Purification and characterization of cutinase from a fluorescent Pseudomonas putida bacterial strain isolated from phyllosphere

Cutinase, an extracellular enzyme, was induced by cutin in a fluorescent Pseudomonas putida strain that was found to be cohabiting with an apparently nitrogen-fixing Corynebacterium. This enzyme was purified from the culture fluid by acetone precipitation followed by chromatography on DEAE-cellulose, QAE-Sephadex, Sepharose 6B, and Sephadex G-100. The purified enzyme showed a single band when subjected to polyacrylamide electrophoresis and the enzymatic activity coincided with the protein band. Sodium dodecyl sulfate-polyacrylamide electrophoresis showed a single band at a molecular weight of 30,000 and gel filtration of the native enzyme through a calibrated Sephadex G-100 column indicated a molecular weight of 30,000, showing that the enzyme is a monomer. The amino acid composition of bacterial cutinase is distinctly different from that of fungal or plant cutinases. This bacterial cutinase showed a broad pH optimum between 8.5 and 10.5 with 3H-labeled apple cutin as the substrate. Linear rates of cutin hydrolysis were measured up to 20 min of incubation time and 4 mg/ml of cutin gave the maximum hydrolysis rate. This cutinase catalyzed hydrolysis of p-nitrophenyl esters of C4 to C16 fatty acids with decreasing V and increasing Km for the longer chain esters. It did not hydrolyze tripalmitoyl glycerol or trioleyl glycerol, indicating that this is not a general lipase. Active serine-directed reagents such as organophosphates and organoboronic acids severely inhibited the enzyme, suggesting that bacterial cutinase is an "active serine" enzyme. Neither thiol-directed reagents nor metal ion chelators had any effect on this enzyme. Antibody raised against purified enzyme gave a single precipitin line on Ouchterlony double diffusion analysis. Western blot analysis of the extracellular fluid of induced Ps. putida showed a single band at 30 kDa. No immunological cross-reactivity was detected between the present bacterial enzyme and the fungal enzyme from Fusarium solani pisi when rabbit antibodies against either enzyme was used.     

Purification and characterization of P fimbriae from an Escherichia coli strain isolated from a septicemic turkey

Abstract A pap + Escherichia coli isolate from a turkey with colisepticemia expressed P fimbriae with a major subunit of an apparent molecular mass of 18 kDa which reacted with anti-F11 serum. This fimbriae was purified and polyclonal antiserum was produced in rabbits. The N-terminal amino acid sequence of the major fimbrial subunit of the avian P fimbriae was identical to that of F11. On immunoblotting, the antiserum against the avian P fimbriae strongly reacted with the major subunit of the homologous fimbriae, with F11, and with F165₁ fimbriae. Some antigenic determinants on the major subunits of F13, F7₁, and F7₂ fimbriae, with a stronger reaction against F13 fimbriae, were also recognized. The F11 antiserum reacted similarly to the antiserum against avian P fimbriae although cross-reactions against F13, F7₁, and F7₂ fimbriae were equivalent. In a competitive enzyme-linked immunosorbent assay, serological differences were observed between the purified avian P fimbriae and F11. Thus, the avian P fimbriae is closely related but not identical to F11 fimbriae which are associated with E. coli isolated from human urinary tract infection.     

Purification and characterization of a novel mannitol dehydrogenase from a newly isolated strain of Candida magnoliae

Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37 degrees C and 10.0 and 40 degrees C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k(cat) = 823 s(-1), K(m) = 28.0 mM, and k(cat)/K(m) = 29.4 mM(-1) s(-1)) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.     

Purification and thermodynamic characterization of glucose oxidase from a newly isolated strain of Aspergillus niger

An intracellular glucose oxidase (GOD) was isolated from the mycelium extract of a locally isolated strain of Aspergillus niger NFCCP. The enzyme was partially purified to a yield of 28.43% and specific activity of 135 U mg(-1) through ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration. The enzyme showed high specificity for D-glucose, with a K(m) value of 25 mmol L(-1). The enzyme exhibited optimum catalytic activity at pH 5.5. Optimum temperature for GOD-catalyzed D-glucose oxidation was 40 degrees C. The enzyme displayed a high thermostability having a half-life (t(1/2)) of 30 min, enthalpy of denaturation (H*) of 99.66 kJ mol(-1), and free energy of denaturation (G*) of 103.63 kJ mol(-1). These characteristics suggest that GOD from A. niger NFCCP can be used as an analytical reagent and in the design of biosensors for clinical, biochemical, and diagnostic assays.     

Purification, physico-chemical characterization and sequence of a heat labile alkaline metalloprotease isolated from a psychrophilic Pseudomonas species

The psychrophilic alkaline metalloprotease (PAP) produced by a Pseudomonas bacterium isolated from Antarctica has been purified and characterized. The gene encoding PAP has been cloned and sequenced and the derived amino acid sequence shows 66% identity with the mesophilic alkaline metalloprotease from Pseudomonas aeruginosa IFO 3455 (AP). Compared to the purified AP, PAP is three times more active at 20 degrees C, is very sensitive to chelating agents and is rapidly inactivated at 45 degrees C. The lower thermostability of PAP can tentatively be explained by a loss of a stabilizing Ca(2+), a decrease in the content of hydrophobic residues and a smaller aliphatic index.     

Purification and properties of soluble cytoplasmic toxin from the mosquito pathogen Bacillus sphaericus strain 1593

Soluble cytoplasmic toxin from broken Bacillus sphaericus 1593 sporulating cells was partially purified by ammonium sulfate precipitation, ion exchange, and gel filtration chromatography. Purification was monitored by electrophoresis. The toxin remained active after incubation in the presence of several enzymes and in buffers from pH 6 to 10, but was destroyed by Pronase and subtilisin, and by heating to 80°C for 30 min. Results indicate that the B. sphaericus 1593 cytoplasm contains a single proteinaceous toxin with a molecular weight of 100,000 daltons.     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Purification and characterization of a cell-associated, soluble mannanase from Bacteroides ovatus.

Bacteroides ovatus, a human colonic anaerobe, utilizes the galactomannan guar gum as a sole source of carbohydrate. Previously, we found that none of the galactomannan-degrading enzymes were extracellular, and we characterized an outer membrane mannanase which hydrolyzes the backbone of guar gum to produce large fragments. We report here the purification and characterization of a second mannanase from B. ovatus. This enzyme is cell-associated and soluble. Using ion-exchange chromatography, gel filtration, and chromatofocusing steps, we have purified the soluble mannanase to apparent homogeneity. The enzyme has a native molecular weight of 190,000 and a monomeric molecular weight of 61,000. It is distinct from the membrane mannanase not only with respect to cellular location but also with respect to stability and isoelectric point (pI of 6.9 for the membrane mannanase and pI of 4.8 for the soluble mannanase). The soluble mannanase, like the membrane mannanase, hydrolyzed guar gum to produce large fragments rather than monosaccharides. However, if galactosyl side chains were removed from the galactomannan fragments by alpha-galactosidase, both the soluble mannanase and the membrane mannanase could degrade guar gum to monosaccharides. Thus either or both of these two enzymes, working together with alpha-galactosidase, appear to be sufficient for the breakdown of guar gum to the level of monosaccharides.     

Leucobacter kyeonggiensis sp. nov., a new species isolated from dye waste water

A Gram-positive, aerobic, non-motile bacterium designated F3-P9^T, was isolated from dye waste water in Korea and was characterized using a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that strain F3-P9^T belongs to genus Leucobacter. The 16S rRNA gene sequence similarities among strain F3-P9^T and validated representatives of the genus Leucobacter ranged from 95.9–97.4%. Strain F3-P9^T exhibited DNA-DNA relatedness values below 48% with respect to Leucobacter species. The G+C content of the genomic DNA was 67.5 mol%. F3-P9^T contained MK-11 as the major respiratory quinone. The major fatty acids were anteiso-C_15:0 (48.5%), anteiso-C_17:0 (22.7%), and iso-C_16:0 (14.5%). The peptidoglycan was composed of L-2,4-diaminbutyric acid, alanine, glycine, and glutamic acid. The polar lipid profile showed a major amount of diphosphatidylglycerol (DPG), a moderate amount of phosphatidylglycerol (PG), and two unknown glycolipids. On the basis of its phenotypic and genotypic properties and its phylogenetic distinctiveness, strain F3-P9^T (KEMC 211-128^T =KACC 16572^T =JCM 17539^T) should be classified in the genus Leucobacter as the type strain of a novel species, for which the name Leucobacter kyeonggiensis sp. nov. is proposed.     

Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota.

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.     

Purification and partial characterization of a soluble hemagglutinin from Yersinia pseudotuberculosis

A soluble hemagglutinin (HA) produced by Yersinia pseudotuberculosis strain Inoue, serotype 5b, was purified by ammonium sulfate precipitation, gel filtration on Sepharose CL-6B and high performance liquid chromatography on a DEAE-5PW anion-exchange column. The purified HA was a 14.5 kDa protein with an isoelectric point of 4.5. Amino acid analysis indicated that the HA consisted of 133 residues, corresponding to the molecular weight of 14,100. The amino acid sequence of N-terminal 38 amino acid residues showed no homology with that of several fimbrial proteins from Escherichia coli.     

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A.

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.     

Purification and characterization of an extremely halophilic acetoacetyl-CoA thiolase from a newly isolated Halobacterium strain ZP-6

The extremely halophilic archaeon ZP-6 was isolated from Ai-Ding salt lake in Xinjiang Uighur Autonomous Region of the People's Republic of China. Based on its physiological properties, 16S rDNA sequence, and DNA-DNA homology with known haloarchaea, the isolate was tentatively identified as a Halobacterium sp. An acetoacetyl-CoA thiolase was purified and characterized from this organism. The native enzyme has a molecular mass of 80 +/- 8 kDa and consists of two identical subunits of 43 +/- 2 kDa each. The N-terminus 14 amino acid residues were sequenced and showed identity with the respective part of a putative thiolase (AcaB1) of Halobacterium sp. NRC-1. The purified enzyme has an optimal pH of 7.9 for acetoacetyl-CoA thiolysis. The thiolytic activity was inhibited by the presence of Mg'- and was stimulated by KCl or NaCl. The thiolysis reaction of Halobacterium sp. ZP-6 thiolase can be inhibited by either substrate when present in excess. The distinct kinetic profile indicates that the thiolase from Halobacterium sp. ZP-6 may have a different catalytic mechanism from the so-called ping-pong mechanism employed by other thiolases. To our knowledge, this is the first report of the purification and characterization of a halophilic thiolase from an archaeal species.     

Purification and some properties of a soluble cytochrome (cytochrome c-551) from Erythrobacter species strain OCh 114

A soluble cytochrome, cytochrome c-551 was purified from an aerobic photosynthetic bacterium Erythrobacter species strain OCh 114 (ATCC No. 33942) by ammonium sulfate fractionation, ion-exchange chromatography and gel-filtration. The cytochrome had absorption maxima at 277, 410, and 524–525 nm in the oxidized form, and at 415, 522, and 550.5 nm in the reduced form. At 77 K, the -band of the absorption spectrum of the reduced form split in two at 547 and 549 nm. The millimolar absorption coefficient at 550.5 nm was 26.8 mM^-1 cm^-1 in the reduced form. This cytochrome was an acidic protein with an isoelectric point of 4.9. Its molecular weight was determined to be 15,000 by gel-filtration on Sephadex G-100 and 14,500 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The midpoint potential of this cytochrome was +250 mV at pH 7.0. This cytochrome did not bind CO.     

Purification and characterization of a novel bromoperoxidase-catalase isolated from bacteria found in recycled pulp white water

A bacterial strain, Pseudomonad EF group 70B, containing a high catalase-like activity was found in process water (white water) from pulp using recycled fibers. The enzyme was purified and characterized, and found to be a hydroperoxidase. The active enzyme has an apparent molecular mass of about 153 kDa with two identical subunits and a pI value of 4.7. It has a rather sharp pH optimum for catalase activity at 6.0 but exhibits catalase, peroxidase and brominating activities over a broad pH range from 4 to 8. It was not inhibited by 3-amino-1,2,4-triazole. Peroxidase-like activity was found when adding o-dianisidine, pyrogallol, guaiacol and 4-aminoantipyrine. Brominating activity was noticed using monochlorodimedone as a substrate. The absorption spectrum exhibited a Soret band at 404 nm. Upon reduction with dithionite the Soret peak decreased and shifted to 436 nm. Pyridine hemochrome spectra indicated the presence of a protophorfyrin IX heme group and the enzyme was inhibited by the known heme ligands cyanide and azide. N-terminal amino acid analysis gave the sequence STEVKLPYAVAGGGTTILDAFPGE, which showed no homology with those of known catalases or peroxidases. It is concluded that the enzyme is a novel type of catalase-peroxidase or, more specifically, a bromoperoxidase-catalase, and that future developments of inhibitors of hydrogen peroxide-degrading activities in white water may be based on this enzyme and other catalase-peroxidases.     

餐厨垃圾厌氧消化产沼气过程中酶学表征

厌氧消化产沼气被认为是餐厨垃圾资源化利用的有效方式之一,其实质是在多种微生物综合作用下的生物化学过程.本文研究了在促进和抑制性因子作用下,餐厨垃圾厌氧发酵的酶学过程,对其中的脱氢酶和水解酶(β-葡萄糖苷酶,BAA-蛋白水解酶,碱性磷酸酶)活性变化进行了分析.研究表明,与空白对照组相比添加酵母粉后脱氢酶的最高活性提高了8...     

Purification and characterization of a soluble polyphosphatase from mitochondria of Saccharomyces cerevisiae

A polyphosphatase with the specific activity 2.2 U/mg was purified to apparent homogeneity from a soluble preparation of mitochondria of Saccharomyces cerevisiae. The polyphosphatase is a monomeric protein of approximately 41 kD. The purified enzyme hydrolyzes polyphosphates with an average chain length of 9 to 208 phosphate residues to the same extent, but its activity is approximately 2-fold higher with tripolyphosphate. ATP, PPi, and p-nitrophenyl phosphate are not substrates of this enzyme. The apparent Km values are 300, 18, and 0.25 microM obtained at hydrolysis of polyphosphates with a chain length of 3, 15, and 188 phosphate residues, respectively. Several divalent cations stimulated the enzyme activity 1.2-27-fold (Mg2+ = Co2+ = Mn2+ > Zn2+). Determination of the protein N-terminal sequence and its comparison with the EMBL data library indicates that the soluble polyphosphatase of mitochondria of S. cerevisiae is not encoded by the gene of the major yeast polyphosphatase PPX1.     

Purification and characterization of an extracellular, uracil specific ribonuclease from a Bizionia species isolated from the marine environment of the Sundarbans

The first ribonuclease (RNase) from the Cytophaga-Flavobacterium-Bacteroides phylum, dominant in the marine environment, and also from the first Bizionia species isolated from the tropics was purified and characterized. Extracellular RNase production occurred when the culture medium contained 5-7% (w/v) NaCl. The 53.0 kDa enzyme was purified 29 folds with a recovery of 4% and specific activity of 630unit/mg protein. The pH and temperature optima are 6.5 and 35 degrees C, respectively and the enzyme retains more than half of its activity (relative to optimal assay conditions) after 1h pre-incubation separately with 5% (w/v) NaCl or from pH 5.0 to 8.5 or at 50 degrees C. Dithiothreitol and beta-mercaptoethanol do not inhibit whereas human placental RNase inhibitor protein halves the RNase activity. While Mg(2+), Ba(2+) and Ca(2+) enhanced the enzyme activity, Fe(2+), Cu(2+) and Hg(2+) inactivated it. This RNase degrades uracil containing nucleic acids only. Our isolate could be a novel renewable source of deoxyribonuclease (DNase)--free RNase enzyme.     

Purification and characterization of an intracellular β-glucosidase from a new strain of Leuconostoc mesenteroides isolated from cassava

The lactic acid bacterium, Leuconostoc mesenteroides, when grown on an arbutin-containing medium, was found to produce an intracellular β-glucosidase. The enzyme was purified by chromatofocusing, ion-exchange chromatography and gel filtration. The molecular mass of the purified intracellular β-glucosidase, as estimated by gel filtration, was 360 kDa. The tetrameric structure of the β-glucosidase was determined following treatment of the purified enzyme with dodecyl sulphate (SDS). The intracellular β-glucosidase exhibited optimum catalytic activity at 50°C and pH 6 with citrate–phosphate buffer, and 5·5 with phosphate buffer. The enzyme was active against glycosides with (1→4)-β, (1→4)-α and (1→6)-α linkage configuration. From Lineweaver–Burk plots, K _m values of 0·07 mmol l⁻¹ and 3·7 mmol l⁻¹ were found for p -nitrophenyl-β- D -glucopyranoside and linamarin, respectively. The β-glucosidase was competitively inhibited by glucose and by D -gluconic acid–lactone and a glucosyl transferase activity was observed in the presence of ethanol. The β-glucosidase of Leuconostoc mesenteroides, with cyanogenic activity, could be of potential interest in cassava detoxification, by hydrolysing the cyanogenic glucosides present in cassava pulp.