Emulsifying Activities of Purified Alasan Proteins from Acinetobacter radioresistens KA53

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. The emulsifying activity of the purified polysaccharide (apo-alasan) is very low. Three of the alasan proteins were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had apparent molecular masses of 16, 31, and 45 kDa. Emulsification assays using the isolated alasan proteins demonstrated that the active components of the alasan complex are the proteins. The 45-kDa protein had the highest specific emulsifying activity, 11% higher than the intact alasan complex. The 16- and 31-kDa proteins gave relatively low emulsifying activities, but they were significantly higher than that of apo-alasan. The addition of the purified 16- and 31-kDa proteins to the 45-kDa protein resulted in a 1.8-fold increase in the specific emulsifying activity and increased stability of the oil-in-water emulsion. Fast-performance liquid chromatography analysis indicated that the 45-kDa protein forms a dimer in nondenaturing conditions and interacts with the 16- and 31-kDa proteins to form a high-molecular-mass complex. The 45-kDa protein and the three-protein complex had substrate specificities for emulsification and a range of pH activities similar to that of alasan. The fact that the purified proteins are active emulsifiers should simplify structure-function studies and advance our understanding of their biological roles.     

Emulsifying activities of purified Alasan proteins from Acinetobacter radioresistens KA53

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. The emulsifying activity of the purified polysaccharide (apo-alasan) is very low. Three of the alasan proteins were purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had apparent molecular masses of 16, 31, and 45 kDa. Emulsification assays using the isolated alasan proteins demonstrated that the active components of the alasan complex are the proteins. The 45-kDa protein had the highest specific emulsifying activity, 11% higher than the intact alasan complex. The 16- and 31-kDa proteins gave relatively low emulsifying activities, but they were significantly higher than that of apo-alasan. The addition of the purified 16- and 31-kDa proteins to the 45-kDa protein resulted in a 1.8-fold increase in the specific emulsifying activity and increased stability of the oil-in-water emulsion. Fast-performance liquid chromatography analysis indicated that the 45-kDa protein forms a dimer in nondenaturing conditions and interacts with the 16- and 31-kDa proteins to form a high-molecular-mass complex. The 45-kDa protein and the three-protein complex had substrate specificities for emulsification and a range of pH activities similar to that of alasan. The fact that the purified proteins are active emulsifiers should simplify structure-function studies and advance our understanding of their biological roles.     

Effect of cations, pH and sulfate content on the viscosity and emulsifying activity of the Halomonas eurihalina exopolysaccharide

The effects of monovalent and divalent cations on the rheological behavior of Halomonas eurihalina exopolysaccharide (EPS) were studied. Sodium, potassium, magnesium and calcium were added and the relative abilities to increase viscosity were as follows: KCl > NaCl > MgCl₂ > CaCl₂. The highest viscosity value was measured in acidic 10⁻⁴ M KCl, in which a gel formed. A loss of sulfate content seemed to correlate with the increase of viscosity. H. eurihalina produced EPS in all growth media. Addition of hydrophobic substrates to culture media produced changes in chemical composition and emulsifying activity of the EPS. Xylene was the most effectively emulsified substance and the EPS produced on tetradecane and on corn oil the most active emulsifier. Received 25 July 1997/ Accepted in revised form 30 January 1998     

The AlnB protein of the bioemulsan alasan is a peroxiredoxin

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high molecular weight complex of a polysaccharide and three proteins (AlnA, AlnB and AlnC). AlnA has previously been shown to be an OmpA-like protein that is largely responsible for the emulsifying activity of alasan. To further elucidate the nature of alasan, the gene coding for AlnB was cloned, sequenced and overexpressed in Escherichia coli. The overall 561 bp sequence of the hypothetical AlnB showed strong homology, including all conserved regions and residues known to be essential for enzymatic activity, to the ubiquitous family of thiol-specific antioxidant enzymes known as peroxiredoxins. Transgenic E. coli overexpressing AlnB exhibited increased resistance to cumene hydroperoxide both in liquid culture and on agar medium. Recombinant AlnB had no emulsifying activity but stabilized oil-in-water emulsion generated by AlnA.     

Formation of early-light-inducible-protein complexes and status of xanthophyll levels under high light and cold stress in barley (Hordeum vulgare L.)

In our previous work we found considerable accumulation of early light-inducible proteins (ELIPs) in barley during adaptation to combined high light and cold stress, an accumulation which occurred preferentially in the apical part of the leaves (M.-H. Montané et al., 1997, Planta 202: 293–302). Here we studied, under the same conditions, the effect of adaptation on the composition of thylakoid membrane proteins and pigments, particularly xanthophylls and chlorophyll, and their distribution within the barley leaf. It was observed that high light fluxes appeared to favour the trimerization of the light-harvesting complex of photosystem II (LHC II) whereas cold appeared to favour the monomers of LHC II. High light, cold or the combination of both factors had only a small effect on the protein composition of the thylakoid membranes except for the proteins of LHC II which were found to decrease under high light to a greater extent at 25 °C than at 5 °C. The total xanthophyll-cycle carotenoid content increased linearly with cellular development, the highest amount being observed in the apical part of the leaf. Cold and high light acted synergistically to induce less than a doubling in the amount of total xanthophylls, while chlorophylls a and b remained nearly constant. The fraction consisting of antheraxanthin plus zeaxanthin was up to 4- to 5-fold higher at 5 °C than at 25 °C. As determined previously (Montané et al. 1997), the same conditions caused a 15-fold increase in the accumulation of ELIPs. Consequently, neither the distribution of total xanthophylls nor that of antheraxanthin plus zeaxanthin along the leaf followed the same pattern as ELIP. Thus, the accumulation of xanthophylls cannot be stoichiometrically correlated with that of ELIPs. Using electrophoresis in the presence of decylmaltoside, we could demonstrate for the first time that ELIPs of 13.5 kDa are contained in high-molecular-mass complexes of >100 kDa, which are located in the unstacked stroma lamellar region of the thylakoid membranes. Received: 6 April 1998 / Accepted: 26 January 1999     

Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto

The callose synthase (UDP-glucose: 1,3-β-d-glucan 3-β-d-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g · ml⁻¹, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets. Received: 24 September 1997 / Accepted: 12 November 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     

The effect of low temperature (5–29 °C) and adaptation on the methanogenic activity of biomass

The influence of low temperature (5–29 °C) on the methanogenic activity of non-adapted digested sewage sludge and on temperature/leachate-adapted biomass was assayed by using municipal landfill leachate, intermediates of anaerobic degradation (propionate) and methane precursors (acetate, H₂/CO₂) as substrates. The temperature dependence of methanogenic activity could be described by Arrhenius-derived models. However, both substrate and adaptation affected the temperature dependence. The adaptation of biomass in a leachate-fed upflow anaerobic sludge-blanket reactor at approximately 20 °C for 4 months resulted in a sevenfold and fivefold increase of methanogenic activity at 11 °C and 22 °C respectively. Both acetate and H₂/CO₂ were methanized even at 5 °C. At 22 °C, methanogenic activities (acetate 4.8–84 mM) were 1.6–5.2 times higher than those at 11 °C. The half-velocity constant (K _s) of acetate utilization at 11 °C was one-third of that at 22 °C while a similar K _i was obtained at both temperatures. With propionate (1.1–5.5 mM) as substrate, meth‐anogenic activities at 11 °C were half those at 22 °C. Furthermore, the residual concentration of the substrates was not dependent on temperature. The results suggest that the adaptation of biomass enables the achievement of a high treatment capacity in the anaerobic process even under psychrophilic conditions. Received: 23 December 1996 / Received last revision: 18 June 1997 / Accepted: 23 June 1997     

Influence of phosphate on rhamnose-containing exopolysaccharide rheology and production by Klebsiella I-714

Physiological conditions enhancing rhamnose-containing polysaccharide synthesis by Klebsiella I-714 were studied in batch culture (0.3-l and 2-l bioreactors). The four carbon sources tested, sucrose, sorbitol, Neosorb and Cerelose, allowed exopolysaccharide production. Larger amounts of polymer were produced when high carbon/nitrogen ratios and complex nitrogen sources were used. Exopolysaccharide synthesis was greatest at 30 °C, which was a suboptimal growth temperature. A reduction in the phosphate content of the medium enhanced rhamnose-containing polysaccharide production. When the initial carbon source concentration was augmented, byproducts other than exopolysaccharide were formed. Rhamnose-containing polysaccharide rheology can be modulated by changing the phosphate content of the medium. Received: 11 April 1997 / Received revision: 19 June 1997 / Accepted: 23 June 1997     

Production and characterization of ferulic acid esterase activity in crude extracts by Streptomyces avermitilis CECT 3339

Streptomyces avermitilis CECT 3339 produces extracellular ferulic acid esterase (FAE) activity during growth on a range of lignocellulose substrates. Maximal levels of FAE activity were detected in culture filtrates from S. avermitilis CECT 3339 grown in media containing wheat bran and yeast extract as carbon and nitrogen sources respectively. Biochemical characterization of this enzyme activity revealed that it was 100-fold higher when wheat bran was pretreated with Celluclast (a mix of hydrolytic enzymes). FAE was found to be end-product-inhibited. Characterization of the properties of the enzyme showed that FAE exhibited an activity optimum pH at 6 with pH stability between pH 6 and 8. The optimum temperature was 50 °C while the temperature stability was between 30 °C and 40 °C, with rapid inactivation at 60 °C and above. The characteristics and stability of FAE from S. avermitilis CECT 3339 suggest a potential role for this enzyme in combination with endoxylanases for the upgrading of plant-residue silage and for biopulping. Received: 17 November 1997 / Received revision: 13 March 1998 / Accepted: 13 April 1998     

β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997     

Evaluation of Candida acidothermophilum in ethanol production from lignocellulosic biomass

A Saccharomyces-cerevisiae-based simultaneous saccharification and fermentation (SSF) of lignocellulosic biomass is limited to an operating temperature of about 37 °C, and even a small increase in temperature can have a deleterious effect. This points to a need for a more thermotolerant yeast. To this end, S. cerevisiae D₅A and a thermotolerant yeast, Candida acidothermophilum, were tested at 37 °C, 40 °C, and 42 °C using dilute-acid-pretreated poplar as substrate. At 40 °C, C. acidothermophilum produced 80% of the theoretical ethanol yield, which was higher than the yield from S.cerevisiae D₅A at either 37 °C or 40 °C. At 42 °C, C. acidothermophilum showed a slight drop in performance. On the basis of preliminary estimates, SSF with C. acidothermophilum at 40 °C can reduce cellulase costs by about 16%. Proportionately greater savings can be realized at higher temperatures if such a high-temperature SSF is feasible. This demonstrates the advantage of using thermophilic or thermotolerant yeasts. Received: 20 February 1997 / Received revision: 24 June 1997 / Accepted: 4 July 1997     

A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles

A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C₃–C₆ dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746. Received: 26 January 1999 / Received revision: 10 June 1999 / Accepted: 27 June 1999     

The Active Component of the Bioemulsifier Alasan from Acinetobacter radioresistens KA53 Is an OmpA-Like Protein

The bioemulsifier of Acinetobacter radioresistens KA53, referred to as alasan, is a high-molecular-weight complex of polysaccharide and protein. Recently, one of the alasan proteins, with an apparent molecular mass of 45 kDa, was purified and shown to constitute most of the emulsifying activity. The N-terminal sequence of the 45-kDa protein showed high homology to an OmpA-like protein from Acinetobacter spp. In the research described here the gene coding for the 45-kDa protein was cloned, sequenced, and expressed in Escherichia coli. Recombinant protein AlnA (35.77 kDa without the leader sequence) had an amino acid sequence homologous to that of E. coli OmpA and contained 70% of the specific (hydrocarbon-in-water) emulsifying activity of the native 45-kDa protein and 2.4 times that of the alasan complex. In addition to their emulsifying activity, both the native 45-kDa protein and the recombinant AlnA were highly effective in solubilizing phenanthrene, ca. 80 microg per mg of protein, corresponding to 15 to 19 molecules of phenanthrene per molecule of protein. E. coli OmpA had no significant emulsifying or phenanthrene-solubilizing activity. The production of a recombinant surface-active protein (emulsification and solubilization of hydrocarbons in water) from a defined gene makes possible for the first time structure-function studies of a bioemulsan.     

The concentration of cryoprotective lectins in mistletoe (Viscum album L.) leaves is correlated with leaf frost hardiness

The leaves of mistletoe (Viscum album L.) contain three galactose- and N-acetylgalactosamine-specific isolectin groups (ML I, II, III). The groups ML I and ML III showed strong cryoprotective activity during freezing and thawing of isolated spinach (Spinacia oleracea L.) thylakoid membranes, while ML II showed no such activity. The cryoprotective efficiency of the proteins was correlated with their relative hydrophobicity, as determined by a fluorescence titration assay. We found that the frost hardiness of mistletoe leaves was seasonally regulated under natural conditions. While leaves harvested in winter were not damaged by freezing to −20 °C, leaves harvested in July had already suffered 70% electrolyte leakage after freezing to −5 °C. Likewise, the amount of ML I and ML III varied during the year, with the highest amounts of these cryoprotective lectins in winter and early spring and the lowest amounts during the summer months. There was no comparable change in the amount of ML II. These data suggest that some lectins may play a role in the stabilization of cellular membranes under environmental stress conditions. Received: 18 December 1996 / Accepted: 29 March 1997     

The adverse effect of nitrogen limitation and excess-cellobiose on Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 cultures that were cellobiose-limited converted cellobiose to succinate and acetate, produced little glucose or cellotriose, maintained an intracellular ATP concentration of 4.1 mM and a membrane potential of 140 mV for 24 h, did not lyse at a rapid rate once they had reached stationary phase, and had a most probable number of viable cells that was greater than 10⁶/ml. When the cellobiose concentration was increased 6-fold (5 mM to 30 mM), ammonia was depleted and the cultures left 10 mM cellobiose. Cultures provided with excess cellobiose produced succinate and acetate while they were growing, but there was little increase in fermentation acids after the ammonia was depleted and growth ceased. The stationary-phase, cellobiose-excess cultures had a lysis rate that was 7-fold faster than that of the cellobiose-limited cultures, and the most probable number was only 3.3 × 10³ cells/ml. The stationary-phase, cellobiose-excess cultures had 2.5 times as much cellular polysaccharide as the cellobiose-limited cultures, but the intracellular ATP and membrane potential were very low (0.1 mM and 40 mV respectively). Methylglyoxal, a potentially toxic end-product of carbohydrate fermentation, could not be detected, and fresh inocula grew rapidly in spent medium that was supplemented with additional ammonia. Stationary-phase, cellobiose-excess cultures converted cellobiose to glucose and cellotriose, but the apparent K _m of cellotriose formation was 15-fold lower than the K _m of glucose production (0.7 mM compared to 10 mM). Received: 26 June 1997 / Received revision: 12 August 1997 / Accepted: 29 August 1997     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers, pH 3.5–7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2–4.6 and 7.5–8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 °C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 °C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 × 10³kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors. Received: 8 November 1996 / Received revision: 31 January 1997 / Accepted: 31 January 1997     

Pullulan production from deproteinized whey by Aureobasidium pullulans

Aureobasidium pullulans P56 was investigated using an adaptation technique and a mixed culture system. The adaptation of A. pullulans and the mixed cultures of A. pullulans and/or Lactobacillus brevisX20, Debaryomyces hansenii 194 and Aspergillus niger did not increase the production of polysaccharide. Enzymic hydrolysis of lactose in deproteinized whey gave a higher polysaccharide concentration and polysaccharide yield than acidic hydrolysed lactose. Maximum polysaccharide concentration (11.0 ± 0.5 g L⁻¹), biomass dry weight (10.5 ± 0.4 g L⁻¹), polysaccharide yield (47.2 ± 1.8%) and sugar utilization (93.2 ± 2.8%) were achieved using enzyme-hydrolysed whey (pH 6.5) containing 25 g L⁻¹ lactose and supplemented with K₂HPO₄ 0.5%, L-glutamic acid 1%, olive oil 2.5%, and Tween 80 0.5%. In this case the pullulan content of the crude polysaccharide was 40%. Received 16 December 1997/ Accepted in revised form 12 March 1999     

Effect of aeration and carbon/nitrogen ratio on the molecular mass of the biodegradable polymer poly-β-hydroxybutyrate obtained from Azotobacter chroococcum 6B

The bacterial polyester poly-β-hydroxybutyrate (PHB) was quantified and characterized on an isolate␣of the nitrogen-fixing bacteria Azotobacter chroococcum 6B on the basis of its average molecular mass, determined from the relative viscosity at different aeration rates and carbon/nitrogen ratios during culture in fermentors. A higher value for the molecular mass (1100 kDa) was obtained with the lower aeration rates investigated, which diminished, significantly at the highest aeration rate of 2.5 vvm (a 100-fold decrease). The yield of PHB relative to the amount of glucose consumed increased with the C/N ratio (a maximum of 0.16 g PHB/g glucose consumed with a carbon/nitrogen ratio of 137.7), but the molecular mass was lowered from 800 kDa to nearly 100 kDa. The maximum PHB content was 63.5% (on a cellular dry-weight basis) after 47 h in fed-batch culture with an initial C/N ratio of 68.9 and aeration at a rate of 0.5 vvm. Calorimetric measurements on the isolated PHB showed a melting point near 175 °C. Received: 25 June 1997 / Accepted: 2 July 1997