Isolation and characterization of a bacterium possessing a novel aldoxime-dehydration activity and nitrile-degrading enzymes

A bacterial strain capable of utilizing E-pyridine-3-aldoxime as a nitrogen source was isolated from soil after a 4-month acclimation period and was identified as Rhodococcus sp. The strain contained a novel aldoxime dehydration activity that catalyzed a stoichiometric dehydration of E-pyridine-3-aldoxime to form 3-cyanopyridine. The enzyme activity was induced by various aldoximes and nitriles. The strain metabolized the aldoxime as follows: E-pyridine-3-aldoxime was dehydrated to form 3-cyanopyridine, which was converted to nicotinamide by a nitrile hydratase, and the nicotinamide was successively hydrolyzed to nicotinic acid by an amidase. Received: 21 January 1998 / Accepted: 12 May 1998     

Purification and properties of cellobiose phosphorylase from Clostridium thermocellum

Cellobiose phosphorylase was purified 111-fold from a cell extract of Clostridium thermocellum ATCC 27405, with a yield of 31.4%, to electrophoretic and column chromatographic homogenity. The molecular weight of the enzyme was estimated to be 150,000 by gel filtration and 85,000 by SDS-PAGE, suggesting that it consisted of two identical subunits. It was suggested by spectrophotometric and chemical analyse that the enzyme contained no pyridoxal phosphate. The enzyme was inactivated by N-ethylmaleimide and activated by dithiothreitol, indicating that the exposed thiol group(s) was important for the enzymatic activity. The enzyme could utilize, so far as examined, d-glucose, d-xylose, 2-deoxy-d-glucose, and d-mannose, as acceptors of glucose in the synthetic reaction of disaccharides. The enzyme could to a low degree utilize d-arabinose and d-fucose, as acceptors.     

Bioremoval of an azo textile dye,Reactive Red 198, by <Emphasis Type="Italic">Aspergillus flavus</Emphasis>

The objective of this paper was to study the potential for bioremoval of a textile dye, Reactive Red 198 (RR198), by a fungus isolated from soil collected from an effluent disposal area near a textile company. The fungus was identified as Aspergillus flavus, and its use as a low-cost live-cell biomass for the biodegradation of RR198 from contaminated water was investigated using batch studies. The effects of time, dye concentration, and pH as variable factors were examined in the process. Results showed that bioremoval of RR198 by A. flavus increased to over 84.96% with increasing time until equilibrium was reached after a period of 24 h. A low pH was the most effective, as were lower levels of dye concentration. The decolorization was determined by the decrease in the absorption maximums of this dye by UV–visible spectroscopy. A. flavus was shown to be an efficient fungus for removal of RR198 from wastewater.     

Partial Hydrolysis of a Synthetic Heptapeptide Related to Gramicidin S

Galactomyces reessii L, isolated as a protopectin-solubilizing enzyme-producing strain, produced protopectin-solubilizing enzyme in the culture filtrate. The enzyme was purified by repeated CM-Sephadex C-50 column chromatography, and isolated as a crystalline form with a yield of 16% of the initial activity. The enzyme was a glycoprotein containing about 2.6% carbohydrate (as pentose). Its isoelectric point was around pH 8.4, and the sedimentation coefficient (s_20,w) was determined to be 3.83 S. The molecular weight was determined to be 30,000 by gel filtration on Sephadex G-75 and 29,300 by ultracentrifugal analysis. The enzyme catalyzed the release of highly polymerized pectin from various protopectins. The enzyme also catalyzed the depolymerization of pectic acid or galacturonic acid oligomers, and was confirmed to be an endo- polygalacturonase.     

Cystine lyase: β-cystathionase from turnip roots

Cystine lyase (EC 4.4.1.-) was purified 277-fold by a combination of ammonium sulfate precipitation, chromatography on calcium phosphate and DEAE-cellulose with a 6% recovery. The MW as measured by gel filtration on Biogel p-300 was ca 150 000. The enzyme catalysed the pyridoxal phosphate-dependent degradation of cystine to pyruvate, ammonia and cysteine persulfide. Cysteine persulfide normally degraded spontaneously to elemental sulfur and cysteine, that further reacted to yield cystine and H₂S. Pyridoxal phosphate stabilized the enzyme. The K_m value for cystine was 0.94 mM. The enzyme was insensitive to thiol reagents but was inhibited by some thiols (which may have reduced the cystine). Cystine lyase degraded many compounds having the L-α-amino propionic acid group with a thioether or disulfide linkage attached to the β-carbon but was inactive towards D-configuration at the α-carbon or L-homocystine. The cystine lyase was also a β-cystathionase as indicated by (1) a constant ratio of β-cystathionase activity to cystine lyase activity throughout a 277-fold purification, (2) the inhibition of cystine lyase activity by cystathionine and inhibition of β-cystathionase activity by cystine and (3) similarity in sensitivity to heat, cyanide and hydroxylamine. Using DL-cystathionine as substrate, the K_m value was 4 mM.     

The inhibition of β-glucosidase activity in Trichoderma reesei C30 cellulase by derivatives and isomers of glucose

The inhibition of β-glucosidase in Trichoderma reesei C30 cellulase by D -glucose, its isomers, and derivatives was studied using cellobiose and ρ-nitrophenyl-β-glucoside (PNPG) as substrates for determining enzyme activity. The enzymatic hydrolysis of both substrates was inhibited competitively by glucose with approximate K_i values of 0.5mM and 8.7mM for cellobiose and PNPG as substrate, respectively. This inhibition by glucose was maximal at pH 4.8, and no inhibition was observed at pH 6.5 and above. The α anomer of glucose inhibited β-glucosidase to a greater extent than did the β form. Compared with D -glucose, L -glucose, D -glucose-6-phosphate, and D -glucose-1-phosphate inhibited the enzyme to a much lesser extent, unlike D -glucose-L -cysteine which was almost as inhibitory as glucose itself when cellobiose was used as substrate. Fructose (2?100mM) was found to be a poor inhibitor of the enzyme. It is suggested that high rates of cellobiose hydrolysis catalyzed by β-glucosidase may be prolonged by converting the reaction product glucose to fructose using a suitable preparation of glucose isomerase.     

Detection of a calcium-activated protein kinase in mougeotia by using synthetic Peptide substrates

By using a synthetic peptide, KM-14, a protein kinase was detected and partially purified from Mougeotia sp. The peptide contains the sequence of the regulatory light chain of smooth muscle myosin that is phosphorylated by calcium-calmodulin-dependent myosin light chain kinase (MLCK). The Mougeotia kinase was stimulated 40-fold by calcium with half-maximal stimulation occurring at 1.5 micromolar. The enzyme was fractionated from calmodulin and was depleted of calmodulin based on enzyme activator analysis. The calmodulin-depleted enzyme was fully active and calcium dependent, and was not stimulated further by exogenous calmodulin nor by the calcium effectors phosphatidylserine and diacylglycerol. The enzyme phosphorylated intact chicken gizzard myosin light chain as well as the KM-14 substrate. KM-13, a peptide analog of KM-14 with a deletion of a glutamine at position 5, was a poor substrate with a V_max/K_m ratio 200-fold lower than KM-14. Thus, similarly to vertebrate MLCK, the Mougeotia enzyme is very sensitive to changes in sequence surrounding the phosphorylation site. Calcium-dependent KM-14 kinase activity also was detected in two other algae, Mesotaenium caldariorum and Spirogyra sp., as well as in pea seedlings. The data suggest that plant and algal tissues possess an enzyme with a substrate specificity similar to MLCK, but unlike MLCK, does not appear to require calmodulin for activity.     

Carbonylation as a Key Reaction in Anaerobic Acetone Activation by Desulfococcus biacutus

Acetone is activated by aerobic and nitrate-reducing bacteria via an ATP-dependent carboxylation reaction to form acetoacetate as the first reaction product. In the activation of acetone by sulfate-reducing bacteria, acetoacetate has not been found to be an intermediate. Here, we present evidence of a carbonylation reaction as the initial step in the activation of acetone by the strictly anaerobic sulfate reducer Desulfococcus biacutus. In cell suspension experiments, CO was found to be a far better cosubstrate for acetone activation than CO₂. The hypothetical reaction product, acetoacetaldehyde, is extremely reactive and could not be identified as a free intermediate. However, acetoacetaldehyde dinitrophenylhydrazone was detected by mass spectrometry in cell extract experiments as a reaction product of acetone, CO, and dinitrophenylhydrazine. In a similar assay, 2-amino-4-methylpyrimidine was formed as the product of a reaction between acetoacetaldehyde and guanidine. The reaction depended on ATP as a cosubstrate. Moreover, the specific activity of aldehyde dehydrogenase (coenzyme A [CoA] acylating) tested with the putative physiological substrate was found to be 153 ± 36 mU mg⁻¹ protein, and its activity was specifically induced in extracts of acetone-grown cells. Moreover, acetoacetyl-CoA was detected (by mass spectrometry) after the carbonylation reaction as the subsequent intermediate after acetoacetaldehyde was formed. These results together provide evidence that acetoacetaldehyde is an intermediate in the activation of acetone by sulfate-reducing bacteria.     

Disintegration Mediated Controlled Release Supersaturating Solid Dispersion Formulation of an Insoluble Drug: Design,Development, Optimization,and In Vitro Evaluation

The objective of this study was to develop a solid dispersion based controlled release system for drug substances that are poorly soluble in water. A wax-based disintegration mediated controlled release system was designed based on the fact that an amorphous drug can crystallize out from hydrophilic matrices. For this study, cilostazol (CIL) was selected as the model drug, as it exhibits poor aqueous solubility. An amorphous solid dispersion was prepared to assist the drug to attain a supersaturated state. Povidone was used as carrier for solid dispersion (spray drying technique), hydrogenated vegetable oil (HVO) as wax matrix former, and sodium carboxymethyl cellulose (NaCMC) as a disintegrant. The extreme vertices mixture design (EVMD) was applied to optimize the designed and developed composition. The optimized formulation provided a dissolution pattern which was equivalent to the predicted curve, ascertaining that the optimal formulation could be accomplished with EVMD. The release profile of CIL was described by the Higuchi’s model better than zero-order, first-order, and Hixson-Crowell’s model, which indicated that the supersaturation state of CIL dominated to allow drug release by diffusion rather than disintegration regulated release as is generally observed by Hixson-Crowell’s model. The optimized composition was evaluated for disintegration, dissolution, XRD, and stability studies. It was found that the amorphous state as well as the dissolution profile of CIL was maintained under the accelerated conditions of 40°C/75% RH for 6 months.KEY WORDS: cilostazol, controlled release, disintegration-mediated controlled release (DMCR), extreme vertices mixture design (EVMD), solid dispersion     

Vitellogenesis-related ovary cathepsin D from Xenopus laevis: Purification and properties in comparison with liver cathepsin D

Cathepsin D was purified from ovaries of Xenopus laevis by both QAE-cellulose and pepstatin-Sepharose chromatography and then characterized and compared with Xenopus liver cathepsin D. Ovary cathepsin D appeared predominantly as a 43-kilodalton (kDa) molecular mass, as revealed by SDS-polyacrylamide gel electrophoresis, whereas the liver enzyme was obtained exclusively as a 36-kDa protein. The purified 43-kDa ovary enzyme cleaved vitellogenin limitedly to produce yolk proteins at pH 5.6. The specific activity of ovary cathepsin D was five to six times lower than that of the liver enzyme, as measured by hemoglobin-hydrolysis at pH 3, but the ovary enzyme was shown to be superior to the liver enzyme in terms of vitellogenin-cleaving activity, as examined at pH 5.6. Ovarian enzyme preparations contained variable amounts of 36-kDa species; this form was considered to be an autolytic product of the 43-kDa form arising during purification, because it was not detected in oocyte extracts but was generated by incubation of the purified 43-kDa enzyme alone in an acid solution. The conversion of the 43-kDa form by hepatic factors was accompanied by a marked increase in hemoglobin-hydrolytic activity.     

Purification and Characterisation of Keratinase from a Thermotolerant Feather-degrading Bacterium

Summary Isolation and identification of a thermotolerant feather-degrading bacterial strain from Thai soil as well as purification and properties of its keratinase were investigated. The thermotolerant bacterium was identified as Bacillus licheniformis. The keratinase was purified to homogeneity by three-step chromatography. The purified enzyme exhibited a high specific activity (218 U mg⁻¹) with 86-fold purification and 25% yield. The enzyme was monomeric and had a molecular mass of 35 kDa. The optimum pH and temperature for the enzyme were 8.5 and 60 °C, respectively. The enzyme activity was significantly inhibited by PMSF and partly inhibited by EDTA and iodoacetamide, but was stimulated by metal ions. It hydrolysed soluble proteins with a relative activity of 4–100% and insoluble proteins, including keratins, with a relative activity of 3–35%. Therefore, the enzyme could improve the nutritional value of meat- and poultry-processing wastes containing keratins, collagen and gelatin.     

Fungal leaching of nickeliferous laterites

Extraction of nickel by microbial leaching of Greek laterites is feasible by using ofAspergillus andPenicillium. The effectiveness was found to depend on the ability of the microorganism to produce hydroxycarboxylic acids, especially citric acid, as well as other metabolites. The nickel recoveries achieved were as high as 60%, in 48 d, when the ore was leached in the presence of the living fungi, in a sucrose medium, and as high as 70%, in a much shorter time, when the ore was leached by their metabolic products after pH adjustment by means of sulfuric acid. The use of much cheaper, factory grade, Greek beet molasses as a growth medium proved promising, giving the possibility of making the process more attractive in economic terms.     

Release factor regulating termination of complete protein in a eukaryotic organism

Polypeptide release reaction was studied using a protein release factor and a physiological substrate containing a complete polypeptide chain attached to monosomes of the insect Tenebrio molitor. The intermediate substrate used for the release reaction was synthesized using a cell-free protein synthesizing system from Tenebrio capable of polypeptide synthesis but not release of the completed chain. This system synthesized predominantly adult cuticular protein. The released product was characterized by chromatography after tryptic digestion; many of the tryptic peptides corresponded to those of cuticule labeled in vivo. The protein release factor was obtained as microsomal wash and was further purified by ammonium sulfate precipitation and column chromatography. It released about 30% of the monosome-bound peptide in the absence of GTP. The remaining 70% of peptidyl-tRNA was released as peptidyl-puromycin in the absence of release factor, but required transferase II and GTP. The peptidyl-puromycin varied in size from dipeptide to almost complete protein. The puromycin reaction was inhibited by diphtheria toxin and NAD and was dependent on GTP, while the release of completed peptide was independent of GTP and not affected by diphtheria toxin and NAD. The release factor was capable of releasing formylmethionine as formylmethionine-puromycin from ribosomes in response to poly(A₃,U). Hence it is suggested that the release factor is responding to UAA as terminating codon.     

Degradation of biphenyl by a Micrococcus species

Summary A bacterium capable of utilizing biphenyl as the sole source of carbon was isolated from soil and identified as a Micrococcus species. The organism also utilized 4-chlorobiphenyl and several other aromatic compounds as growth substrates. 2,3-Dihydroxybiphenyl and benzoic acid were identified as intermediates by physico-chemical methods. The bacterium degraded biphenyl to 2,3-dihydroxybiphenyl followed by its meta-ring cleavage to yield 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, which was then hydrolysed to give benzoic acid. Benzoate was further metabolised via a catechol meta-cleavage pathway by a Micrococcus sp.Correspondence to: H. Z. Ninnekar     

Depolarization-independent net uptake of calcium into clonal insulin-releasing cells exposed to glucose

Insulin release, net fluxes of Ca²⁺, and glucose metabolism were studied in a clonal cell line (RINmSF) established from a transplantable rat islet tumor. The insulin content amounted to only 0.03% of that of the total protein and decreased even further with subsequent passages. The insulin secretion was as high as 10 to 20% of the total hormone content per hour. Insulin release was stimulated by K⁺ depolarization but not by exposure to glucose. In contrast to this secretory pattern, glucose but not K⁺ stimulated the net uptake of Ca²⁺ at micromolar concentrations of the ion. The glucose effect was not mimicked by 20 mM 3-O-methylglucose. It was as pronounced at 1 mM as at 20 mM of the sugar and corresponded to an uptake of 119 fmol cm^–2 s^–1. Glucose metabolism was typical for tumor cells with a high glycolytic flux and an oxidationtoutilization ratio as low as 0.05–0.15. Maximal oxidative degradation was attained already at l mM. This concentration was also equivalent to the K_m for glucose utilization, indicating a substantial left-hand shift of the normal dose-response curve. It is suggested that glucose induces a depolarizationindependent net uptake of Ca²⁺ by favouring intracellular buffering of the cation.     

In vitro tolerability of human nasal mucosa: histopathological and scanning electron-microscopic evaluation of nasal forms containing Sandostatin®

Anin vitro human nasal model was developed as a tool to study the local tolerabiliity of nasal powder forms using excised nasal mucosa in a diffusion chamber. The suitability of this model was tested using Sandostatin^® (SMS) an octapeptide analog of somatostatin, as a reference drug enhanced by Avicel^® (microcrystalline cellulose) or lactose (100 mesh). The standard nasal spray vehicle was taken as a harmless control and 1% chenodeoxycholate (CDC) as a harmful control in terms of local tolerability. The extent of peptide permeation was determined by measuring SMS concentration in the receiving chamber. The labeling of SMS was detected by immunoperoxidase staining on cross sections. The local tolerability for all tested forms was assessed by histopathological examination and scanning electron microscopy. The apparent permeation coefficient allowed us to rank the absorption of the tested drug forms as Avicel > spray=lactose>1%CDC. For all formulations, SMS was detected in the epithelium. No changes of the nasal mucosa could be observed with Avicel, lactose or nasal spray vehicle in the presence or absence of SMS. 1%CDC with or without drug showed an immediate destruction of the nasal epithelium. The validation of thisin vitro model using human nasal mucosa will be further discussed as a tool for assessing the local tolerability of intranasally applied test substances.Abbreviations CDC chenodeoxycholate - SMS Sandostatin^® or octreotide     

A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris

A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris. The screening was performed with a heterologous probe from carrot. The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)₄ repeats and a high content in Tyr and Lys residues. The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links. The analysis of genomic DNA gel blots using both the N. sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N. sylvestris as well as in related Nicotianeae including a laboratory hybrid. This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe. The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific. The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Enrichment of new alkane oxidizing bacterial strains for human drug metabolite production

An extracellular xylanase produced by Streptomyces matensis DW67 was purified from the culture supernatant by ammonium sulfate precipitation, ion exchange and gel filtration chromatography and characterized. The xylanase was purified to 14.5-fold to homogeneity with a recovery yield of 14.1%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of 21.2 kDa. However, it had a very low apparent molecular mass of 3.3 kDa as determined by gel filtration chromatography. The N-terminal sequence of first 15 amino acid residues was determined as ATTITTNQTGYDGMY. The optimal temperature and pH for purified xylanase was 65 °C and pH 7.0, respectively. The enzyme was stable within the pH range of 4.5–8.0 and was up to 55 °C. The xylanase showed specific activity towards different xylans and no activity towards other substrates tested. Hydrolysis of birchwood xylan by the xylanase yielded xylobiose and xylotriose as principal products. The enzyme hardly hydrolyzed xylobiose and xylotriose, but it could hydrolyze xylotetraose and xylopentaose to produce mainly xylobiose and xylotriose through transglycosylation. These unique properties of the purified xylanase make this enzyme attractive for biotechnological applications, such as bioblenching in paper and pulp industries, production of xylooligosaccharides. This is the first report of the xylanase from S. matensis.     

Bacterial Growth as a Practical Indicator of Extensive Biodegradability of Organic Compounds

The proportionality of growth, as indicated by turbidity of cultures of Pseudomonas C12B, to the initial concentration of sodium dodecyl sulfate, dodecanol, or a mixture of C₁₀-C₂₀ secondary alcohol sulfates, each provided as sole carbon source in basal mineral salts medium, was demonstrated. Subsequently, the direct correlation of culture turbidity as a growth indicator and degradation of sodium dodecyl sulfate and the C₁₀-C₂₀ compounds was established. Degradation of these detergents was measured by the rise in surface tension and the decrease in methylene blue values, respectively. Turbidimetry was found to be a poor indicator of degradation of dodecanol in the early hours of culture, however, and did not correlate over a significant range with degradation of substrate. Viable cell counts did parallel dodecanol degradation as measured by gas-liquid chromatography. The use of bacterial growth as a reliable, quantitative, and easily measured parameter indicating biodegradability was suggested for those organic compounds which can be shown to serve as a carbon source for a bacterium.