A covalent two-step immobilization technique using itaconic anhydride

β-Glucosidase from almonds (EC 3.2.1.21) was covalently immobilized by a two-step technique. In the first step, double bonds were introduced into the β-glucosidase by derivatization with itaconic anhydride. In separate studies with α-N-protected l-amino acids, it was established that itaconic anhydride acylated mainly primary amino groups of lysines and, to a much lesser extent hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. The acylated β-glucosidase showed no loss of activity and the K _m decreased from 3.6 mM to 2.6 mM when p-nitrophenyl β-d-glucopyranoside was used as the substrate. In the second step, the derivatized β-glucosidase was co-polymerized radically with N,N′-methylenebisacrylamide in buffer solution. The resulting acrylamide immobilizate possessed a much better storage stability at 30–56 °C when compared to β-glucosidase immobilized on Eupergit C. However, the specific activity was higher with the Eupergit immobilizate. Free and acrylamide-immobilized β-glucosidase were used for glucosylation of chloramphenicol by transglucosylation in 20% (v/v) acetonitrile at 37 °C. The acrylamide immobilizate demonstrated a great enhancement of stability and approximately 50% more chloramphenicol β-glucoside was obtained after 5 h. Received: 22 September 1997 / Accepted: 28 October 1997     

High-density Escherichia coli cultures for continuous l(−)-carnitine production

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. Received: 10 December 1998 / Received revision: 19 February 1999 / Accepted: 20 February 1999     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Induction of xylanase in Aspergillus tamarii by methyl β-d-xyloside

Aspergillus tamarii produced extracellular xylanase and intracellular β-xylosidase inductively in washed glucose-grown mycelia incubated with xylan and methyl β-d-xyloside, a synthetic glycoside. Methyl β-d-xyloside was a more effective inducer than xylan at the same concentration for both enzymes. Glucose and cycloheximide were found to inhibit xylanase production by methyl β-d-xyloside. Methyl β-d-xyloside was hydrolyzed to xylose by mycelial extract in vitro. Received: 23 May 1996 / Received revision: 5 September 1996 / Accepted: 13 October 1996     

A new route to l-threo-3-[4-(methylthio)phenylserine], a key intermediate for the synthesis of antibiotics: recombinant low-specificity d-threonine aldolase-catalyzed stereospecific resolution

A new enzymatic resolution process was established for the production of l-threo-3-[4-(methylthio)phenylserine] (MTPS), an intermediate for synthesis of antibiotics, florfenicol and thiamphenicol, using the recombinant low-specificity d-threonine aldolase from Arthrobacter sp. DK-38. Chemically synthesized dl-threo-MTPS was efficiently resolved with either the purified enzyme or the intact recombinant Escherichiacoli cells overproducing the enzyme. Under the optimized experimental conditions, 100 mM (22.8 g l⁻¹) l-threo-MTPS was obtained from 200 mM (45.5 g l⁻¹) dl-threo-MTPS, with a molar yield of 50% and a 99.6% enantiomeric excess. Received: 2 September 1998 / Received revision: 27 October 1998 / Accepted: 29 November 1998     

Production of d-ribose by fermentation

The production of d-ribose by fermentation has received much attention lately, possibly because of the use of this pentose to synthesize antiviral and anticancer drugs. This review briefly outlines the methods that have been used to synthesize d-ribose since it was identified in yeast RNA, and focuses in particular on the latest developments in d-ribose fermentation, which have led to d-ribose yields that exceed 90 g/l. Furthermore, the various transketolase-deficient d-ribose-producing mutants that are used, and the biochemical and genetic rationales applied to select them or to enhance their d-ribose productivities, are dealt with. Attention is also drawn to the unusual pleiotropic characteristics of the mutant strains, as well as to the industrial and academic applications of d-ribose. Received: 29 January 1997 / Received revision: 13 March 1997 / Accepted: 15 March 1997     

A self-consistent cell flux expression for simultaneous chemotaxis and contact guidance in tissues

During wound healing, both chemotaxis and contact guidance can contribute to the migration of blood and tissue cells to the wound. In order to understand the wound healing process, we must thus understand how cells respond to both these simultaneous directional cues, which are not necessarily coaligned. Although chemotaxis and contact guidance have been studied individually, the interaction between them has not been addressed. We extend a stochastic cell movement model, developed by Dickinson and Tranquillo (1995) [6] for individual cues, for simultaneous chemotaxis and contact guidance by a two-parameter perturbation analysis in terms of the two associated cues, a chemotactic factor gradient and aligned tissue fibers. We present results from analysis of the first-order perturbation, which includes the cell flux expression heuristically proposed by others, but reveals paradoxical results for other indices of cell movement, such as the mean-squared displacement. We then present second-order perturbation results that resolve these paradoxical results. Finally, we relate these results to a continuum mechanical model developed by Barocas and Tranquillo (1997) [3] that predicts fiber alignment due to cell traction induced tissue contraction. Received: 30 April 1999 / Revised version: 30 October 1999 / Published online: 14 September 2000     

A new arabinofuranohydrolase from Bifidobacterium adolescentis able to remove arabinosyl residues from double-substituted xylose units in arabinoxylan

An arabinofuranohydrolase (AXH-d3) was purified from a cell-free extract of Bifidobacterium adolescentis DSM 20083. The enzyme had a molecular mass of approximately 100 kDa as determined by gel filtration. It displayed maximum activity at pH 6 and 30 °C. Using an arabinoxylan-derived oligosaccharide containing double-substituted xylopyranosyl residues established that the enzyme specifically released terminal arabinofuranosyl residues linked to C-3 of double-substituted xylopyranosyl residues. In addition, this arabinofuranohydrolase released arabinosyl groups from wheat flour arabinoxylan polymer but showed no activity towards p-nitrophenyl α-l-arabinofuranoside or towards sugar-beet arabinan, soy arabinogalactan, arabino-oligosaccharides and arabinogalacto-oligosaccharides. Received: 15 July 1996 / Received revision: 18 October 1996 / Accepted: 18 October 1996     

Enzymatic synthesis of alkyl-α-glucoside catalysed by a thermostable α-transglucosidase in solvent-free organic medium

α-Transglucosidase from Talaromycesduponti was used to synthesize different alkyl-α-d-glucosides from α(1-4) linked carbohydrate donors. The enzymatic preparation, purified by a single step, consisting of hydrophobic interaction chromatography, was sufficiently pure for very stereospecific synthesis to be achieved. Biphasic and homogeneous organic media could be compared for such purposes. Yields appeared to be two- to threefold higher in low-water biphasic media. High concentrations of the glucosyl donor were present in the aqueous phase, while water-immiscible alcohols were used as glucosyl acceptors. The high efficiency of the method was attributed to the shift of the thermodynamic equilibrium thanks to the extraction of the product from the aqueous phase, where the reaction occurs, into the organic phase. Operated in a continuous biphasic reactor, T. dupontiα-transglucosidase showed a good thermostability with a half-life of 23 days at 30 °C. Received: 26 January 1998 / Received revision: 15 April 1998 / Accepted: 19 April 1998     

Enzymatic synthesis of silicon-containing dipeptides with 3-trimethylsilylalanine

In this study, the enzymatic synthesis of silicon-containing dipeptides with a silicon-containing amino acid, 3-trimethylsilylalanine (TMS-Ala), was attempted in ethyl acetate, and the effects of TMS-Ala on thermolysin-catalyzed dipeptide synthesis are also discussed. Benzyloxycarbonyl(Z)-TMS-Ala was recognized by thermolysin as a better substrate than Z-Leu, and various silicon-containing dipeptides, Z-(TMS-Ala)-Xaa-OMe (Xaa = Leu, Ile, Phe, etc.), could be obtained. The acceleration of the reaction rate in the synthesis of Z-(TMS-Ala)-Leu-OMe compared with Z-Leu-Leu-OMe synthesis was explained by the higher hydrophobicity of the side-chain of TMS-Ala containing a trimethylsilyl group. On the other hand, TMS-Ala-OMe was not accepted as the amino component because of the bulkiness of the trimethylsilyl group. The enantioselectivity of thermolysin was very high. Z-d-TMS-Ala was not a substrate, while Z-l-TMS-Ala served as a good substrate. Received: 5 October 1998 / Received last revision: 4 December 1998 / Accepted: 26 December 1998     

Characterization of a xylitol dehydrogenase and a d-arabitol dehydrogenase from the thermo- and acidophilic red alga Galdieria sulphuraria

Galdieria sulphuraria (Galdieri) Merola can grow heterotrophically on at least ten different polyols. We investigated their metabolic path to glycolysis/gluconeogenesis and identified two NAD-dependent polyol dehydrogenases. Activity of other enzymes metabolizing mannitol or sorbitol could not be detected. The two dehydrogenases had a broad substrate specificity and were termed xylitol dehydrogenase (EC 1.1.1.14; substrate specificity: xylitol > d-sorbitol > d-mannitol > l-arabitol) and d-arabitol dehydrogenase (EC 1.1.1.11; substrate specificity: d-arabitol > l-fucitol > d-mannitol > d-threitol) according to the substrate with the lowest K _m value. The xylitol dehydrogenase was stable during purification. In contrast, the d-arabitol dehydrogenase was thermolabile and depended on divalent ions for stability and activity, preferentially Mn²⁺ and Ni²⁺. The molecular mass of the xylitol dehydrogenase was estimated to be 295 kDa by size-exclusion chromatography and 220 kDa by rate-sedimentation centrifugation. The d-arabitol dehydrogenase had a molecular mass of 105 kDa as determined by rate-sedimentation centrifugation. The specific activity of both enzymes increased about fourfold when cells were transferred from autotrophic to heterotrophic conditions regardless of whether sugars or polyols were supplied as substrates. The significance of polyol metabolism in Galdieria sulphuraria with regard to the natural habitat of the alga is discussed. Received: 15 January 1997 / Accepted: 12 February 1997     

Production of d-amino acids using immobilized d-hydantoinase from lentil, Lens esculenta, seeds

d-Hydantoinase from the lentil, Lens esculenta, seed is quite unstable, and has been immobilized on Diethyl amino ethyl (DEAE) cellulose by an adsorption and cross-linking method. The immboilized d-hydantoinase exhibited 80% enzyme activity and contained 86% protein. The immobilization of the enzyme preparation does not change its optimum pH, temperature or affinity constant, but increases its shelf-life, thermostability and stability in various organic solvents. This immobilized d-hydantoinase can be used effectively for the production of d-amino acids from the corresponding hydantoins and may therefore be of use in the chemical and pharmaceutical industries. Received: 28 April 1998 / Received last revision: 10 July 1998 / Accepted: 10 July 1998     

Isolation and characterization of Enterobacter cloacae capable of metabolizing asparagine

A gram-negative, rod-shaped bacterium capable of utilizing l-asparagine as its sole source of carbon and nitrogen was isolated from soil and identified as Enterobacter cloacae. An intracellularly expressed l-asparaginase was detected and it deaminated l-asparagine to aspartic acid and ammonia. High-pressure liquid chromatography analysis of a cell-free asparaginase reaction mixture indicated that 2.8 mM l-asparagine was hydrolyzed to 2.2 and 2.8 mM aspartic acid and ammonia, respectively, within 20 min of incubation. High asparaginase activity was found in cells cultured on l-fructose, d-galactose, saccharose, or maltose, and in cells cultured on l-asparagine as the sole nitrogen source. The pH and temperature optimum of l-asparaginase was 8.5 and 37–42 °C, respectively. The half-life of the enzyme at 30 °C and 37 °C was 10 and 8 h, respectively. Received: 19 February 1998 / Received last revision: 4 June 1998 / Accepted: 10 July 1998     

Novel l-specific cleavage of the urethane bond of t -butoxycarbonylamino acids by whole cells of Corynebacterium aquaticum

Microorganisms capable of cleaving the urethane bond of t-butoxycarbonyl (Boc) amino acids in a whole-cell reaction were screened among stock cultures, and Corynebacterium aquaticum IFO12154 was the most promising. The conversion of Boc-Ala to Ala was stimulated by CoSO₄ in the medium and reaction mixture. The optimum whole-cell concentration was 25 mg lyophilized cells/ml. Boc-l-Met was the best substrate for this reaction, and other Boc-L-amino acids, as well as benzyloxycarbonyl-l-amino acids with hydrophobic residues, were also good substrates. Boc-d- and Z-d-amino acids were inert. When the reactions had proceeded for 24 h with each substrate at 10 mM, the molar conversion rates from Boc-l-, dl- and d-Met were 100%, 50%, and 0% respectively. From 150 mM Boc-l-Met, 143 mM l-Met was formed at a molar yield of 95.3%. Received: 3 September 1996 / Received last revision: 7 April 1997 / Accepted: 19 April 1997     

Biotechnological production of <Emphasis Type="SmallCaps">l</Emphasis>-ribose from <Emphasis Type="SmallCaps">l</Emphasis>-arabinose

l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g⁻¹ h⁻¹ (1.84 ± 0.03 g l⁻¹ h⁻¹) and 0.27 ± 0.01 g g⁻¹ h⁻¹ (1.91 ± 0.1 g l⁻¹ h⁻¹) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose.     

Isolation of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> mutants for heterologous protein production from a double proteinase gene disruptant

The recombinant Pichia pastoris harboring an improved methionine adenosyltransferase (MAT) shuffled gene was employed to biosynthesize S-adenosyl-l-methionine (SAM). Two l-methionine (l-Met) addition strategies were used to supply the precursor: the batch addition strategy (l-Met was added separately at three time points) and the continuous feeding strategies (l-Met was fed continuously at the rate of 0.1, 0.2, and 0.5 g l⁻¹ h⁻¹, respectively). SAM accumulation, l-Met conversion rate, and SAM productivity with the continuous feeding strategies were all improved over the batch addition strategy, which reached 8.46 ± 0.31 g l⁻¹, 41.7 ± 1.4%, and 0.18 ± 0.01 g l⁻¹ h⁻¹ with the best continuous feeding strategy (0.2 g l⁻¹ h⁻¹), respectively. The bottleneck for SAM production with the low l-Met feeding rate (0.1 g L⁻¹ h⁻¹) was the insufficient l-Met supply. The analysis of the key enzyme activities indicated that the tricarboxylic acid cycle and glycolytic pathway were reduced with the increasing l-Met feeding rate, which decreased the adenosine triphosphate (ATP) synthesis. The MAT activity also decreased as the l-Met feeding rate rose. The reduced ATP synthesis and MAT activity were probably the reason for the low SAM accumulation when the l-Met feeding rate reached 0.5 g l⁻¹ h⁻¹.     

Artificially increased ascorbate content affects zeaxanthin formation but not thermal energy dissipation or degradation of antioxidants during cold-induced photooxidative stress in maize leaves

Infiltrating detached maize (Zeamays L.) leaves with L-galactono-1,4-lactone (L-GAL) resulted in a 4-fold increase in the content of leaf ascorbate. Upon exposure to high irradiance (1000 μmol photons m⁻² s⁻¹) at 5 °C, L-GAL leaves de-epoxidized the xanthophyll-cycle pigments faster than the control leaves; the maximal ratio of de-epoxidized xanthophyll-cycle pigments to the whole xanthophyll-cycle pool was the same in both leaf types. The elevated ascorbate content, together with the faster violaxanthin de-epoxidation, did not affect the degree of photoinhibition and the kinetics of the recovery from photoinhibition, assayed by monitoring the maximum quantum efficiency of photosystem II primary photochemistry (F_v/F_m). Under the experimental conditions, the thermal energy dissipation seems to be zeaxanthin-independent since, in contrast to the de-epoxidation, the decrease in the efficiency of excitation-energy capture by open photosystem II reaction centers (F_v′/F_m′) during the high-irradiance treatment at low temperature showed the same kinetic in both leaf types. This was also observed for the recovery of the maximal fluorescence after stress. Furthermore, the elevated ascorbate content did not diminish the degradation of pigments or α-tocopherol when leaves were exposed for up to 24 h to high irradiance at low temperature. Moreover, a higher content of ascorbate appeared to increase the requirement for reduced glutathione. Received: 20 May 1999 / Accepted: 29 October 1999     

Production of d-hydantoinase by halophilic Pseudomonas sp. NCIM 5109

About 1000 bacterial colonies isolated from sea water were screened for their ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine as a criterion for the determination of hydantoinase activity. The strain M-1, out of 11 hydantoinase-producing strains, exhibited the maximum ability to convert dl-5-phenylhydantoin to d(−)N-carbamoylphenylglycine. The strain M-1 appeared to be a halophilic Pseudomonas sp. according to morphological and physiological characteristics. Optimization of the growth parameters revealed that nutrient broth with 2% NaCl was the preferred medium for both biomass and enzyme production. d-Hydantoinase of strain M-1 was not found to be inducible by the addition of uracil, dihydrouracil, β-alanine etc. The optimum temperature for enzyme production was about 25 °C and the organism showed a broad pH optimum (pH 6.5–9.0) for both biomass and hydantoinase production. The organism seems to have a strict requirement of NaCl for both growth and enzyme production. The optimum pH and temperature of enzyme activity were 9–9.5 and 30 °C respectively. The biotransformation under the alkaline conditions allowed the conversion of 80 g l⁻¹ dl-5-phenylhydantoin to 82 g l⁻¹ d(−)N-carbamoylphenylglycine within 24 h with a molar yield of 93%. Received: 15 September 1997 / Received revision: 5 January 1998 / Accepted: 6 January 1998     

Searching for Spatial Patterns in a Pollinator–Plant–Herbivore Mathematical Model

This paper deals with the spatio-temporal dynamics of a pollinator–plant–herbivore mathematical model. The full model consists of three nonlinear reaction–diffusion–advection equations defined on a rectangular region. In view of analyzing the full model, we firstly consider the temporal dynamics of three homogeneous cases. The first one is a model for a mutualistic interaction (pollinator–plant), later on a sort of predator–prey (plant–herbivore) interaction model is studied. In both cases, the interaction term is described by a Holling response of type II. Finally, by considering that the plant population is the unique feeding source for the herbivores, a mathematical model for the three interacting populations is considered. By incorporating a constant diffusion term into the equations for the pollinators and herbivores, we numerically study the spatiotemporal dynamics of the first two mentioned models. For the full model, a constant diffusion and advection terms are included in the equation for the pollinators. For the resulting model, we sketch the proof of the existence, positiveness, and boundedness of solution for an initial and boundary values problem. In order to see the separated effect of the diffusion and advection terms on the final population distributions, a set of numerical simulations are included. We used homogeneous Dirichlet and Neumann boundary conditions.     

3-Ketoglycoside-mediated metabolism of sucrose in E. coli as conferred by genes from Agrobacterium tumefaciens

Escherichia coli strains that did not have the ability to use sucrose as a sole carbon source gained this ability after receiving a cloned fragment of DNA from Agrobacterium tumefaciens. No invertase was detected in the sucrose-metabolizing E. coli, but evidence for the activity of certain enzymes, known to be produced by biotype 1 strains of Agrobacterium, were found. Evidence was found for the presence of d-glucoside 3-dehydrogenase (G3DH) and α-3-ketoglucosidase. The activity of enzyme extracts on 3-ketosucrose also indicated that 3-ketoglucose reductase, or some enzyme that acts on 3-ketoglucose, was present in the Suc⁺ E. coli as well. The fragment was found to complement a G3DH mutant of A. tumefaciens and was also found to confer chemotaxis towards sucrose in E. coli. Received: 13 September 1996 / Received revision: 15 January 1997 / Accepted: 24 January 1997