Ferroxidase activity in a laccase-like multicopper oxidase from Liriodendron tulipifera.

Ferroxidase activity was detected in a laccase-like multicopper oxidase (LMCO) produced in transgenic tobacco cells expressing an LMCO cDNA (Ltlacc2.2) cloned from yellow-poplar (Liriodendron tulipifera). This marks the first report of ferroxidase activity associated with a plant laccase and suggests that some members of this plant enzyme family may have physiological functions based on activities other than their more widely recognized phenoloxidase activity. Recent work with LMCOs from bacteria, yeast and mammals has shown that metal oxidase activities in these enzymes can be important for their primary physiological functions, With respect to ferroxidase activity in certain plant LMCOs, it is proposed that the high levels of LMCO expression in plant vascular tissues may reflect the need for high-efficiency iron uptake pumps in tissues that undergo lignification during normal development. Such iron uptake pumps would function to minimize levels of free iron so that reactive oxygen species do not reach toxic levels when H2O2 is generated for peroxidase-mediated monolignol coupling during lignin deposition.     

Accelerated decolorization of structurally different azo dyes by newly isolated bacterial strains

Wastewater effluents from the textile and other dye-stuff industries contain significant amounts of synthetic dyes that require treatment to prevent groundwater contamination. In research aimed at biotechnology for treatment of azo dyes, this study examined 288 strains of azo-dye degrading bacteria to identify efficient strains and determine incubation times required for decolorization. Initial enrichment cultures were carried out using a mixture of four structurally different dyes (Acid Red 88, Reactive Black 5, Direct Red 81, and Disperse Orange 3) as the sole source of C and N to isolate the bacteria from soil, activated sludge, and natural asphalt. Six strains were selected for further study based on their prolific growth and ability to rapidly decolorize the dyes individually or in mixtures. Treatment times required by the most efficient strain, AS96 (Shewanella putrefaciens) were as short as 4 h for complete decolorization of 100 mg l⁻¹ of AR-88 and DR-81 dyes under static conditions, and 6 and 8 h, respectively, for complete decolorization of RB-5 and DO-3. To our knowledge, these bacterial strains are the most efficient azo-dye degrading bacteria that have been described and may have practical application for biological treatment of dye-polluted wastewater streams.     

Production, purification and characterization of cholesterol oxidase from a newly isolated Streptomyces sp.

Cholesterol oxidase production (COD) by a new isolate characterized as Streptomyces sp. was studied in different production media and fermentation conditions. Individual supplementation of 1 % maltose, lactose, sucrose, peptone, soybean meal and yeast extract enhanced COD production by 80–110 % in comparison to the basal production medium (2.4 U/ml). Supplementation of 0.05 % cholesterol (inducer) enhanced COD production by 150 %. COD was purified 14.3-fold and its molecular weight was found to be 62 kDa. V_max (21.93 μM/min mg) and substrate affinity K_m (101.3 μM) suggested high affinity of the COD for cholesterol. In presence of Ba²⁺ and Hg²⁺ the enzyme activity was inhibited while Cu²⁺ enhanced the activity nearly threefold. Relative activity of the enzyme was found maximum in triton X-100 whereas sodium dodecyl sulfate inactivated the enzyme. The enzyme activity was also inhibited by the thiol-reducing reagents like Dithiothreitol and β-mercaptoethanol. The COD showed moderate stability towards all organic solvents except acetone, benzene and chloroform. The activity increased in presence of isopropanol and ethanol. The enzyme was most active at pH 7 and 37 °C temperature. This organism is not reported to produce COD.     

A multicopper oxidase is essential for manganese oxidation and laccase-like activity in Pedomicrobium sp. ACM 3067

Pedomicrobium sp. ACM 3067 is a budding-hyphal bacterium belonging to the alpha-Proteobacteria which is able to oxidize soluble Mn2+ to insoluble manganese oxide. A cosmid, from a whole-genome library, containing the putative genes responsible for manganese oxidation was identified and a primer-walking approach yielded 4350 bp of novel sequence. Analysis of this sequence showed the presence of a predicted three-gene operon, moxCBA. The moxA gene product showed homology to multicopper oxidases (MCOs) and contained the characteristic four copper-binding motifs (A, B, C and D) common to MCOs. An insertion mutation of moxA showed that this gene was essential for both manganese oxidation and laccase-like activity. The moxB gene product showed homology to a family of outer membrane proteins which are essential for Type I secretion in Gram-negative bacteria. moxBA has not been observed in other manganese-oxidizing bacteria but homologues were identified in the genomes of several bacteria including Sinorhizobium meliloti 1021 and Agrobacterium tumefaciens C58. These results suggest that moxBA and its homologues constitute a family of genes encoding an MCO and a predicted component of the Type I secretion system.     

Characterization of cholesterol oxidase from a marine Streptomyces sp. and its cytotoxicity

A marine actinobacterial strain (designated as AKHSS) capable of producing cholesterol oxidase on the enzyme indicator plates was identified as Streptomyces sp. The cell-free lysate of the strain was used for monitoring the production of cholesterol oxidase and the maximal enzyme yields were recorded at 72 h post inoculation. The cholesterol oxidase was purified using polyethylene glycol 4000 precipitation, diethylaminoethyl Sephacel anionic column chromatography and Superdex-200 gel filtration to near homogeneity. Through electron-spray ionization mass spectrometry, molecular mass of the purified enzyme was recorded as 42.84 kDa. The optimum pH of the enzyme was found to be 9 and it was stable up to 60 °C. Metal salts like MgSO₄ and ZnSO₄ stimulated the enzyme activity. The V_max and K_m of the purified enzyme with cholesterol as substrate were found to be 1.22 μmoles/min/mL and 0.54 mM respectively. The enzyme showed significant cytotoxicity on breast (MCF-7), nasopharyngeal (KB) and ovarian (OVCAR) cancer cell lines at very low concentrations ranging from 0.093 to 0.14 μM, as evident from MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell viability assay. Besides, the enzyme exhibited relatively less cytotoxicity on primary mouse embryonic fibroblast (3T3) cells. Thus, cholesterol oxidase of Streptomyces sp. AKHSS could be a potential anticancer agent.     

An alkali-thermotolerant extracellular protease from a newly isolated Streptomyces sp. DP2

Of six alkalitolerant, extracellular protease producing bacterial strains isolated, DP2 displayed maximum activity. This organism was designated as Streptomyces sp. DP2 and identified as Streptomyces ambofaciens. Maximum protease yield was observed after 48 hours of submerged fermentation using various carbon and nitrogen sources. Fructose was found to be the best substrate for protease production, followed by maltose, lactose and wheat bran. Mustard cake is reported for the first time as the most ideal nitrogen source although soybean meal also gave comparable yield. The protease produced by Streptomyces sp. DP2 exhibited extensive activity over a broad pH range (4–12) with maximum activity at pH 8, and was active over a broad range of elevated temperatures (50–100°C), and possessed thermostability at 60–90°C for up to 1 hour. Enzyme activity was reduced by EDTA (25%), SDS (16%), and PMSF (6%). This novel alkaline protease has both alkali- and thermostability that may have industrial significance.     

Production of a cellulase-free xylanase from agricultural waste materials by a thermotolerant Streptomyces sp.

1444 microorganisms were isolated from soil samples from the northern Thai and screened at 55 °C by using basal medium supplemented with 1% carboxymethyl cellulose as a sole carbon source. One isolate, Streptomyces Ab106, had a high activity of a cellulase-free xylanase also without mannanase activity. The maximum cellulase-free xylanase activities of 3.5, 3.3, 3.1 and 2.7 IU were after growth of the organism with 1% (w/v) corn hull, corncob, bagasse and oat spelt xylan, respectively, at 55 °C for 6 days, respectively. The activity was more than 5 times higher than that at 35 °C.     

Rapid decolorization of azo dyes by crude manganese peroxidase from Schizophyllum sp. F17 in solid-state fermentation

The production of ligninolytic enzymes by the fungus Schizophyllum sp. F17 using a cost-effective medium comprised of agro-industrial residues in solid-state fermentation (SSF) was optimized. The maximum activities of the enzymes manganese peroxidase (MnP), laccase (Lac), and lignin peroxidases (LiP) were 1,200, 586, and 109 U/L, respectively, on day 5 of SSF. In vitro decolorization of three structurally different azo dyes by the extracellular enzymes was monitored to determine its decolorization capability. The results indicated that crude MnP, but not LiP and Lac, played a crucial role in the decolorization of azo dyes. After optimization of the dye decolorization system with crude MnP, the decolorization rates of Orange IV and Orange G, at an initial dye concentration of 50 mg/L, were enhanced to 76 and 57%, respectively, after 20 min of reaction at pH 4 and 35°C. However, only 8% decolorization of Congo red was observed. This enzymatic reaction system revealed a rapid decolorization of azo dyes with a low MnP activity of 24 U/L. Thus, this study could be the basis for the production and application of MnP on a larger scale using a low-cost substrate.     

Gene cloning, protein purification, and enzymatic properties of multicopper oxidase, from Klebsiella sp. 601

A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat//Km values of 0.49 mmol/L, 1.08 x 103 s-1, and 2.23 x 103 s-1.mmol/L-1, respectively, for DMP, 5.63 mmol/L, 6.64 x 103 s-1, and 1.18 x 103 s-1.mmol/L-1 for ABTS, and 0.023 mmol/L, 11 s-1, and 4.68 x 102 s-1.mmol/L-1 for SGZ.     

Production of cholesterol oxidase by a newly isolated Rhodococcus sp.

Fifteen strains of microorganisms with ability to degrade cholesterol were isolated. Among them a Gram-positive, non-motile, non-sporing bacterium with meso-DAP in the cell wall and with a rod-coccus cycle showed the highest ability for cholesterol degradation. It was identified as Rhodococcus sp. strain 2C and was deposited by code 1633 in Persian type culture collection (PTCC). This strain was able to produce high levels of both extracellular and cell-bound cholesterol oxidases in media containing cholesterol as a sole carbon source. The effects of medium composition and physical parameters on cholesterol oxidase production were studied. The optimized medium was found to contain cholesterol 0.15% (w/v), yeast extract 0.3% (w/v), diammonium hydrogen phosphate 0.1% (w/v), Tween 80 (0.05%). The optimum pH and temperature for cholesterol oxidase production in optimized medium were found to be 8–30 °C respectively. Triton X-100 showed the greatest effect in releasing the cell-bound enzyme. The first and most probably the main metabolite of cholesterol degradation was purified and identified as 4-cholestene-3-one.     

Decolorization of azo, triphenylmethane and anthraquinone dyes by a newly isolated Trametes sp. SQ01 and its laccase

A white-rot fungus, strain SQ01, was isolated from decayed wood in a temperate forest. The strain was identified as a member of genus Trametes, based on the morphological characteristics and a complete sequence analysis of its 18S rRNA gene and ITS region. Strain SQ01 was capable of decolorizing a variety of synthetic dyes, including azo, triphenylmethane, and anthraquinone dyes, with an optimal efficiency of decolorization obtained when dyes added after 5 days of culture, with the exception of Cresol Red, showing that the point of dye addition was an important influencing factor for decolorization by this fungus. All of the tested dyes were decolorized by the purified laccase in the absence of any redox mediators, but only a few were completely removed, while others were not completely degraded even with increased decolorization time.     

A neutral lipase applicable in biodiesel production from a newly isolated Streptomyces sp. CS326

In an attempt to isolate a biocatalyst able to catalyze biodiesel production from microbial source, Streptomyces sp. CS326 was screened from hundreds of soil isolates collected from various parts of Korea. In 16S rRNA sequence analysis, the strain showed high degree of similarity with Streptomyces xanthocidicus (99.79%); therefore, it is classified as Streptomyces sp. CS326. An extracellular lipase produced by the strain (LP326) was purified using a single step gel permeation chromatography on Sepharose CL-6B. Molecular weight of LP326 was estimated to be 17,000 Da by SDS-PAGE. The activity was optimum at 40 °C and pH 7.0 and was stable at pH 5.0-8.0 and below 50 °C. It preferred p-nitrophenyl palmitate (C16), a long chain substrate; and K (m) and V (max) for the substrate were determined to be 0.24 mM and 4.6 mM/min mg, respectively. First 10 N-terminal amino acid sequences were APDLVALQSE, which are different from so far reported lipases. LP326 catalyzed biodiesel production using methanol and various oils; therefore, the enzyme can be applicable in the field of biofuel.     

Accelerated decolorization of reactive azo dyes under saline conditions by bacteria isolated from Arabian seawater sediment

Presence of huge amount of salts in the wastewater of textile dyeing industry is one of the major limiting factors in the development of an effective biotreatment system for the removal of azo dyes from textile effluents. Bacterial spp. capable of thriving under high salt conditions could be employed for the treatment of saline dye-contaminated textile wastewaters. The present study was aimed at isolating the most efficient bacterial strains capable of decolorizing azo dyes under high saline conditions. Fifty-eight bacterial strains were isolated from seawater, seawater sediment, and saline soil, using mineral salt medium enriched with 100?mg?l^?1 Reactive Black-5 azo dye and 50?g NaCl l^?1 salt concentration. Bacterial strains KS23 (Psychrobacter alimentarius) and KS26 (Staphylococcus equorum) isolated from seawater sediment were able to decolorize three reactive dyes including Reactive Black 5, Reactive Golden Ovifix, and Reactive Blue BRS very efficiently in liquid medium over a wide range of salt concentration (0–100?g NaCl l^?1). Time required for complete decolorization of 100?mg dye l^?1 varied with the type of dye and salt concentration. In general, there was an inverse linear relationship between the velocity of the decolorization reaction (V) and salt concentration. This study suggested that bacteria isolated from saline conditions such as seawater sediment could be used in designing a bioreactor for the treatment of textile effluent containing high concentration of salts.     

Decolorization of textile azo dyes by newly isolated halophilic and halotolerant bacteria

Studies were carried out on the decolorization of textile azo dyes by newly isolated halophilic and halotolerant bacteria. Among the 27 strains of halophilic and halotolerant bacteria isolated from effluents of textile industries, three showed remarkable ability in decolorizing the widely utilized azo dyes. Phenotypic characterization and phylogenetic analysis based on 16S rDNA sequence comparisons indicate that these strains belonged to the genus Halomonas. The three strains were able to decolorize azo dyes in a wide range of NaCl concentration (up to 20%w/v), temperature (25-40 degrees C), and pH (5-11) after 4 days of incubation in static culture. They could decolorize the mixture of dyes as well as pure dyes. These strains also readily grew in and decolorized the high concentrations of dye (5000 ppm) and could tolerate up to 10,000 ppm of the dye. UV-Vis analyses before and after decolorization and the colorless bacterial biomass after decolorization suggested that decolorization was due to biodegradation, rather than inactive surface adsorption. Analytical studies based on HPLC showed that the principal decolorization was reduction of the azo bond, followed by cleavage of the reduced bond.     

Purification and characterization of veratryl alcohol oxidase from Comamonas sp. UVS and its role in decolorization of textile dyes

In the present work, we have purified veratryl alcohol oxidase (VAO) enzyme from Comamonas UVS to evaluate its potential to decolorize textile dyes. VAO was purified (13.9 fold) by an ion exchange followed by the size exclusion chromatography. Molecular weight of the VAO was estimated to be about 66 kDa by SDS-PAGE. The optimum pH and temperature of oxidase were 30°C and 65°C, respectively. VAO showed maximum activity with n-propanol among the various substrates (n-propanol, veratryl alcohol, L-dopa, tryptophan, etc.). Under standard assay conditions, K_m value of the enzyme was 2.5 mM towards veratrole. The enzyme activity was completely inhibited by 0.5 mM sodium azide. L-cysteine, dithiothreitol, and the metal chelator, EDTA had a slight inhibitory effect. The purified enzyme was able to decolorize textile dyes, Red HE7B (57.5%) and Direct Blue GLL (51.09%) within 15 h at 40 μg/mL concentration. GC-MS analysis of the metabolites suggested oxidative cleavage and desulphonation of these dyes.     

Characterization of a new alginate lyase from newly isolated Flavobacterium sp. S20

Alginate lyase is a promising biocatalyst because of its application in saccharification of alginate for the production of biochemicals and renewable biofuels. This study described the isolation of a new alginate metabolizing bacterium, Flavobacterium sp. S20, from sludge samples and the characterization of its alginate lyase Alg2A. The alginate lyase gene, alg2A, was obtained by constructing and screening the genomic library of the strain S20 and overexpressed in Escherichia coli. Substrate specificity assays indicated Alg2A preferred poly-α-l-guluronate as a substrate over poly-β-d-mannuronate. In the saccharification process of a high content (10 %, w/v) of sodium alginate, the recombinant alginate lyase Alg2A yielded 152 of mM the reducing sugars after 69 h of reaction, and the amounts of oligosaccharides with a different degree of polymerization (DP) generated by Alg2A gradually accumulated without significant variation in the distribution of oligosaccharide compositions. These results indicated that Alg2A possessed high enzymatic capability for saccharifying the alginate, which could be used in saccharifying the alginate biomass prior to the main fermentation process for biofuels. In addition, Alg2A had a different endolytic reaction mode from both the two commercial alginate lyases and other alginate lyases from polysaccharide lyase family 7 owing to high yields of penta-, hex-, and hepta-saccharides in the hydrolysis products of Alg2A. Thus, Alg2A could be a good tool for the large-scale preparation of alginate oligosaccharides with high DP.     

Characterization of a xylanolytic complex from Streptomyces sp.

Streptomyces sp. DSM 41796 produced four major extracellular xylanases with Mr of 145, 120, 60 and 45 kDa. Those of 145 and 60 kDa formed a heterodimer. All xylanases, except that of 120 kDa, were induced by xylose, d-arabinose or sucrose, while commercial xylans induced the 60 kDa xylanase in a major proportion than others, and sugar-cane bagasse pith or lemon peel induced predominantly the 45 kDa xylanase.     

Endoglucanase S, a novel endocellulase exhibiting exoglucanase activity from a newly isolated Streptomyces sp. LX

A novel endocellulase, designated as endoglucanase S, was purified to homogeneity from culture supernatant fluids of the newly isolated Streptomyces sp. LX, and shown not to be identical with previously described endo-β-1,4-glucanases. Both endo- and exo-cellulase activities were found to reside on a monomeric protein of 48 kDa. Its temperature optimum is 50 °C, and it was stable at 60 °C. The optimum pH is 5·5, and it has a broad pH stability from pH 3·0–7·0. The action of the enzyme on carboxymethylcellulose (CMC) suggested that the enzyme behaved as endoglucanase, whereas it was also active on crystalline cellulose with cellodextrin as end-product. Fragmentation of filter paper revealed that the degree of polymerization of residual cellulose decreased with time but only 5·2% of filter paper was converted into soluble carbohydrate.