Hydrocarbon pseudosolubilizing and emulsifying proteins produced by Pseudomonas cepacia N1

Significant specific pseudosolubilizing activity of the n-hexadecane pseudosolubilizing factor (C₁₆PSF), n-octadecane pseudosolubilizing factor (C₁₈PSF) and pristane pseudosolubilizing factor (PrisPSF) was found. From the partial chemical analysis of the chromatographically purified samples the pseudosolubilizing factors (PSFs) and n-hexadecane emulsifying factor (HEF-C₁₆) were tentatively identified as glycoprotein and lipoprotein, respectively. Each of the purified PSFs showed molecular weight 14,000 in SDS-PAGE but they had different amino acid compositions. The carbohydrate component of the C₁₆PSF consists of 93% glucose residues. C₁₂-, C₁₃-, C₁₄- and C₁₆- are the major fatty acids constituting 64% of the total fatty acids of HEF-C₁₆.     

Isolation and functional characterization of hydrocarbon emulsifying and solubilizing factors produced by a Pseudomonas species

Pseudomonas PG-1 cultivated on pristane produced in good amount a heat-stable polymeric substance which showed strong hydrocarbon emulsifying and solubilizing properties. The substance was isolated in crude form and was found to contain 34% protein, 16% carbohydrate, and 40% lipid. The hydrocarbon solubilizing activity of the isolate was strongly inhibited by EDTA but the chelating agent had no effect on the hydrocarbon emulsifying activity. Both activities of the isolate were strongly inhibited by chymotrypsin treatment indicating the importance of the protein moiety for its activity. Hydrocarbon solubilization by the isolate showed a certain degree of specificity to pristane in modest agitation generally used in microbial cultivation, but this specificity was lost by vigorous agitation in a Waring blender. It was proposed that in the first case, solubilization was effected by a solubilizing factor specific to pristane, whereas in the latter case, nonspecific solubilization occurred due to the action of the emulsifying factor. The rate of pristane solubilization by heat-treated culture broth under the conditions of agitation used in cultivation (rotary shaker, 120 rpm) was found to be ca. 750 mg L(-1) h(-1) which was much larger than the maximal pristane uptake rate of 170 mg L(-1) h(-1) observed during microbial growth on the substrate. It was concluded that hydrocarbon solubilization could satisfactorily account for the substrate uptake and growth.     

An Exocellular Protein from the Oil-Degrading Microbe Acinetobacter venetianus RAG-1 Enhances the Emulsifying Activity of the Polymeric Bioemulsifier Emulsan

The oil-degrading microorganism Acinetobacter venetianus RAG-1 produces an extracellular polyanionic, heteropolysaccharide bioemulsifier termed emulsan. Emulsan forms and stabilizes oil-water emulsions with a variety of hydrophobic substrates. Removal of the protein fraction yields a product, apoemulsan, which exhibits much lower emulsifying activity on hydrophobic substrates such as n-hexadecane. One of the key proteins associated with the emulsan complex is a cell surface esterase. The esterase (molecular mass, 34.5 kDa) was cloned and overexpressed in Escherichia coli BL21(DE3) behind the phage T7 promoter with the His tag system. After overexpression, about 80 to 90% of the protein was found in inclusion bodies. The overexpressed esterase was recovered from the inclusion bodies by solubilization with deoxycholate and, after slow dialysis, was purified by metal chelation affinity chromatography. Mixtures containing apoemulsan and either the catalytically active soluble form of the recombinant esterase isolated from cell extracts or the solubilized inactive form of the enzyme recovered from the inclusion bodies formed stable oil-water emulsions with very hydrophobic substrates such as hexadecane under conditions in which emulsan itself was ineffective. Similarly, a series of esterase-defective mutants were generated by site-directed mutagenesis, cloned, and overexpressed in E. coli. Mutant proteins defective in catalytic activity as well as others apparently affected in protein conformation were also active in enhancing the apoemulsan-mediated emulsifying activity. Other proteins, including a His-tagged overexpressed esterase from the related organism Acinetobacter calcoaceticus BD4, showed no enhancement.     

Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

Some properties and immunological relationship of acid phosphatases from gramineae

Two acid phosphatases have been found in crude extracts of seeds, coleoptiles and leaves of various grass species by means of crossed immunoelectrophoresis.The enzymes, cross-reacting with antibodies raised against proteins of Poa pratensis seeds differ in their binding to con A. The use of affinity chromatography on con A-Sepharose has separated the acid phosphatases into two fractions: the non-binding (acid phosphatase A) and the con A-binding (acid phosphatase B). The con A-binding acid phosphatase B from all tissues was further purified by gel filtration on Biogel P-100 and hydrophobic interaction chromatography on phenyl-Sepharose. Two isoenzymes: acid phosphatase B₁ and B₂ were obtained. The isoenzymes are glycoproteins containing D-mannose or D-glucose in their carbohydrate moiety. They retained the enzyme activity after binding in macromolecular complex with antibodies or con A. The purified acid phosphatases from all tissues cross-react with monospecific antibodies raised against P. pratensis seeds acid phosphatase B₁ indicating the antigenic relationship between the enzymes of various grass species.     

Protein-like Activator for n-Alkane Oxidation by Pseudomonas aeruginosa S7B1

During the study of the n-alkane oxidation by Pseudomonas aeruginosa S₇B₁, a nondialysable activator for n-alkane oxidation was discovered in the culture broth of the strain. The activator was purified, as judged by cellulose acetate membrane electrophoresis, by ammonium sulfate precipitation and chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-75 columns.

The purified activator, which was positive in protein color reactions, remarkable stimulated only the oxidation of n-hexadecane, though it was not observed in case of palmitic acid or glucose oxidation.

Co-operative action between the activator and rhamnolipid, which had been isolated as a growth stimulant of P. aeruginosa S₇B₁ on n-hexadecane by the authors, was observed not only in the oxidation of n-hexadecane but also in the growth on n-hexadecane.     

The minor anionic form of arylsulphatase B (arylsulphatase Bm) of monkey brain. Purification and phosphoprotein nature

The anionic form of arylsulphatase B (arylsulphatase Bm) was purified to apparent homogeneity from monkey brain through steps involving chromatography on diethylaminoethyl-cellulose, Blue-Sepharose, Biogel HTP and finally Biogel P-300 gel filtration. The molecular weight of the purified enzyme as deduced by gel filtration on Biogel P-300 and by sodium dodecylsulphate gel electrophoresis was ∼ 30,000.Escherichia coli alkaline phosphatase treatment of arylsulphatase Bm resulted in the conversion of upto 84% of the enzyme into a less charged form of enzyme, that could not bind to diethylaminoethyl cellulose. Potassium phosphate an inhibitor of alkaline phosphatase prevented this conversion. Upon acid hydrolysis the purified enzyme yielded approximately 7.0 mol of inorganic phosphate per mol of protein.Vibrio cholerae neuraminidase treatment did not alter the charge on arylsulphatase Bm.     

Purification and characterization of an organic solvent-tolerant lipase from Pseudomonas aeruginosa LX1 and its application for biodiesel production

An organic solvent-tolerant lipase from newly isolated Pseudomonas aeruginosa LX1 has been purified by ammonium sulfate precipitation and ion-exchange chromatography leading to 4.3-fold purification and 41.1% recovery. The purified lipase from P. aeruginosa LX1 was homogeneous as determined by SDS-PAGE, and the molecular mass was estimated to be 56 kDa. The optimum pH and temperature for lipase activity were found to be 7.0 and 40 °C, respectively. The lipase was stable in the pH range 4.5–12.0 and at temperatures below 50 °C. Its hydrolytic activity was found to be highest towards p-nitrophenyl palmitate (C16) among the various p-nitrophenol esters investigated. The lipase displayed higher stability in the presence of various organic solvents, such as n-hexadecane, isooctane, n-hexane, DMSO, and DMF, than in the absence of an organic solvent. The immobilized lipase was more stable in the presence of n-hexadecane, tert-butanol, and acetonitrile. The transesterification activity of the lipase from P. aeruginosa LX1 indicated that it is a potential biocatalyst for biodiesel production.     

Purification and Characterization of Polygalacturonase-3 from Jamaica cherry (Muntingia calabura Linn)

Endo-polygalacturonase-3 (PG-3), the key enzyme of fruit ripening was purified to near homogeneity as judged by native PAGE from the fruit tissues of Jamaica cherry (Muntingia calabura) using ammonium sulphate fractionation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the PG-3 enzyme was determined as 85 kD, by size exclusion chromatography. SDS-PAGE of PG-3 revealed two dissimilar bands of 62 and 21 kD as heterogenous subunits. The optimum pH of PG-3 was found to be 4.0. The enzyme had an optimum temperature of 40°C and was relatively stable at 50°C and 60°C. K_m for the substrate polygalacturonic acid was found to be 0.27%. The purified enzyme was a glycoprotein with 6.6 % carbohydrate content.     

Affinity purification of a 65-kilodalton parasporal protein from Bacillus thuringiensis PG-14 that shows mosquitocidal activity

By using antibody-mediated affinity chromatography, a highly mosquito larvicidal but nonhemolytic fraction was obtained from alkali-solubilized, silkworm (Bombyx mori) larval gut juice-treated parasporal inclusions of Bacillus thuringiensis strain PG-14 (serotype 8a : 8b). This fraction contained a 65-kDa protein only but not a 25-kDa protein, the main component in the flow through fraction unbound to the affinity column. The 25-kDa protein purified from the unbound fraction by CM-cellulose chromatography demonstrated a high hemolytic activity against sheep red blood cells but very low mosquito larvicidal activity.     

Cytochrome P-450 in the sophorose-lipid-producing yeast Candida (Torulopsis) apicola

The appearance of cytochrome P-450 and of cytochrome oxidase aa₃ were determined in the sophorose lipid producing yeast Candida (Torulopsis) apicola IMET 43 747 grown on a mixture of glucose and n-hexadecane. Cytochrome P-450, detectable in both the logarithmic and the stationary growth phase was not repressed by glucose. At the end of the logarithmic growth phase the content of cytochrome P-450 was three- to fivefold increased, which was connected with initiation of sophorose lipid biosynthesis. After that it dropped to the basal level, which remained constant during sophorose lipid biosynthesis. Cytochrome P-450 from logarithmic cells was cross-reactive with an antibody derived against cytochrome P-450alk from C. tropicalis. With microsomal proteins of stationary cells no cross-reactivity was obtained. The microsomal hydroxylase system of stationary cells seem to be regulated by the carbohydrate used as carbon source. Correspondence to: R. K. Hommel     

Isolation and characterization of a lectin from the seeds of Erythrina Edulis

A galactose-specific lectin was isolated from the seeds of Erythrina edulis. The protein was purified by affinity chromatography of the globulin fraction on an allyl-galactoside polyacrylamide gel. The hemagglutination properties, amino acid composition, A₂₈₀, MW of the protein and of its subunits, carbohydrate content, electrophoretic pattern and isoelectric point were determined. Comparison of its properties with those of other Erythrina lectins shows that the protein is a distinct member of this group of lectins.     

Detection of peptide, an activator of cyclic nucleotide phosphodiesterases, in uterine tissue

The soluble fraction of uterine tissue was found to contain a factor which is a potent activator of various cyclic nucleotide phosphodiesterases (with the exception of the retinal photoreceptor cell enzyme). The protein origin of this factor was established, using proteolysis, precipitation with trichloroacetic acid. The activator was purified by gel filtration on Sephadex G-25 and Biogel P-4 as well as by a highly effective liquid chromatography. The activator was shown to be a peptide with Mr = 1150 Da. The peptide was thermostable and stable within a broad range of pH. Besides phosphodiesterases, the peptide activated 5'-nucleotidase and Mg2+-ATPase of photoreceptor membranes.     

Solubilization and partial purification of cytochrome P-450 from rat lung microsomes

Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino-$\text{n}$-octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome $\text{b}$₅ and NADPH-cytochrome $\text{c}$ reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome $\text{c}$ reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found.     

Characterization of hydrocarbon emulsification and solubilization occurring during the growth of Endomycopsis lipolytica on hydrocarbons

A comparative characterization of the phenomena of hydrocarbon emulsification and solubilization taking place during the growth of Endomycopsis lipolytica on n-alkanes and alkenes was made. Evidence was obtained for the cellular production of different factors involved in emulsification and solubilization of hydrocarbons. It was shown that the production of these factors closely followed cell growth. The inducible nature of the alkane solubilizing factor was demonstrated using actidione as inhibitor. Whereas emulsifying factor was demonstrated using actidione as inhibitor. Whereas emulsifying factors showed a broad affinity to some particular hydrocarbons, solubilizing factor was found to be highly specific for the particular hydrocarbon on which the cells were grown. The emulsifying factor was heat stable whereas the solubilizing factor was highly unstable even at ?4°C. Metal-ion chelating agents strongly inhibited the activity of both of the factors. A crude isolate of the alkane emulsifying factor was obtained and its peptide characteristics were demonstrated. Using EDTA as an inhibitor for the emulsification–solubilization activity, evidence was obtained for the predominent role played by the emulsification–solubilization mechanism in the uptake of alkane by yeast cells.     

The bioadhesive of Mytilus byssus: A protein containing L-DOPA

L-DOPA was identified in hydrolysates of $\text{Mytilus}$ byssal adhesive discs and is present at about $\text{10}\text{res}\text{1000}$. The compound was isolated and purified by ion exchange on cellulose phosphate and Biogel P-2 gel filtration. Identity with standard DOPA was demonstrated using thin-layer chromatography, the effect of pH on UV absorbance, fluorescence spectrophotometry, amino acid analysis, and the preparation of ethylenediamine derivatives. Contrary to earlier reports, dityrosine was not detected. A sodium dodecylsulfate-insoluble protein containing $\text{48}\text{res}\text{1000}$ of DOPA was isolated from the gland that secretes the disc adhesive. This protein is presumed to be a precursor of the adhesive.     

A novel xylan degrading β-d-xylosidase: purification and biochemical characterization

Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular ??-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. ??-xylosidase is a glycoprotein (39?% carbohydrate content) and has a molecular mass of 137?kDa by SDS-PAGE, with optimal temperature and pH at 70?°C and 3.0?C5.5, respectively. ??-xylosidase was stable in acidic pH (3.0?C6.0) and 70?°C for 1?h. The enzyme was activated by 5?mM MnCl₂ (28?%) and MgCl₂ (20?%) salts. The ??-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-??-d-xylopyranoside, exhibiting apparent K_m and V_max values of 0.66?mM and 39?U (mg protein)^?1 respectively, and to a lesser extent p-nitrophenyl-??-d-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel ??-d-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by ??-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.     

Bovine aortic chondroitin sulphate- and dermatan sulphate-containing proteoglycans. Isolation, fractionation and chemical characterization.

1. Guanidinium chloride (4M) in the presence of proteinase inhibitors extracted 90% of bovine aorta galactosaminoglycans as proteoglycans that were subsequently purified by ion-exchange and gel chromatography. 2. Fractionation of the calcium salts of the purified proteoglycans with increasing concentration of ethanol yielded fractions PG-25 (28%), PG-35 (45%) and PG-50 (37%). 3. Fraction PG-50 contained proteochondroitin 6-sulphate, whereas fractions PG-25 and PG-35 were proteodermatan sulphates of greatly different carbohydrate composition; the molar proportions of L-iduronate-N-acetylgalactosamine 4-sulphate, D-glucuronate-N-acetyl-galactosamine 4-sulphate and D-glucuronate-N-acetylgalactosamine 6-sulphate were 75: 18 :7 in fraction PG-25 and 14 :46 :40 in fraction PG-35. 4. The presence of alternating or mixed sequences with L-iduronate- and D-glucuronate-containing repeating disaccharides was indicated by the formation of tetrasaccharides after chondroitinase AC digestion (single L-iduronate residues) and by the release of fragments containing four or five consecutive D-glucuronate-N-acetylgalactosamine repeats after periodate oxidation and alkaline elimination. 5. The amino acid compositions of fractions PG-25 and PG-35 were similar and markedly different from that of fraction PG-50, which also contained more side chains.     

Properties of rat liver plasma membrane adenylate cyclase after chromatography on O-diethylaminoethyl-cellulose and agarose-hexane-GTP: Sensitivity of partially purified and purified enzyme to Lubrol-PX, 5′-guanylylimidodiphosphate,and glucagon

Partially purified rat liver plasma membranes were enriched to yield a more glucagon-sensitive membrane fraction which was solubilized with Lubrol-PX. The supernate obtained after centrifugation at 165,000g was subjected to O-diethylaminoethyl anion exchange chromatography. An adenylate cyclase fraction was eluted and purified further by chromatography on agarose-hexane-GTP. The enzyme adsorbed to the affinity resin and was eluted with 0.5 m Tris-HCl. The protein isolated by chromatography on the affinity resin was homogenous by conventional acrylamide gel electrophoresis; one band was observed in sodium dodecyl sulfate. The purified enzyme was free of nucleotide phosphohydrolases found in the parent solubilized membrane preparation. The anion exchange product was not sensitive to glucagon; Lubrol-PX and 5′-guanylylimidodiphosphate [Gpp(NH)p] decreased the activity of this fraction. In the presence of detergent or guanyl nucleotide, glucagon, at 10^?6m, increased enzyme activity by 30 and 21%, respectively, to a statistically significant degree, but not above basal levels. Adenylate cyclase was also purified by subjecting the 165,000g supernate directly to agarose-hexane-GTP; agarose-hexane-ATP or agarose-hexane was not effective. The affinity-derived material was associated with 85 nmol of Lubrol-PX/mg of protein. When calculated on the basis of a molecular weight of 150,000 for detergent-free protein after gel filtration on Bio-Gel A-0.5 m, there was 13 mol of detergent/mol of the enzyme obtained by chromatography on the affinity resin. The direct affinity product was insensitive to glucagon and Gpp(NH)p; enzyme activity varied as a function of Lubrol concentration.     

Long-Chain Aldehyde Dehydrogenase That Participates in n-Alkane Utilization and Wax Ester Synthesis in Acinetobacter sp. Strain M-1

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C₄ to C₁₄), with a preference for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Δ) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.