J.M. Obón

High-density Escherichia coli cultures for continuous L(-)-carnitine production.

Abstract The use of a biological procedure for l-carnitine production as an alternative to chemical methods must be accompanied by an efficient and highly productive reaction system. Continuous l-carnitine production from crotonobetaine was studied in a cell-recycle reactor with Escherichia coli O44 K74 as biocatalyst. This bioreactor, running under the optimum medium composition (25 mM fumarate, 5 g/l peptone), was able to reach a high cell density (26 g dry weight/l) and therefore to obtain high productivity values (6.2 g l-carnitine l−1 h−1). This process showed its feasibility for industrial l-carnitine production. In addition, resting cells maintained in continuous operation, with crotonobetaine as the only medium component, kept their biocatalytic capacity for 4 days, but the biotransformation capacity decreased progressively when this particular method of cultivation was used.

Comparative thermostability of glucose dehydrogenase from Haloferax mediterranei. Effects of salts and polyols

The effect of temperature and pH on thermoinactivation kinetics of glucose dehydrogenase from Haloferax mediterranei has been studied in the presence of different monovalent salts (LiCl, LiBr, NaCl, NaBr, KCl, KBr, NH4Cl, and NH4Br) and polyols (glycerol, erythrytol, xylitol, and sorbitol) concentrations. The stabilization degree of salts followed the rank of the Hofmeister series, and the product of the Setchenov constant (Ks) times the concentration of solute (Cs) was useful to predict the enzyme stability in the presence of salt solutions. Polyols stabilized the halophilic enzyme as much as salts. For an equal polyol concentration, the thermostability increased in the range glycerol < erythritol < xylitol < sorbitol. The overall hydroxyl group concentration proved to be a good parameter for correlating the protective effect of polyols with the polyol nature. Thermoinactivation of the halophilic glucose dehydrogenase in the presence of NaCl and sorbitol was compared with that of a nonhalophilic glucose dehydrogenase in terms of the transition state theory. The free activation energy was, in all cases, enthalpy driven, and hydrogen-bond and/or ionic-binding interactions are the main forces involved in protein stabilization. The halophilic enzyme showed, in general, lower free activation energies for the deactivation process. The adaptation of the enzyme to a halophilic environment led to an enzyme with higher activity at high salt concentrations, but such an increase in enzyme activity was not related to an enhancement in enzyme thermostability.

Increased activity of glucose dehydrogenase co-immobilized with a redox mediator in a bioreactor with electrochemical NAD+ regeneration

An electrochemical bioreactor with glucose dehydrogenase immobilized on to the electrode surface produced gluconic acid from glucose with concomitant recycling of the NAD+ coenzyme at 0.7 V. Since the enzyme is deactivated during operation at this redox potential, co-immobilization of 3,4-dihydroxybenzaldehyde as mediator allowed the system to operate at 0.2 V and increased both the activity (2.4-times) and the stability of the immobilized enzyme (2.2-times). The different effective electrochemical surfaces resulting from the different mediator immobilization modes are important in determining these three properties.

l(−)-Carnitine production with immobilized Escherichia coli cells in continuous reactors

Abstract Escherichia coli 044 K74 was able to biotransform trans-crotonobetaine to l (−)-carnitine in continuous packed-bed reactors with cells immobilized in glass beads or polyurethane foams. Fumarate was added to the medium used for growth to increase both the carnitine production rate and carnitine yield. Although high productivities close to 2 g l−1h−1 were obtained with both supports, carnitine yields of 26% are still low for its large-scale application. Biotransformation rates with immobilized resting cells took place to the same extent as that with growing cells, but enhanced the carnitine yield; however, after a week of continuous l (−)-carnitine production, the biotransformation capacities diminished due to a progressive cell washout. The reactors with immobilized resting cells completely recovered the l (−)-carnitine production rate after feeding the systems with complex medium for 12 h. Continuous reactors with immobilized resting cells look promising for bioconversions when run under continuous production/regeneration cycles.

L(-)-carnitine production using a recombinant Escherichia coli strain

The L(-)-carnitine production by biotransformation using the recombinant strain Escherichia coli pT7-5KE32 has been studied and optimized with crotonobetaine and D(+)-carnitine as substrates. A resting rather than a growing cells system for L(-)-carnitine production was chosen, crotonobetaine being the best substrate. High biocatalytic activity was obtained after growing the cells under anaerobic conditions at 37 degrees C and with crotonobetaine or L(-)-carnitine as inducer. The growth incubation temperature (37 degrees C) was high enough as to activate the heat-inducible lambdap(L) promoter inserted in the plasmid pGP1-2. The best biotransformation conditions were with resting cells, under aerobiosis, with 4 g l(-1) and 100 mM biomass and substrate concentrations respectively. Under these conditions the biotransformation time (1 h) was shorter and the L(-)-carnitine yield (70%) higher than previously reported. Consequently productivity value (11.3 g l(-1)h(-1)) was highly improved when comparing with other published works. The resting cells could be reused until eight times maintaining product yield levels well over 50% that meant to increase ten times the L(-)-carnitine obtained per gram of biomass.

β‐Galactosidase immobilization for milk lactose hydrolysis: a simple experimental and modelling study of batch and continuous reactors

The experiment described in this paper introduces students to the practical use of an enzyme (beta-galactosidase, or lactase) acting on a natural substrate. The enzyme is immobilized onto a cheap support, and the immobilized derivative is used in a packed-bed reactor for continuous milk lactose hydrolysis. The results are compared to those obtained for discontinuous batch reactors with soluble enzyme. A mathematical model of the two types of reactors is run, and its results are compared with the experimental data obtained.

Continuous retention of native NADP(H) in an enzyme membrane reactor for gluconate and glutamate production

A new concept of membrane reactor for continuous retention of NADP(H) with non-charged ultrafiltration membranes is proposed. The presence of the high molecular weight polymer polyethyleneimine (PEI) in the reaction medium allow to achieve high retainment ratios for the native coenzyme when the molar ratio PEI/NADP(H) in the reactor is > 1. Nad concentrations > 0.3 M decrease significantly the retainment ratio of NADP + in the reactor. Therefore, this system is applicable for enzyme reactions requiring low ionic strength media. This reactor configuration has been applied to obtain simultaneously gluconate and glutamate by coupling two enzymes for coenzyme regeneration, glucose dehydrogenase and glutamate dehydrogenase. Sorbitol stabilization of the halophilic enzymes allows to compensate the reduced NaCl concentration required for coenzyme retention. Experimental performance of the reaction is compared successfully with a theoretical model that considers the kinetic mechanisms of the enzymes involved.

Effect of temperature and long-term operation on passively immobilizedZymomonasmobilis for continuous ethanol production

SummaryA fibrous support was used forZ. mobilis immobilization. The system showed a broad optimum temperature range (25–35°C) for highest ethanol productivity, ethanol yield and glucose conversion during continuous fermentation of a 100 g/L glucose medium. Ethanol production and glucose conversion kept steady during two months of continuous operation at D=1h−1.

pH influence on ethanol production and retained biomass in a passively immobilizedZymomonasmobilis system

SummaryA broad pH range of 4.5–7.5 for maximum ethanol productivity and ethanol yield was observed with a passively immobilizedZ.mobilis system. Total retained biomass (as suspended flocs and entrapped cells) was >50 g/l for medium pH values between 4.0–8.0. The entrapped cells to suspended flocs ratio was highest at pH 4.0, whereas at pH above 5.2 it was close to 1.0. The observed enhancement of cell immobilization on the packing support at low pH seemed to be related to formation of bacterial filaments.

Water Concentration and Activity Effects on Aminoacylase in Aqueous/Organic One-Liquid-Phase Systems

Aminoacylase has been employed as a model system to study its catalytic properties at low water concentrations/water activities with different water-miscible organic cosolvents. Cosolvents assayed were alcohols and polyols with pure logarithm of the partition coefficient (log P) values, on the standard water/octanol system, ranging between -5.2 and 0.24.Experimental hydrolysis equilibrium constants (Kapp), at a constant water concentration, decreased with the fall in log P of the cosolvent, as well as with reduction of the water concentration/water activity, as would be expected. The enzyme hydrolytic and synthetic activities, measured at a constant water concentration/water activity value, followed a sigmoidal dependence on log P of the cosolvent employed when the water concentration or water activity values were lower than 50% (w/w) or 0.66, respectively. This became a hyperbolic relationship at higher water concentration/water activity values. A linear relationship between the logarithm of the limiting...