the neutralization tank (M) - S ₀ initial substrate concentration (M) - S _T substrate concentration in neutralization tank (M) - t time (min) - v _i initial reactor rate (mol min^–1 gcat^–1) - V _s superficial velocity (cm seg^–1) - V _T volume of neutralization tank (cm³) - X _E substrate conversion entering tank - X _T substrate conversion in neutralization tank - X conversion - Z reactor length (cm) - z axial position in reactor (cm) - z ^* non-dimensional axial position in reactor - biocatalyst's density (gcat cm^–3) - p pressure drop in the packed-bed reactor     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans has been proposed to oxidize acetate to CO₂ via an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway rather than via the citric acid cycle. We report here the presence of the enzyme activities required for the operation of the novel pathway. In cell extracts the following activities were found (values in brackets=specific activities and apparent K _m; 1 U·mg^-1=1 mol·min^-1·mg protein^-1 at 37°C): Acetate kinase (6.3 U·mg^-1; 2 mM acetate; 2.4 mM ATP); phosphate acetyltransferase (60 U·mg^-1, 0.4 mM acetylphosphate; 0.1 mM CoA); carbon monoxide dehydrogenase (29 U·mg^-1; 13% carbon monoxide; 1.3 mM methyl viologen); 5,10-methylenetetrahydrofolate reductase (3 U·mg^-1, 0.06 mM CH₃–FH₄); methylenetetrahydrofolate dehydrogenase (3.6 U·mg^-1, 0.9 mM NAD, 0.1 mM CH₂=FH₄); methenyltetrahydrofolate cyclohydrolase (0.3 U·mg^-1); formyltetrahydrofolate synthetase (3 U·mg^-1, 1.4 mM FH₄, 0.4 mM ATP, 13 mM formate); and formate dehydrogenase (10 U·mg^-1, 0.4 mM formate, 0.5 mM NAD). The specific activities are sufficient to account for the in vivo acetate oxidation rate of 0.26 U·mg^-1.Non-standard abbreviations FH₄ Tetrahydrofolate - CHO-FH₄ N¹⁰-formyltetrahydrofolate - CHFH₄ N⁵,N¹⁰-methenyltetrahydrofolate - CH₂=FH₄ N⁵,N¹⁰-methylenetetrahydrofolate - CH₃–FH₄ N⁵-methyltetrahydrofolate - MOPS morpholinopropane sulfonic acid - DTT d,l-1,4-dithiothreitol - TRIS tris-(hydroxymethyl)-aminomethane - Ap₅A p¹,P⁵-di(adenosine-5)pentaphosphate - MV methyl viologen     

Utilization of l-alanine by Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 can satisfy all its nitrogen and carbon requirements from l-alanine. Addition of 100 M methionine sulfoximine to alanine grown cultures had no effect on growth rate indicating that deamination of alanine via alanine dehydrogenase and re-assimilation of the released NH ₄ ⁺ by glutamine synthetase/glutamate synthase was an insignificant route of nitrogen transfer in this bacterium. Determination of aminotransferase activities in cell-free extracts failed to demonstrate the presence of direct routes from alanine to either aspartate or glutamate. The only active aminotransferase involving l-alanine was the alanine-glyoxylate enzyme (114–167 nmol·min^–1·mg^–1 protein) which produced glycine as end-product. The amino group of glycine was further transaminated to yield aspartate via a glycineoxaloacetate aminotransferase (117–136 nmol·min^–1 ·mg^–1 protein). No activity was observed when 2-oxoglutarate was substituted for oxaloacetate. The formation of glutamate from aspartate was catalysed by aspartate-2-oxoglutarate aminotransferase (85–107 nmol·min^–1·mg^–1 protein). Determinations of free intracellular amino acid pools in alanine and alanine+100 M methionine sulfoximine grown cells showed the predominance of glutamate, glycine and aspartate, providing further evidence that in alanine grown cultures R. acidophila satisfies its nitrogen requirements for balanced growth by transamination.Abbreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulfoximine - GOT glutamate-oxaloacetate aminotransferase - GPT glutamate-pyruvate amino-transferase - AGAT alanine-glyoxylate aminotransferase - GOAT glycine-oxaloacetate aminotransferase - GOTAT glycine-2-oxoglutarate aminotransferase - AOAT alanine-oxaloacetate aminotransferase     

Optimal temperature and concentration profiles in a cascade of CSTR's performing Michaelis-Menten reactions with first order enzyme deactivation

A necessary condition is found for the intermediate temperatures and substrate concentrations in a series of CSTR's performing an enzyme-catalyzed reaction which leads to the minimum overall volume of the cascade for given initial and final temperatures and substrate concentrations. The reaction is assumed to occur in a single phase under steady state conditions. The common case of Michaelis-Menten kinetics coupled with first order deactivation of the enzyme is considered. This analysis shows that intermediate stream temperatures play as important a role as intermediate substrate concentrations when optimizing in the presence of nonisothermal conditions. The general procedure is applied to a practical example involving a series of two reactors with reasonable values for the relevant five operating parameters. These parameters are defined as dimensionless ratios involving activation energies (or enthalpy changes of reaction), preexponential factors, and initial temperature and substrate concentration. For negligible rate of deactivation, the qptimality condition corresponds to having the ratio of any two consecutive concentrations as a single-parameter increasing function of the previous ratio of consecutive concentrations.List of Symbols C _E,0 mol.m^–3 Initial concentration of active enzyme - C _E,i mol.m^–3 Concentration of active enzyme at the outlet of the i-th reactor - C _S,0 mol.m^–3 Initial concentration of substrate - C _S,i mol.m^–3 Concentration of substrate at the outlet of the i-th reactor - Da _i Damköhler number associated with the i-th reactor ((V _i.k_v,0.C_E,0)/(Q.C_S,0)) - Da _min Minimum value of the overall Damköhler number - Da _tot Overall Damköhler number - E _d J.mol^–1 Activation energy of the step of deactivation of the enzyme - E _m J.mol^–1 Standard enthalpy change of the step of binding of substrate to the enzyme - E _v J.mol^–1 Activation energy of the step of enzymatic transformation of substrate - i Integer variable - j Dummy integer variable - k Dummy integer variable - k _d,i s^–1 Kinetic constant associated with the deactivation of enzyme in the i-th reactor (k _d,o·exp{–E _d/(R.T _i}) - k _d,0 s^–1 Preexponential factor of the kinetic constant associated with the deactivation of the enzyme - K _m,i mol.m^–3 Equilibrium constant associated with the binding of substrate to the enzyme in the i-th reactor, (k _m,o·exp{–E _m}(R.T _i}) - K _m,0 mol.m^–3 Preexponential factor of the Michaelis-Menten constant associated with the binding of substrate to the enzyme - k _v,i s^–1 Kinetic constant associated with the transformation of the substrate by the enzyme in the i-th reactor (k _v,o·exp{–E _v/(R.T _i})) - k _v,0 s^–1 Preexponential factor of the kinetic constant associated with the transformation of the substrate by the enzyme - N Number of reactors in the series - Q m³.s^–1 Volumetric flow rate of reacting liquid through the reactor network - R J.K^–1.mol^–1 Ideal gas constant - T _i K Absolute temperature at the outlet of the i-th reactor - T ₀ K Initial absolute temperature - V _i m³ Volume of the i-th reactor - v _max mol.m^–3.s^–1 Maximum rate of reaction under saturation conditions of substrate - x _i Normalized concentration of substrate (C_S,i/C_{S, 0}) - x _i,opt Optimum value of the normalized concentration of substrate - y _i Dimensionless temperature (exp{–T ₀/T _i}) - y _i,opt Optimum value of the dimensionless temperature Greek Symbols Dimensionless preexponential factor associated with the Michaelis-Menten constant (K _m,0/C_s,0) - Dimensionless activation energy of the step of enzymatic transformation of substrate (E _v/R.T₀)) - Dimensionless standard enthalpy change of the step of binding of substrate to the enzyme (E _m/(R.T₀)) - Dimensionless activation energy of the step of deactivation of the enzyme (E _d/(R.T₀)) - Dimensionless deactivation preexponential factor ((k _d,0.C_S,0)/(k_v,0.C_E,0)     

Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

Purification and characterization of an (S)-3-hydroxycarboxylate oxidoreductase from Clostridium tyrobutyricum

An NADP⁺ —dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified. As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps. Its molecular mass was estimated to be 40–43 kDa. The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate. Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate. Except for the latter all substrates were converted enantioselectively to (S)-3-hydroxy acids in the presence of NADPH. 4-Chloro-3-oxobutyrate was reduced to the (R)-3-hydroxy acid. The specific activity of the enzyme was about 1400 mol min^–1 mg^–1 protein for the reduction of 3-oxobutyrate at pH 5.0. The Michaelis constant (K _m) values for 3-oxobutyrate, 3-oxovalerate and 3-oxocaproate were determined to be 0.22, 1.6 and 3.0 mM, respectively. The K _m values for dehydrogenation of (S)-3-hydroxybutyrate, (S)-3-hydroxyvalerate and (S)-3-hydroxycaproate were found to be 2.6, 1.1 and 5.2 mM, respectively. The identity of 43 of the first 45 N-terminal amino acid residues has been determined. So far such enzyme activities have been described in eucaryotes only.Dedicated to Prof. A. Trebst on the occasion of his 65th birthday     

Stroke volume response to progressive exercise in athletes engaged in different types of training

Using the impedance cardiography method, heart rate ( _c) matched changes on indexed stroke volume (SI) and cardiac output (CI) were compared in subjects engaged in different types of training. The subjects consisted of untrained controls (C), volleyball players (VB) who spent about half of their training time (360 min · week^–1) doing anaerobic conditioning exercises and who had a maximal oxygen uptake ( ) 41% higher than the controls, and distance runners (D) who spent all their training time (366 min·week^–1) doing aerobic conditioning exercises and who had a 26% higher than VB. The subjects performed progressive submaximal cycle ergometer exercise (10 W·min^–1) up to _c of 150 beats·min^–1. In group C, SI had increased significantly (P<0.05) at _c of 90 beats·min^–1 ( + 32%) and maintained this difference up to 110 beats·min^–1, only to return to resting values on reaching 130 beats·min^–1 with no further changes. In group VB, SI peaked (+ 54%) at _c of 110 beats·min^–1, reaching a value significantly higher than that of group C, but decreased progressively to 22010 of the resting value on reaching 150 beats·min^–1. In group D, SI peaked at _c of 130 beats·min^–1 (+ 54%), reaching a value significantly higher than that of group VB, and showed no significant reduction with respect to this peak value on reaching 150 beats·min^–1. As a consequence, the mean CI increase per _c unit was progressively higher in VB than in C (+46%) and in D than in VB (+ 105%). It was concluded that thef _c value at which SI ceased to increase during incremental exercise was closely related to the endurance component in the training programme.     

Ventilatory responses to exercise and carbon dioxide in elderly and younger humans

The present investigation examined the relationship between CO₂ sensitivity [at rest (S _R) and during exercise (S _E)] and the ventilatory response to exercise in ten elderly (61–79 years) and ten younger (17–26 years) subjects. The gradient of the relationship between minute ventilation and CO₂ production ( _E/ CO₂) of the elderly subjects was greater than that of the younger subjects [mean (SEM); 32.8 (1.6) vs 27.3 (0.4); P<0.01]. At rest, S _R was lower for the elderly than for the younger group [10.77 (1.72) vs 16.95 (2.13) 1 · min^–1 · kPa^–1; 1.44 (0.23) vs 2.26 (0.28) 1 · min^–1 · mmHg^–1; P<0.05], but S _E was not significantly different between the two groups [17.85 (2.49) vs 19.17 (1.62) l · min^–1 · kPa^–1; 2.38 (0.33) vs 2.56 (0.21) 1 · min^–1 · mmHg^–1]. There were significant correlations between both S _R and S _E, and _E/ CO₂ (P<0.05; P<0.001) for the younger group, bot none for the elderly. The absence of a correlation for the elderly supports the suggestion that _E/ CO₂ is not an appropriate index of the ventilatory response to exercise for elderly humans.     

Cost minimization in the predesign of an enzymatic CSTR: an overall approach

The balance equations pertaining to the modelling of a CSTR performing an enzyme-catalyzed reaction in the presence of enzyme deactivation are developed. Combination of heuristic correlations for the size-dependent cost of equipment and the purification-dependent cost of recovery of product with the mass balances was used as a basis for the development of expressions relating a (suitably defined) dimensionless economic parameter with the optimal outlet substrate concentration under the assumption that overall production costs per unit mass of product were to be minimized. The situation of Michaelis-Menten kinetics for the substrate depletion and first order kinetics for the deactivation of enzyme (considering that the free enzyme and the enzyme in the enzyme/substrate complex deactivate at different rates) was explored, and plots for several values of the parameters germane to the analysis are included.List of Symbols C _E mol m^–3 concentration of active enzyme - C _E,0 mol m^–3 initial concentration of active enzyme - C _p mol m^–3 concentration of product of interest - C _s mol m^–3 concentration of substrate - C _s,0 mol m^–3 initial concentration of substrate - I $ capital cost of equipment - k _d s^–1 deactivation constant of free enzyme - k _d s^–1 deactivation constant of enzyme in enzyme/substrate complex - K _m mol m^–3 Michaelis-Menten constant - K _m dimensionless counterpart of K _m - k _r s^–1 rate constant associated with conversion of enzyme/substrate complex into product - M _w kg mol^–1 molecular weight of product of interest - P $ kg^–1 cost of recovery of product of interest in pure form - Q m³s^–1 volumetric flow rate - V m³ volume of reactor - X $ kg^–1 global manufacture cost of product of interest in pure form - X dimensionless counterpart of X Greek Symbols ₁ $ m^–1.8 constant - ₂ $ m^–3 constant - t s useful life of CSTR - ₀ ratio of initial concentrations of enzyme and substrate - ratio of deactivation constant of free enzyme to rate constant of depletion of substrate - ratio of deactivation constants - univariate function expressing the dependence of the rate of enzyme deactivation on C _S - univariate function expressing the dependence of the rate of substrate depletion on C _S - dimensionless economic parameter     

Studies on the variables and kinetics of SCP production from nopal fruit juice

Summary Submerged batch cultivation under controlled environmental conditions of pH 3.8, temperature 30°C, and K_La200 h^–1 (above 180 mMO₂ l ^–1 h^–1 oxygen supply rate) produced a maximum (12.0 g·l ^–1) SCP (Candida utilis) yield on the deseeded nopal fruit juice medium containing C/N ratio of 7.0 (initial sugar concentration 25 g·l ^–1) with a yield coefficient of 0.52 g cells/g sugar. In continuous cultivation, 19.9 g·l ^–1 cell mass could be obtained at a dilution rate (D) of 0.36 h^–1 under identical environmental conditions, showing a productivity of 7.2 g·l ^–1·h^–1. This corresponded to a gain of 9.0 in productivity in continuous culture over batch culture. Starting with steady state values of state variables, cell mass (C_X–19.9 g·l ^–1), limiting nutrient concentration (C_ln–2.5 g·l ^–1) and sugar concentration (C_S–1.5 g·l ^–1) at control variable conditions of pH 3.8, 30°C, and K_La 200 h^–1 keeping D=0.36 h^–1 as reference, transient response studies by step changes of these control variables also showed that this pH, temperature and K_La conditions are most suitable for SCP cultivation on nopal fruit juice. Kinetic equations obtained from experimental data were analysed and kinetic parameters determined graphically. Results of SCP production from nopal fruit juice are described.Nomenclature C_ln concentration of ammonium sulfate (g·l ^–1) - C_S concentration of total sugar (g·l ^–1) - C_X cell concentration (g·l ^–1) - D dilution rate (h^–1) - K_ln Monod's constant (g·l ^–1) - m maintenance coefficient (g ammonium sulfate cell^–1 h^–1) - m_(S) maintenance coefficient (g sugar g cell^–1 h^–1) - t time, h - Y yield coefficient (g cells/g ammonium sulfate) - Y_m maximum of Y - Y_S yield coefficient based on sugar consumed (g cells · g sugar^–1) - Y_S(m) maximum value of Y_S - ^µm maximum specific growth rate constant (h^–1)