Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens

Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l⁻¹) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l⁻¹ for B. adusta and I. benzoinum and 100 mg l⁻¹ for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l⁻¹). Received: 10 December 1996 / Received revision: 17 February 1997 / Accepted: 22 February 1997     

Lysozyme reactivation using immobilized molecular chaperonin GroEL

The molecular chaperonin, GroEL, was immobilized to a porous matrix and used to reactivate denatured lysozyme. The maximum reactivation yield was obtained at 37°C and pH 6–8 and about 90% activity of the denatured lysozyme was restored under the conditions. The coupling density of GroEL had little effect on the chaperoning activity of GroEL up to 48 mg g^–1 gel. The immobilized GroEL was reusable, indicating the possibility of using it on a large scale for the refolding of proteins.     

Characterization of galactose-induced extracellular and intracellular pectolytic activities from the exo-1 mutant strain of Neurospora crassa

Pectolytic enzymes from the hyperproducer exo-1 mutant of Neurospora crassa are induced either by pectin or galactose. Galactose-induced pectinases, in contrast with pectin-induced enzymes, are not affected by glucose repression. Here, the pectolytic enzymes induced by galactose were purified and characterized. Extracellular pectolytic activities were separated into two main fractions. Pool I contained lyases, and a polygalacturonase (PG) copurifying as a complex of about 80 kDa (gel filtration). Pool II contained PG only. Under urea-SDS-PAGE the lyases and polygalacturonase from pool I migrated with an apparent MW of 56.2 kDa, and 34.3 kDa, respectively. PG from pool II exhibited an apparent MW of 44.7 kDa. Cell extracts contained PG free of lyase activities. Purified intracellular PG migrated (SDS-PAGE) as a single band of apparent MW of 31.5 kDa. All pectinases were glycoproteins (18.5–39% carbohydrate), with stability and optimum pH at 5–6 and 9–10 for PG and lyases, respectively. Temperature optima were 40–50°C, respectively. All enzymes were inactivated at 60°C, with a half-life from 1.5 to 5 min. Activation energy (Ea) values for extracellular and intracellular PG varied between 0.45 and 2.0 Kcal mol⁻¹. Pool II and intracellular PG and lyases, exhibited a random mechanism of hydrolysis. Pool I PG exhibited an exo character. Received 20 October 1997/ Accepted in revised form 28 February 1998     

Bioconversion of 2-cyanopyrazine to 5-hydroxypyrazine-2-carboxylic acid with Agrobacterium sp. DSM 6336

5-Hydroxypyrazine-2-carboxylic acid, a versatile building block for the synthesis of new antituberculous agents, was prepared by whole-cell biotransformation from 2-cyanopyrazine via pyrazinecarboxylic acid using Agrobacterium sp. DSM 6336. By developing a fermentation process for this two-enzyme-step bioconversion, a product concentration of 286 mM (40 g/l) was obtained. After the isolation method had been optimized the total yield was 80%. Received: 28 February 1997 / Received revision: 28 April 1997 / Accepted: 4 May 1997     

Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1

The dibenzo-p-dioxin(DD)- and dibenzofuran(DF)-degrading bacterium, Sphingomonas sp. strain RW1, was tagged by insertion of a mini-Tn5 lacZ transposon in order to follow its fate in complex laboratory soil systems. The tagged strain was tested for its ability to survive in soil and degrade DF and DD applied at a concentration of 1 mg/g. Bacteria pre-adapted to soil conditions were found to survive better in DF- and DD-amended soil and degrade the substrate more efficiently than bacteria that had not been subjected to pre-adaptation. The concentration of soil-applied DF and DD, individually and in combination, decreased to less than 2% of the original concentrations within 3 weeks of addition of the RW1 derivative, accompanied by a short, but significant exponential increase in RW1 viable cells. During the same period the native bacterial population in soil was stable while viable fungi declined. Received: 12 November 1996 / Received revision: 21 February 1997 / Accepted: 22 February 1997     

Microorganism inactivation using high-pressure generation in sealed vessels under sub-zero temperature

In order to test the possibility of utilizing high pressure in bioscience and biotechnology, a simple method for high-pressure generation and its use for microbial inactivation have been studied. When a pressure vessel was filled with water, sealed tightly and cooled to sub-zero temperatures, high pressure was generated in the vessel. The pressure generation was 60 MPa at −5 °C, 103 MPa at −10 °C, and 140 MPa at −15 °C, −20 °C, and −22 °C. The high pressure generated inactivated microorganisms effectively: yeasts (Saccharomyces cerevisiae and Zygosaccharomyces rouxii), bacteria (Lactobacillus brevis and Eschericia coli), and fungi (Aspergillus niger and Aspergillus oryzae) were completely inactivated when stored in sealed vessels −20 °C for 24 h. However, Staphylococcus aureus was only partly inactivated under the same conditions. This method opens up a new application of high pressure for storing, transporting, and sterilizing of foods and biological materials. Received: 28 July 1997 / Received last revision: 12 June 1998 / Accepted: 19 June 1998     

On-line enzyme activity determination using the stopped-flow technique: application to laccase activity in pulp mill waste-water treatment

An automated system for on-line measurement of enzyme activity is proposed. The system uses a flow injection manifold in the stopped-flow mode to measure initial reaction rates. The time during which the flow is halted is selected in such a way as to optimise the enzyme/substrate ratio for the correct determination of activity values. The proposed system was used to determine the activity of laccase produced by the fungus Trametes versicolor immobilised on nylon in a fixed-bed reactor used for treating pulp mill waste water. Received: 17 February 1997 / Received revision: 23 April 1997 / Accepted: 27 April 1997     

A third xylanase from Trichoderma reesei PC-3-7

A third xylanase (Xyn III) from Trichoderma reesei PC-3–7 was purified to electrophoretic homogeneity by gel filtration and ion-exchange chromatographies. The enzyme had a molecular mass of 32 kDa, and its isoelectric point was 9.1. The pH optimum of Xyn III was 6.0, similar to that of Xyn II, another basic xylanase of  T. reesei. The purified Xyn III showed high activity with birchwood xylan but no activity with cellulose and aryl glycoside. The hydrolysis of birchwood xylan by Xyn III produced mainly xylobiose, xylotriose and other xylooligosaccharides. The amino acid sequences of the N-terminus and internal peptides of Xyn III exhibited high homology with the family F xylanases, showing that they were distinct from those of Xyn I and Xyn II of  T. reesei, which belong to family G. These results reveal that Xyn III is a new specific endoxylanase, differing from Xyn I and Xyn II in  T. reesei. It is noteworthy that this novel xylanase was induced only by cellulosic substrates and l-sorbose but not by xylan and its derivarives. Furthermore,  T. reesei PC-3-7 produced Xyn III in quantity when grown on Avicel or lactose as a carbon source, while  T. reesei QM9414 produced little or no Xyn III. Received: 7 November 1997 / Received last revision: 2 February 1988 / Accepted: 23 February 1998     

One-step transformation of the dimorphic yeast Yarrowia lipolytica

An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10⁵ transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997     

Glucose-induced inactivation of isocitrate lyase in Aspergillus nidulans

     The existence of a second mechanism of catabolite control of isocitrate lyase of Aspergillus nidulans, in addition to the carbon catabolite repression phenomenon recently reported was analysed. Isocitrate lyase was rapidly and specifically inactivated by glucose. The inactivation was irreversible at all stages in the presence of cycloheximide, showing that reactivation depends on de novo protein synthesis. In addition, analysis of glucose-induced inactivation of isocitrate lyase in a creA ^d -30 strain showed that the creA gene is not involved in this process. Received: 13 May 1994 / Accepted 12 August 1994     

Bioconversion of limonene to α-terpineol by immobilized Penicillium digitatum

Bioconversion of (4R)-(+)-limonene to (4R)-(+)-α-terpineol by immobilized fungal mycelia of Penicillium digitatum was investigated in batch, repeated-batch and continuously fed systems. The fungi were immobilized in calcium alginate beads. These beads remained active for at least 14 days when they were stored at 4 °C. Three different aeration rates were tested. The highest yield was obtained at a dissolved oxygen level of 50.0 μmol/l. α-Terpineol production by this fungus was 12.83 mg (g beads)⁻¹ day⁻¹, producing a 45.81% bioconversion of substrate. Repeated-batch bioconversion showed yield decreases in the second and the third cycles. Regeneration with nutrient media after the third cycle improved the bioconversion yields. With continuous bioconversion, the half-life was dependent on the aeration. The optimum conditions with a continuous reactor were at an aeration rate of 0.3 standard l/min and a dilution rate of 0.0144 h⁻¹. Received: 10 June 1997 / Received revision: 18 August 1997 / Accepted: 11 September 1997     

Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp

Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers). Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released (K _v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from Penicillium simplicissimum (K _v = 0.15 l mPa⁻¹s⁻¹g⁻¹) and a mannanase from Sclerotium rolfsii (K _v = 0.12 l mPa⁻¹s⁻¹g⁻¹) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO respectively. A less significant brightness increase was obtained with enzymes showing lower K _v values, such as a xylanase from Schizophyllum commune (K_v = 0.051  l mPa⁻¹s⁻¹g⁻¹, 0.2% ISO) and a bacterial mannanase (K _v = 0.061 l mPa⁻¹s⁻¹g⁻¹,0.5% ISO). Received: 19 December 1996 / Received revision: 20 February 1997 / Accepted: 22 February 1997     

Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme

By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4. The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1. Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-l bioreactor at 37 °C and pH 7.0. Overexpression of MDH in Escherichia coli BL21(DE3) [pASFG1] was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Under standard growth conditions, E. coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low. Decreasing the growth temperature to 27 °C and omitting pH regulation resulted in a significant increase in the formation of soluble and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26 000 U/l, which corresponds to 0.38 g/l MDH. This was an 87-fold overexpression of MDH compared to that of the natural host R. sphaeroides Si4, and a 236-fold improvement of the volumetric yield. MDH was purified from E. coli BL21(DE3) [pASFG1] with 67% recovery, using ammo-nium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration. Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme. Received: 18 February 1997 / Received revision: 27 March 1997 / Accepted: 27 March 1997     

A para-site-specific hydroxylation of aromatic compounds by Mycobacterium sp. strain 12523: stabilization of the hydroxylation activity

Mycobacterium sp. strain 12523 has a para-site-specific hydroxylation activity, which produce para-substituted phenols from various aromatic compounds. However, the activity is unstable and the reactions are inactivated within 24 h. In order to extend the reaction period, the factors that affected reaction stability were examined. The hydroxylation activity of the cells incubated in buffer was significantly stabilized by the inclusion of an inducer such as methyl ethyl ketone. It is suggested that a regulatory mechanism is involved in controlling the activity. This study resulted in the development of a convenient method to stabilize the hydroxylation activity, involving the addition of an inducer, such as acetone, to the reaction system. This method permitted the hydroxylation reaction to continue for more than 67 h. Received: 27 January 1997 / Received revision: 18 March 1997 / Accepted: 13 April 1997     

Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination

The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27–43 days) and the volumetric productivity was maximum (18.7 mg day⁻¹ l⁻¹ gel) at the highest dilution rate (0.25 day⁻¹) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer. Received: 22 October 1999 / Received revision: 14 February 2000 / Accepted: 18 February 2000     

Biotransformation of linoleic acid with the Candida tropicalis M25 mutant

Linoleic acid was transformed by mutant Candida tropicalis M25 and transformations were studied in batch and fed-batch cultures. Cofermentations with palmitic acid as inducer of the fatty acid degradation pathway were performed. Besides the (Z),(Z)-octadeca-6,9-dienedioic acid, (Z),(Z)-3-hydroxyoctadeca-9,12-dienedioic acid and (Z),(Z)-3-hydroxytetradeca-5,8-dienedioic acid were obtained as the main fermentation products. The maximum concentrations of (Z),(Z)-octadeca-6,9-dienedioic acid and (Z),(Z)-3-hydroxyoctadeca-9,12-dienedioic acid reached values of 6.4 g/l and 6.9 g/l respectively. The structures of the products were characterized by chemical and spectroscopic methods. The configuration of the double bonds was not changed during bioconversion. As only one regioisomer of the hydroxylated fatty acid was detected, the hydroxylation is site-specific. Received: 11 November 1996 / Received revision: 11 February 1997 / Accepted: 24 February 1997     

Efficient secretion of Trichoderma reesei cellobiohydrolase II in Schizosaccharomyces pombe and characterization of its products

A cbh2 cDNA encoding Trichoderma reesei QM9414 cellobiohydrolase II, located on the expression vector whose copy number is controlled by the level of gentamicin, was successfully expressed under the control of a human cytomegalovirus promoter in the fission yeast, Schizosaccharomyces pombe. The 24-amino-acid leader peptide of the cbh2 gene was recognized by the yeast, enabling the efficient secretion of the heterologous cellobiohydrolase. The transformed S. pombe strain produced over 115 μg cellobiohydrolase proteins/ml rich medium supplemented with malt extract and 100 μg/ml gentamicin. The molecular masses of the recombinant cellobiohydrolases, secreted as two molecular species, were estimated to be 70 kDa and 72 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Deglycosylation treatments revealed that the recombinant enzymes were overglycosylated and scarcely susceptible to α-mannosidase. The recombinant enzymes showed no carboxymethylcellulase activity, but showed similar characteristics to those of a native enzyme purified from T. reesei in their optimum pH and temperature, pH and temperature stabilities, and V _max values toward phosphoric-acid-swollen cellulose as substrate, except that their K _m values were about fourfold higher than that of the native enzyme. Received: 4 August 1997 / Received revision: 13 October 1997 / Accepted: 31 October 1997     

Removal of organic pollutants and of nitrate from wastewater from the dairy industry by denitrification

The aim of this work was to remove nitrate-N and organic pollutants from wastewater of the dairy industry by denitrification. An artificially prepared wastewater, containing 250 mg/l nitrate-N and 1.5 g/l whey powder, was completely denitrified with removal of 90%–93% of the chemical oxygen demand (COD) of the whey powder by suspended or immobilized mixed cultures and by a suspended or immobilized pure culture that was isolated from the mixed culture inoculum. For the above COD/nitrate-N ratio of 6:1, the results indicated that the organic compounds of the wastewater served as electron donors for complete denitrification and that there was no need to add an external carbon source. In batch denitrification assays the suspended or immobilized mixed cultures proved to be more active and reacted faster than the isolated pure cultures. In continuous denitrification processes with immobilized pure or mixed cultures, the alginate beads, used for immobilization, were not stable for more than 12 days of incubation. The mixed free cultures removed the nitrate-N and COD continuously with no change of their activity for at least 15 days at an optimum hydraulic retention time of 0.27 days with a loading rate of 900 mg nitrate-N l⁻¹ day⁻¹. Received: 13 October 1997 /  Received revision: 16 December 1997 / Accepted: 19 December 1997     

Respiratory activity of biofilms: measurement and its significance for the elimination of n-butanol from waste gas

A reaction chamber was developed to determine the respiratory activity of microorganisms immobilized on various support materials for waste gas treatment. The volumetric respiration rate was identified as a suitable parameter for estimating the degradative activity of waste gas treatment plants. A laboratory trickle-bed reactor was filled with either granular clay, polyamide beads, or sintered styrofoam. n-Butanol was used as model solvent to determine the efficiency of its elimination from the gas phase. This crucial parameter was correlated with the volumetric degradation rate, determined from the overall material balance under steady-state operating conditions. The volumetric respiration rate of n-butanol was determined with the reaction chamber, and exceeded the volumetric degradation rate of n-butanol determined from the reactor 16- to 26-fold, depending on the support material. The respiration rate was correlated to the degradation rate by the stoichiometry of n-butanol oxidation and a correlation factor of 2.6–4.3. The volumetric respiration rate appeared to be a suitable parameter to determine the degradative activity of the trickle-bed reactor used. The volumetric respiration rate can be ultimately applied to estimate the efficiency of elimination of an organic pollutant and to calculate the dimensions of a reactor required to eliminate a given organic load from waste gas. Received: 20 February 1997 / Received revision: 20 May 1997 / Accepted: 20 May 1997