Purification and Characterization of the Enzyme Cholesterol Oxidase from a New Isolate of Streptomyces sp.

An extracellular cholesterol oxidase (cho) enzyme was isolated from the Streptomyces parvus, a new source and purified 18-fold by ion exchange and gel filtration chromatography. Specific activity of the purified enzyme was found to be 20 U/mg with a 55 kDa molecular mass. The enzyme was stable at pH 7.2 and 50 °C. The enzyme activity was inhibited in the presence of Pb(2+), Ag(2+), Hg(2+), and Zn(2+) and enhanced in the presence of Mn(2+). The enzyme activity was inhibited by the thiol-reducing reagents (DTT, β-mercaptoethanol), suggesting that disulfide linkage is essential for the enzyme activity. The enzyme activity was found to be maximum in the presence of Triton X-100 and X-114 detergents whereas sodium dodecyl sulfate fully inactivated the enzyme. The enzyme showed moderate stability towards all organic solvents except acetone, benzene, chloroform and the activity increased in the presence of isopropanol and ethanol. The K(m) value for the oxidation of cholesterol by this enzyme was 0.02 mM.     

Ligase-Independent Cloning of Amylase Gene from a Local Bacillus subtilis Isolate and Biochemical Characterization of the Purified Enzyme

Five hundred ninety-seven bacterial isolates from Turkish hot spring water sources were screened for their ability to produce extracellular α-amylase. Among them, a high enzyme-producing Bacillus subtilis isolate, A28, was selected, and its α-amylase gene was cloned and expressed in Escherichia coli by a ligase-independent method. α-Amylase from the recombinant strain was purified to homogeneity by Q-Sepharose anion exchange and Sephacryl S-100 gel filtration chromatographies. The final yield of the enzyme was about 22.5 % of the initial activity, with a 16.4-fold increase in specific activity compared with the culture lysate. The optimum temperature and pH of the enzyme were 70 °C and 6.0, respectively. The enzyme was highly active at acidic-neutral pH range of 4.5–7.0. The amy28 α-amylase retained 100 % of its activity after incubation at 50 °C for 90 min. Co⁺², Cu²⁺, Fe²⁺, Fe³⁺, Ni⁺², and Zn⁺² caused significant inhibition in enzyme activity, which was not affected by Na⁺, Mg²⁺, Li⁺, and Ba²⁺. The activity was inhibited about 70 % upon treatment of the enzyme with 10 mM ethylenediaminetetraacetic acid. However, Ca²⁺ ions known as high temperature stabilizer for other amylases did not stimulate the activity of the enzyme. Due to pH stability and thermostability of the recombinant amylase, this enzyme may be suitable in starch processing, brewing, and food industries.     

Gene Cloning, Expression, and Characterization of a Cyclic Nucleotide Phosphodiesterase from Arthrobacter sp. CGMCC 3584

Based on thermal asymmetric interlaced polymerase chain reaction, the arpde gene encoding a cyclic nucleotide-specific phosphodiesterase was cloned from Arthrobacter sp. CGMCC 3584 for the first time. The 930-bp region encoded a 309-amino-acid protein with a molecular weight of 33.6 kDa. The recombinant ArPDE was able to hydrolyze 3′,5′-cAMP, 3′,5′-cGMP, and 2′,3′-cAMP. The K _m values of ArPDE for 3′,5′-cAMP and 3′,5′-cGMP were 6.82 and 12.82 mM, respectively. ArPDE was thermostable and displayed optimal activity at 45 °C and pH 7.5. The enzyme did not require any metal cofactors, although its activity was stimulated by 2 mM Co²⁺ and inhibited by Zn²⁺. Nucleotides, reducing agents, and sulfhydryl reagents had different inhibitory effects on the activity of ArPDE. NaF, the actual compound used to improve the industrial yield of cAMP, exhibited 62 % inhibitions at concentrations of 10 mM.     

Characterization of a Thermotolerant and Alkalotolerant Xylanase from a Bacillus sp.

In a recent screening for thermophilic bacteria from Azores hot springs, a Bacillus sp strain 3M, exhibiting cellulase-free extracellular xylanolitic activity, was isolated. Further enzyme characterization from liquid cultures grown on birchwood xylan revealed that the endo-l,4-βxylanase retains 100% of activity for at least 3 d at 55°C. At 80°C, it retains 47% of its maximal activity, and the enzyme is still active at 90°C. The optimum pH of the enzyme has a broad pH range, between 6.0 and 7.5, and it is remarkably active for the alkaline region, exhibiting 89% of relative activity at pH 9.O. The enzyme was partially inactivated by different divalent metal ions. Because of its tolerance for high temperature and pH conditions, and the absence of contaminating cellulase activity, the xylanase produced byBacillus sp 3M appears to be attractive for use in the pulp and paper industry. Indeed, the efficiency of the enzyme application to the kraftEucalyptus pulp was studied for bleaching pretreatment, resulting in a moderate increase of pulp bleachability.     

Characterization of a kerationlytic metalloprotease from Bacillus sp. SCB-3

A keratinolytic protease-producing microorganism was isolated from soybean paste waste and was identified as a strain of Bacillus sp. The keratinase was purified by polyethylene glycol precipitation and two successive column chromatographies with DEAE-Toyopearl 650C and Sephacryl S-200 HR. The purified enzyme had overall 11 purification folds with an 18% yield. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel filtration on Sephacryl G-200 indicated that the purified enzyme was monomeric and had a molecular weight of 134 kDa. The optimum temperature and pH were 40°C and 7.0, respectively. This enzyme was completely inhibited by EDTA and EGTA, and it was restored by the addition of Ca⁺² and Mg⁺². These results suggested that it is a metalloprotease. The stimulated enzyme activity by reducing agents indicated that the reducing condition was important in the expression of the activity.     

Characterization of a New Cold-adapted Lipase from Pseudomonas sp. TK-3

A psychrotrophic Pseudomonas sp. TK-3 was isolated from dirty and cool stream water in Toyama, Japan from which we cloned and characterized the bacterial lipase LipTK-3. The sequenced DNA fragment contains an open reading frame of 1,428?bp that encoded a protein of 476 amino acids with an estimated molecular mass of 50,132?Da. The lipase showed high sequence similarity to those of subfamily ??.3 lipase and had a conserved GXSXG motif around the catalytic Ser residue. Its optimal temperature was 20?C25?°C, lower than in most other subfamily ??.3 lipases. The lipase exhibited about 30?% of maximal activity at 5?°C. The optimal pH value was 8.0. The activity was strongly inhibited by EDTA and was highly dependent on Ca²⁺. Tricaprylin and p-nitrophenyl caprylate were the most favorable substrates among the triglycerides and p-nitrophenyl esters, respectively. LipTK-3 also showed high activity towards natural substrates including edible vegetable oils and animal fats. Furthermore, LipTK-3 was very active and stable in the presence of several detergents, metal ions, and organic solvents. This cold-adapted lipase may prove useful for future applications.     

Recombinant Expression and Characterization of l-Asparaginase II from a Moderately Thermotolerant Bacterial Isolate

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5?°C, was isolated from cow dung; l-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37?°C and in a buffer system of pH?6 to 7. The enzyme exhibited thermostability at 50?°C with a 33% and 28% of activity retention after 45 and 60?min. The kinetic parameters for the enzyme were calculated from Lineweaver?CBurk plot, and K _m and V _max were 0.89?mM and 0.18?U/mg, respectively.     

Biosynthesis of Benzoylformic Acid from Benzoyl Cyanide with a New Bacterial Isolate of Brevibacterium sp. CCZU12-1

Brevibacterium sp. CCZU12-1 with high nitrilase activity could effectively hydrolyze benzoyl cyanide into benzoylformic acid. After the culture optimization, the preferred carbon sources, nitrogen sources, and inducer were glucose (10 g/L), a composite of peptone (10 g/L) plus yeast extract (2.5 g/L), and ε-caprolactam (2.0 mM), respectively. After the reaction optimization, the optimum reaction temperature, reaction pH, organic cosolvent, and metal ion were 30 °C, 7.0, ethanol (2 %, v/v), and Ca²⁺ (0.1 mM), respectively. At biotransformation of 120-mM benzoyl cyanide for 24 h, the yield of benzoylformic acid reached 91.8 %. Moreover, the microbial nitrilase from Brevibacterium sp. CCZU12-1 could hydrolyze various nitriles, and it significantly exhibited high nitrilase activity against benzoyl cyanide, 3-cyanopyridine, and α-cyclohexyl-mandelonitrile.     

Ubiquinone Q9 from a marine isolate of an actinobacterium Nocardia sp.

The major component of a nonpolar fraction of an extract of an actinobacterium Nocardia sp. KMM 3749 isolated from an unidentified marine ascidian was shown, using NMR spectroscopy and mass spectrometry, to be 2,3-dimethoxy-5-methyl-6-polyprenyl-1,4-benzoquinone (ubiquinone Q₉).     

Optimization of a Sequential Extraction Scheme for the Characterization of Heavy Metal Mobility in Iron Oxide Rich Sediments

Abstract

Several parameters governing the extraction of metals from iron oxide rich sediments, using sequential extraction schemes were optimized. The mode of shaking, soil/extractant ratio and concentrations of MgCl₂ and NH₂OH·HCl, for samples collected in the Odiel Marshes Natural Park (SW Spain), were considered. The concentration of NH₂OH·HCl deserved particular attention due to the nature of the studied sediments. A 0.4 M concentration of this extractant was needed to avoid readsorption of Cu and As in the samples. In addition, readsorption processes were studied using a candidate to reference material with a high organic matter content that was previously analyzed in an interlaboratory study.     

Purification and Characterization of Novel Halo-Acid-Alkali-Thermo-stable Xylanase from Gracilibacillus sp. TSCPVG

An aerobic xylanolytic moderately halophilic and alkali-tolerant bacterium, Gracilibacillus sp. TSCPVG, produces multiple xylanases of unusual halo-acid-alkali-thermo-stable nature. The purification of a major xylanase from TSCPVG culture supernatant was achieved by hydrophobic and gel permeation chromatographic methods followed by electroelution from preparatory PAGE. The molecular mass of the purified xylanase was 42 kDa, as analyzed by SDS-PAGE, with a pI value of 6.1. It exhibited maximal activity in 3.5 % NaCl and retained over 75 % of its activity across the broad salinity range of 0–30 % NaCl, indicating a high halo-tolerance. It showed maximal activity at pH 7.5 and had retained 63 % of its activity at pH 5.0 and 73 % at pH 10.5, signifying the tolerance to broad acid to alkaline conditions. With birchwood xylan as a substrate, K _m and specific activity values were 21 mg/ml and 1,667 U/mg, respectively. It is an endoxylanase that degrades xylan to xylose and xylobiose and had no activity on p-nitrophenyl-β-d-xylopyranoside, p-nitrophenyl-β-d-glucopyranoside, p-nitrophenyl acetate, carboxymethylcellulose, and filter paper. Since it showed remarkable stability over different salinities, broad pH, and temperature ranges, it is promising for application in many industries.     

Performance of a Bismuth Bulk Rotating Disk Electrode for Heavy Metal Analysis: Determination of Lead in Environmental Samples

The bismuth bulk electrode is proposed here for the first time in the rotating configuration (BiB‐RDE) as the electrode of choice for voltammetric analysis of selected heavy metal ions. Optimization of chemical and instrumental parameters was carried out to develop a reliable and convenient method for the determination of Zn(II), Cd(II) and Pb(II) by SWASV. Appropriate detection limits were found for environmental monitoring applications in the medium – low µg/L range. The method was validated for Pb(II) determination by certified reference materials. Successful application to the determination of Pb(II) in samples of fortified rainwater and sewage sludge from a steel industry is described.     

The Application of a Sol-Gel Technique to Preparation of a Heavy Metal Biosorbent from Yeast Cells

In this work we compared biosorbents obtained by encapsulation of polysaccharides, isolated from waste brewing biomass, in sol-gel derived silicates and an organic polymer. Biosorbents were prepared by mixing cross-linking-agents—organic or siliceous—with dried cells envelopes. Siliceous prepolymers were synthezised via transesterification and hydrolysis from tetraethoxysilane (TEOS) and methanol. Sorption of Cd2+, Cu2+ and Ag+ by biosorbent granules (0.25–0.6 mm) was examined in batch and in a packed column. The biosorbent prepared by interesterification of TEOS showed a 2–3 times higher intensity of sorption than the biosorbent cross-linked with epichlorohydrin (the most effective cross-linking organic agent) while the sorption capacity of both biosorbents was equal. The specific surface area of the silica matrix was 597 m2/g but only traces of metals were sequestered from solution with a concentration of Cd2+ of 50 mg/l. The biosorbent with a silica matrix is a heterogeneous material containing microporous matrix inclusions of thin cell walls. Its high sorption intensity and good mechanical strength will be useful in continuous metal uptake of low concentrations of metals.     

Formation of Lutein, β-Carotene and Astaxanthin in a Coelastrella sp. Isolate

In this study, the effect of media composition, N/P ratio and cultivation strategy on the formation of carotenoids in a Coelastrella sp. isolate was investigated. A two-stage process utilizing different media in the vegetative stage, with subsequent re-suspension in medium without nitrate, was employed to enhance the formation of carotenoids. The optimal growth and carotenoid content (β-carotene and lutein) in the vegetative phase were obtained by cultivation in M-8 and BG11 media. Use of a N/P ratio of 37.5 and low light intensity of 40 μmol m⁻² s⁻¹ (control conditions) led to optimal biomass production of up to 1.31 g L⁻¹. Low concentrations of astaxanthin (maximum of 0.31 wt. %) were accumulated under stress conditions (nitrogen-deficient medium containing 1.5 % of NaCl and light intensity of 500 μmol m⁻² s⁻¹), while β-carotene and lutein (combined maximum of 2.12 wt. %) were produced under non-stress conditions. Lipid analysis revealed that palmitic (C16:0) and oleic (C18:1) constituted the main algal fatty acid chains (50.2 ± 2.1% of the total fatty acids), while esterifiable lipids constituted 17.2 ± 0.5% of the biomass by weight. These results suggest that Coelastrella sp. could also be a promising feedstock for biodiesel production.     

Phytotoxins. II. Characterization of a phytotoxic fraction from alternaria alternata f. sp. lycopersici

A host-specific phytotoxic fraction obtained from the cell-free culture filtrate of A. alternata f. sp. lycopersici is shown to consist of two esters of 1,2,3-propanetricarboxylic acid and 1-amino-11,15-dimethylheptadeca-2,4,5,13,14-pentol 1. The sites of esterification are a terminal carboxyl of the acid and C₁₃ (major component 2a) and C₁₄ (2b) of 1.     

软珊瑚Sinularia sp.中次生代谢产物的结构鉴定

细胞毒性;软珊瑚Sinularia sp.中次生代谢产物的结构鉴定     

Purification and Biochemical Characterization of a Novel Thermo-stable Carboxymethyl Cellulase from Azorean Isolate Bacillus mycoides S122C

Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76?%, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62?kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by ??-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50?°C and pH?7.0, respectively. The purified CMcellulase was stable with about 60?% activity in broad pH ranges from 5 to 10 and temperature of 40 to 60?°C. However, purified CMcellulase was stable at about 70?% at 70?°C and also stable overall at 78?% for surfactants. CMcellulase activity was inhibited by ions such as HgCl₂, followed by CuSo₄, FeCl₂, and MnCl₂, while CoCl₂ activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.     

Gene Cloning,Overexpression, and Characterization of a Xylanase from <Emphasis Type="Italic">Penicillium</Emphasis> sp. CGMCC 1669

A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml⁻¹. After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40°C, was stable at acidic buffers of pH 4.5–9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and α-chymotrypsin). The specific activity, K _m, and V _max for oat spelt xylan substrate was 7,988 U mg⁻¹, 22.2 mg ml⁻¹, and 15,105.7 μmol min⁻¹ mg⁻¹, respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.