Biodegradation of aliphatic and aromatic hydrocarbons by bacteria isolated from Bahregan area.

Environmental pollution with aromatic and aliphatic hydrocarbons caused by oil and petrochemical industries has very toxic and carcinogenic effects on living organisms and should be removed from the environment. In this research, after analyzing the oil sludge of the Bahregan area, it was found that most aliphatic paraffin compounds are related to octadecane, most liquid aliphatic compounds are related to hexadecane, and most aromatic compounds are related to naphthalene, phenanthrene, fluoranthene, and anthracene. Then, we investigated the ability of native bacteria from this area, such as Thalassospira, Chromohalobacter, and a bacterial consortium, to biodegrade the dominant aromatic and aliphatic hydrocarbons found in oil sludge. The results of Gas Chromatography-Mass Spectrometry analysis showed that among the tested hydrocarbon sources, Thalassospira can completely remove octadecane and hexadecane, and Chromohalobacter can reduce hexadecane from 15.9 to 9.9%. The bacterial consortium can completely remove octadecane and reduce hexadecane from 15.9 to 5.1%, toluene from 25.6 to 0.6%, and phenanthrene from 12.93 to 6%. According to the obtained results, the bacterial consortium effectively plays a role in the biodegradation of aromatic and aliphatic hydrocarbons, making it a viable solution for treating hydrocarbon pollutants in various environments.

Production of biosurfactant by salt-resistant Bacillus in lead-supplemented media: application and toxicity.

A group of biosurfactants are lipopeptides that are produced by some microorganisms, especially Bacillus strains. They are new bioactive agents with anticancer, antibacterial, antifungal, and antiviral activities. Also, they are used in sanitation industries. In this study, a lead-resistant strain of Bacillus halotolerans was isolated for lipopeptide production. This isolate exhibited metal resistance (lead, calcium, chromium, nickel, copper, manganese, and mercury), salt tolerance (12%), and antimicrobial activities against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Saccharomyces cerevisiae. The production of lipopeptide was optimized, concentrated, and then extracted from the polyacrylamide gel in a simple way for the first time. The nature of the purified lipopeptide was determined by FTIR, GC/MS, and HPLC analyses. The purified lipopeptide indicated significant antioxidant properties (90.38% at a concentration of 0.8 mg ml-1). Also, it had anticancer activity by apoptosis (flow cytometry analysis) in MCF-7 cells, while it had no cytotoxicity on HEK-293 normal cells. Therefore, Bacillus halotolerans lipopeptide has the potential to be used as an antioxidant, antimicrobial, or anticancer agent in the medical and food industries.

Biosynthesis of Calcite Nanocrystal by a Novel Polyextremophile Bhargavaea cecembensis-Related Strain Isolated from Sandy Soil.

Urease-producing bacteria are abundant in soils, which can precipitate calcium carbonate nanocrystals by enzymatic hydrolysis of urea in the presence of calcium ions. This process is known as microbially induced calcium carbonate precipitation (MICP), and it has received much attention in recent years as an eco-friendly technology. Therefore, the purpose of the present study was to isolate local extremophile bacterial strains capable of producing calcium carbonate. Among a total of 44 isolated urease-producing strains from sandy soils, one strain with a high level of urease activity (8.16 U/ml) and production of a large amount of calcium carbonate (410 mg/100 ml) was selected for further investigation. 16S rRNA gene sequencing showed that this strain had 99.66% sequence identity to Bhargavaea cecembensis. The SEM-EDX and XRD analyses indicated that irregular vaterite and aggregated nanocalcite were the dominant polymorphs produced by this strain. The size of these nanocalcite crystals ranged between 25 and 42 nm. The selected strain showed high levels of tolerance to different conditions of temperature, pH, and salinity. This strain grows at high temperatures up to 50 °C, alkaline pH (9-11), and high concentrations of NaCl (20-25% w/v). Flow cytometry analysis demonstrated 96% cell viability of the isolated strain after desiccation stress. Bhargavaea was first reported in 2009 as a new genus, and it belongs to the Firmicutes. So far, there has been no report on its MICP potential. The present study is the first one to report nanocrystal calcium carbonate precipitation in polyextremophile Bhargavaea cecembensis, which makes it a suitable candidate for bio-cementation under extreme circumstances.

Predatory and biocontrol potency of Bdellovibrio bacteriovorus toward phytopathogenic strains of Pantoea sp. and Xanthomonas campestris in the presence of exo-biopolymers: in vitro and in vivo assessments.

Bdellovibrios are predatory bacteria that invade other live Gram-negative bacterial cells for growth and reproduction. They have recently been considered as potential living antibiotics and biocontrol agents. In this study, the predatory activity and biocontrol potency of Bdellovibrio bacteriovorus strain SOIR-1 against Pantoea sp. strain BCCS and Xanthomonas campestris, two exo-biopolymer-producing phytopathogens, was evaluated. Plaque formation assays and lysis analysis in the broth co-cultures were used for the in vitro evaluation of bacteriolytic activity of strain SOIR-1. The in vivo biocontrol potential of strain SOIR-1 was evaluated by pathogenicity tests on the onion bulbs and potato tuber slices. The phytopathogens were also recovered from the infected plant tissues and confirmed using biochemical tests and PCR-based 16S rRNA gene sequence analysis. Typical bdellovibrios plaques were developed on the lawn cultures of Pantoea sp. BCCS and X. campestris. The killing rate of strain SOIR-1 toward Pantoea sp. BCCS and X. campestris was 84.3% and 76.3%, respectively. Exo-biopolymers attenuated the predation efficiency of strain SOIR-1 up to 10.2-18.2% (Pantoea sp. BCCS) and 12.2-17.3% (X. campestris). The strain SOIR-1 significantly reduced rotting symptoms in the onion bulbs caused by Pantoea sp. BCCS (69.0%) and potato tuber slices caused by X. campestris (73.1%). Although more field assessments are necessary, strain SOIR-1 has the preliminary potential as a biocontrol agent against phytopathogenic Pantoea sp. BCCS and X. campestris, especially in postharvest storage. Due to the particular physicochemical properties of evaluated exo-biopolymers, they can be used in the designing encapsulation systems for delivery of bdellovibrios.

Expression regulation of chitin-binding protein and metal-binding peptide in new Bacillus velezensis: MALDI-TOF MS/MS analysis.

Metallothionein and metal-binding peptides are small cysteine-rich proteins produced by different organisms in stress conditions. In this study, the metal-binding peptide was detected in extracellular proteins of a new Bacillus velezensis strain, isolated from metal contaminated soil, and grown on the lead-enriched medium, for the first time. The presence of sulfide peptide was assayed by two simple tests (lead sulfide and Ellman's reagent test) for preliminary, and subsequently confirmed using polyacrylamide gel electrophoresis at media with different lead concentrations that the low-molecular-weight protein fragments (≈10 kDa) were observed while none were detected in the medium containing sodium chloride or calcium salt. The amino acids of the observed fragments were analyzed by matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF MS/MS). Also, the metal adsorption was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by staining with chromium solution. The results showed that the putative sulfide peptide is metallothionein, which is induced in stress conditions. It was interesting that in all SDS profiles, one protein fragment (≈18 kDa) was inhibited in lead-enriched media. The data from MALDI-TOF MS/MS analysis showed that this fraction was a chitin-binding protein whose production was regulated by metal contamination. It is anticipated that these two proteins regulate the toxicity of lead.

Detection of simultaneous production of kurstakin, fengycin and surfactin lipopeptides in Bacillus mojavensis using a novel gel-based method and MALDI-TOF spectrometry.

Bacterial lipopeptides have become a research focus of many studies owing to their industrial and pharmaceutical importance. Although such studies focused on researching purification procedures and qualitative analysis, much remains to be explored and developed to improve the current methods. To enable thorough studies of lipopeptides, this paper describes a new method for purification and characterization of in-gel anionic lipopeptides. Specifically, lipopeptides attributed to the anti-staphylococcal activity of Bacillus mojavensis HF were separated using SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) and subsequently characterized using mass spectrometry. Lipopeptide band obtained by gel electrophoresis was first visualized using three different staining methods. Next, the lipopeptide isomers were efficiently recovered from the gel band and structural characterization of the extracted lipopeptides was carried out by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). MS analysis revealed that Bacillus mojavensis HF produced three types of lipopeptides including surfactin, fengycin, and kurstakin. 14 clusters of ion peaks were identified as fengycin A with fatty acid of C15-C17, fengycin B (C16, C17), surfactin (C13-C16), and kurstakin (C9-C12). Moreover, tandem mass spectrometric analysis (MS/MS) revealed the sequences of fengycin A and surfactin. In this study, we identified a high variety and number of surfactin and fengycin isomers, which previous reports lacked. To the best of our knowledge, we are the first to report the presence of kurstakin in Bacillus mojavensis species. Finally, we demonstrated that our gel-based study of lipopeptides allowed for a precise and reproducible investigation of these molecules.

Generation of non-toxic, chemical functional bio-polymer for desalination, metal removal and antibacterial activities from animal meat by-product.

The meat industry produces a lot of waste, which contains large amounts of the organics and nutrients. Animal by-products have potential for biomaterial extraction. The use of bio-material, which can be obtained from plant sources, microorganisms, agricultural, and animal waste are nowadays favored because of their compatible, cost-effective, and low-risk for removal of pollutants, compared to chemical and physical methods. In this study, a biopolymer from meat by-product extracted by methanol-chloroform and characterized by FTIR, GC-MS, HPLC, and SDS-PAGE analyzes. The extracted biomaterial was useful in water desalination by calcium carbonate precipitation and heavy metals removal, which was confirmed by FTIR and ICP analyzes. The extracted biomaterial also has antibacterial properties against Pseudomonas aeruginosa and Escherichia coli without toxicity to human blood cells, which can make it useful in industries such as its application in fish ponds.

Inhibition of quorum sensing-controlled virulence factors with natural substances and novel protease, obtained from Halobacillus karajensis.

INTRODUCTION: In recent years, a challenge in clinical treatment has developed due to bacterial resistance to antibiotics. One of the new mechanisms against infections is virulence factor inhibition. Many virulence factors are controlled by quorum sensing pathways such as biofilm formation and pyocyanin production. The goal of the present study was to investigate the effect of an obligate halophilic bacterial strain on Pseudomonas aeruginosa and Staphylococcus aureus, due to its halo-tolerant substances and enzymes. METHODS: The effect of Halobacillus karajensis on bacterial growth and production of virulence factors was studied in this work. The obligate halophile cells and supernatant fractions were extracted by the methanol/chloroform method and characterized by Fourier Transform Infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Gas Chromatography-Mass Spectrometry (GC-MS), and zymography. The effects of these fractions were studied on biofilm formation in P. aeruginosa and S. aureus as well as on pyocyanin production in P. aeruginosa. The effective protein in the fraction was analyzed by the SDS-PAGE method, and all protein fragments were studied for pyocyanin inhibition. RESULTS: The crude supernatant extract, MMS fraction, from H. karajensis was effective for the biofilm reduction in S. aureus (74%) and P. aeruginosa (27%). Two proteases in this fraction, which were recognized by zymography on skim milk, were the probable causes for extracellular polymeric substances (EPS) hydrolysis in the biofilm matrix. Also, halide crystals and branched fatty acids, 12methyl-tetradecanoic acid, in the other fractions decreased the biofilm by 18% in S. aureus. The results showed that a new 25 kD protein, which was obtained from MMS fraction, inhibited pyocyanin production by 60% in P. aeruginosa. The zymogram and bioinformatics studies showed that this protein was a serine alkaline metalloprotease and had an interaction with AHL molecules. CONCLUSION: The inhibitory effects of the non-toxic natural substances and proteases on biofilm formation and pyocyanin production, specifically the 25 kD protease, are novel in this study and make them a good candidate for infected wound healing and inhibiting the virulence factors.

Chitooligosaccharides and Thermostable Chitinase Against Vulvovaginal Candidiasis and Saprophyte Fungi: LC Mass Studies of Shrimp Shell Fermentation by Bacillus altitudinis.

Some patients with candidiasis seek alternatives drug to treat vaginal yeast infection like herbal preparations and probiotics. However, the effectiveness of such treatments has not received much study. In this research, the unique chitinotrophic Bacillus was isolated on shrimp shell from salt lakes and identified as Bacillus altitudinis by 16SRNA sequencing. This strain produced a novel chitin-oligosaccharide material and thermostable chitinase (5.1 units/ml) during 4 days incubation on shrimp shell medium; nevertheless, its growth on nutrient agar was negative. The zymogram showed less than 50 kD protein responsible for chitinase activities. The LC/MS detection of concentrate fermented products showed the production of oligosaccharide during chitin fermentation. As results of shrimp shell degradation, 65.6 mg/l protein, 73.4 mg/l N-acetyl glucose amine, and oligosaccharide were produced. Synergism activities of chitooligosaccharide and chitinase from this strain against fungi and pathogen candida (staining with methylene blue showed that almost 50% of 106 cells were died during 6 h) are promising for new anti-fungal drug with no side effect.

MALDI-TOF analysis of a novel extremophile peptide purified from Halarchaeum acidiphilum ASDL78 with antiarchaeal and antibacterial activities.

In hypersaline environments, halophilic archaea synthesize antimicrobial substances called halocins. There is a promise to make new drugs for antibiotic-resistant strains. Here, we report the antibacterial activity of a new haloarchaea selected from Lut Desert, Iran. A total of 38 isolated halophilic bacteria and archaea were screened for the antagonistic activity test of each strain against other bacterial and archaeal strains. Finally, a strain, recognized as Halarchaeum acidiphilum, with a fast grown strain and high antagonistic potential against different strains was identified by morphological, physiological, and molecular characteristics. The halocin was produced in a semisolid submerge medium and partially purified by heat treatments and molecular weight ultrafiltration cutoff (3, 50, and 10 kDa). It was a cell-free, heat-resistant (85°C for 2 h) protein with a molecular mass near to 20 kDa produced at the endpoint of logarithmic growth. The molecular weight of halocin was 17 kDa, and indicated no apparent homology with known halocins, suggesting that this might be a new halocin. Therefore, a new strain belonging to Halarchaeum genus was isolated and characterized here that produced an antimicrobial and anti-haloarchaea halocin.

Production of glycolipid biosurfactant during crude oil degradation by the novel indigenous isolated Achromobacter kerstersii LMG3441.

This study aimed to find biosurfactant producing and crude oil-degrading bacteria able to decontaminate crude oil from wastewater. The bacteria that were isolated from contaminated sites in an oil refinery plant in Isfahan, Iran, were identified by 16S rDNA sequencing as Achromobacter kerstersii strain LMG3441, Klebsiella pneumonia strain SKBA6, and Klebsiella variicola strain SKV2. According to the results obtained from different tests for the production of biosurfactant among three strains, only Achromobacter kerstersii strain LMG3441 was selected for further study. The pattern of residual hydrocarbons was analyzed by high-resolution gas chromatography-mass spectrometry (GC-MS). This novel and indigenous strain was capable of producing the highest amount of a glycolipid biosurfactant (7.81 g/L) in MSM (mineral salt medium) with 1% (v/v) crude oil as the only source of carbon and energy. The compound showed high surface activation capacity with reduction of surface tension from 40 mN m-1 in the control to 23.3 mN m-1 by the bacterium. The results of GC-MS for assessment of residual hydrocarbons in the MSM and comparison with crude oil as a control showed that 53% of the hydrocarbons in the crude oil were consumed by this novel strain.

Molecular investigation of two novel bacteriophages of a facultative methylotroph, Raoultella ornithinolytica: first report of Raoultella phages.

Bacteria of the genus Raoultella are known to inhabit aquatic environments and can be found in medical samples. The pathogenicity of Raoultella ornithinolytica isolates in human has become increasingly important, and several cases of infections have been reported recently. However, there are no reports of isolation of bacteriophages infecting this bacterium. In this study, two novel phages (ISF3 and ISF6) of a methylotrophic Raoultella strain were isolated from sewage. To characterize the isolated phages, morphological features, protein profiles, restriction digestion patterns, and partial genome sequences were studied. Despite morphological differences, electron microscopy revealed that both phages had an icosahedral capsid connected to a contractile tail, suggesting that ISF3 and ISF6 both belong to the family Myoviridae. Partial nucleotide sequences of the ISF3 genome showed 99% to 100% identity to DNA of Klebsiella pneumonia phages KP15, KP27 and BMBT1; however, the restriction digestion profiles of ISF3 genome digested by EcoRI and EcoRV differed from those of Klebsiella phages KP15 and KP27. A partial sequence alignment showed that ISF6 can be classified as a member of a new viral genus due to its significant differences from other previously identified phages. To the best of our knowledge, this is the first report of the isolation and characterization of the specific Raoultella phages that have potential to be used as new pharmaceuticals against R. ornithinolytica.

LC/MS detection of oligogalacturonic acids obtained from tragacanth degradation by pectinase producing bacteria.

Tragacanth, a highly branched carbohydrate polymer isolated from Astragalus, is one of the most commonly used gums in food industry. The primary structure of tragacanth is composed of galacturonic acid monomers connected with α 1-4 links, and it is very similar to the pectin. Tragacanth degradation by microorganisms is significant in two aspects: first, food preservation and microbial growth control due to too much use of tragacanth in the food industry, second, therapeutic and pharmaceutical potential of obtained oligosaccharides. In the present study, we report three new strains of bacteria, Acinetobacter guillouiae strain TD1, Kosakonia sacchari strain TD2, and Bacillus vallismortis strain PD1 with the capability of growing in tragacanth as an only source of carbon and energy. The evolutionary history of the isolated strains was analyzed based on 16S rRNA gene sequences in MEGA7 using the neighbor-joining method. The production of di and tri galacturonic acid due to pectinase activities of the strains were detected by thin layer chromatography (TLC) and liquid chromatography/Mass spectroscopy (LC/MS) analysis. Here is the first report of the ability to grow in tragacanth and pectinase activity monitoring in bacteria. Our results revealed that all of the isolated strains are capable of degrading pectin and tragacanth to oligo-galacturonic acids. The obtained products, which have different structures depending on the tragacanth structures and types of pectinolytic enzymes, would show therapeutic and pharmaceutical potentials.

Bioremediation of chromium contaminated water by diatoms with concomitant lipid accumulation for biofuel production.

Hexavalent chromium compounds such as chromate and dichromate, commonly designated as Cr (VI) compounds, are widely used heavy metals in different industries and are considered highly toxic to most life forms. Unfortunately, they have become a major pollutant of groundwater and rivers around dichromate using industries. Bioremediation is widely used to decrease the amount of dichromate in wastewater but requires large amounts of precious fresh water. Here we tested two marine micro-algal species, Phaeodactylum tricornutum strain CCY0033 and Navicula pelliculosa strain CCMP543, for their ability of dichromate bioremediation and concomitantly producing lipids that can serve as biofuel. Dichromate tolerance of the strains was investigated under different growth conditions in order to obtain high biomass yields, high lipid accumulation and high dichromate removal from the medium. Both algal strains grew well and produced high biomass in media containing up to 1 mg of dichromate per liter. Variations in growth conditions revealed that dichromate removal from the medium correlated positively with biomass yield. Dichromate removal using living cells was in the same order of magnitude as with autoclaved dead cells or when using extracted extracellular polymeric substances (EPS). This suggests biosorption of dichromate to cell-associated polymeric substances as the major mechanism of the bioremediation process. For both strains, optimal dichromate removal and lipid production were achieved at a light intensity of 55 µmol m-2s-1 and at a sodium nitrate concentration of 3 mM. The optimal temperature for dichromate removal and lipid production was 23 °C for P. tricornutum and 27 °C for N. pelliculosa. Compared to P. tricornutum strain CCY0033, N. pelliculosa strain CCMP543 produced an overall higher lipid yield under these conditions.

Natronoarchaeum persicum sp. nov., a haloarchaeon isolated from a hypersaline lake.

A novel halophilic archaeon, designated strain WIIAL99T, was isolated from Lake Meyghan, a hypersaline lake in Iran. Cells of strain WIIAL99T were non-motile, catalase-positive and oxidase-negative. Strain WIIAL99T required at least 2.5 M NaCl and 0.05 M MgCl2 for growth. Optimal growth was achieved at 3.5 M NaCl and 0.1 M MgCl2. The optimum pH and temperature for growth were pH 7.0 and 37-40 °C; it was able to grow at pH 6.0-8.5 and 20-55 °C. Cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was 8 % (w/v). The major polar lipids of strain WIIAL99T were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, disulfated diglycosyl diether and one unidentified glycolipid. The DNA G+C content of strain WIIAL99T was 66.7 mol%. The closest relative was Natronoarchaeum rubrum JCM 17119T with 98.2 % similarity in the orthologous 16S rRNA gene sequence. Analysis of 16S rRNA and rpoB' gene sequences indicated that strain WIIAL99T is a member of the genus Natronoarchaeum in the family Halobacteriaceae and forms a distinct cluster. It was concluded that strain WIIAL99T (=IBRC-M 11062T=LMG 29814T) represents a novel species of the genus Natronoarchaeum, for which the name Natronoarchaeum persicum sp. nov. is proposed.

Heterotrophic Bioleaching of Sulfur, Iron, and Silicon Impurities from Coal by Fusarium oxysporum FE and Exophiala spinifera FM with Growing and Resting Cells.

Coal is the most abundant fossil fuel containing sulfur and other elements which promote environmental pollution after burning. Also the silicon impurities make the transportation of coal expensive. In this research, two isolated fungi from oil contaminated soil with accessory number KF554100 (Fusarium oxysporum FE) and KC925672 (Exophiala spinifera FM) were used for heterotrophic biological leaching of coal. The leaching were detected by FTIR, CHNS, XRF analyzer and compared with iron and sulfate released in the supernatant. The results showed that E. spinifera FM produced more acidic metabolites in growing cells, promoting the iron and sulfate ions removal while resting cells of F. oxysporum FE enhanced the removal of aromatic sulfur. XRF analysis showed that the resting cells of E. spinifera FM proceeded maximum leaching for iron and silicon (48.8, 43.2 %, respectively). CHNS analysis demonstrated that 34.21 % of sulfur leaching was due to the activities of resting cells of F. oxysporum FE. Also F. oxysporum FE removed organic sulfur more than E. spinifera FM in both growing and resting cells. FTIR data showed that both fungi had the ability to remove pyrite and quartz from coal. These data indicated that inoculations of these fungi to the coal are cheap and impurity removals were faster than autotrophic bacteria. Also due to the removal of dibenzothiophene, pyrite, and quartz, we speculated that they are excellent candidates for bioleaching of coal, oil, and gas.

Tolerance Induction of Temperature and Starvation with Tricalcium Phosphate on Preservation and Sporulation in Bacillus amyloliquefaciens Detected by Flow Cytometry.

The Bacillus species have many applications in the preparation of various enzymes, probiotic, biofertilizer, and biomarkers for which the survival of resting cells and spore formation under different conditions are important. In this study, water and saline along with different mineral substances such as calcium carbonate, calcium phosphate, and silica were used for the detection of survival and preservation of Bacillus amyloliquefaciens. The results showed intensive death of resting cells at 8 °C, but significant survival at 28 °C after one month. However, preservation by minerals significantly decreased the rate of death and induced sporulation at both the temperatures. The resting cells were maintained at room temperature (about 60 % of the initial population survived after a month) in the presence of tricalcium phosphate. The results showed that temperature has more effect on sporulation compare with starvation. The sporulation in normal saline at 28 °C was 70 times more than that at 8 °C; meanwhile, addition of tricalcium phosphate increases sporulation by 90 times. Also, the FTIR data showed the interaction of tricalcium phosphate with spores and resting cells. The discrimination of sporulation from non-sporulation state was performed by nucleic acid staining with thiazole orange and detected by flow cytometry. The flow cytometric studies confirmed that the rates of sporulation in pure water were significantly more at 28 °C. This is the first report on the detection of bacterial spore with thiazole orange by flow cytometry and also on the interaction of tricalcium phosphate with spores by FTIR analyses.

Enhancement of production and activity of alkaline zinc metalloprotease from Salinivibrio proteolyticus using low intensity direct electric current and zinc nanoparticles.

OBJECTIVES: To improve the production and activity of an alkaline zinc metalloprotease from Salinivibrio proteolyticus in response to ZnSO4 (ionic and nanoparticle forms) and low intensity direct electric current (LIDC). RESULTS: A DC of 50 µA for 10 min increased enzyme production from 35 to 53 U ml(-1) when applied to the stationary phase bacterial cells. Zn(2+) improved enzyme production better than zinc nanoparticles (52 vs. 43.5 U ml(-1)). Zinc nanoparticles (0.5 mM) added to an enzyme reaction mixture containing casein (0.65 %) and 20 mM Tris/HCl buffer (pH 8) improved enzyme activity more than Zn(2+) (42 vs. 36 U ml(-1)). CONCLUSION: LIDC exposure (50 µA, 10 min) to the stationary phase bacterial cells increases metalloprotease production in Salinivibrio. A low concentration of zinc nanoparticles (0.5 mM) increases maximum enzyme activity.

Surface display of bacterial tyrosinase on spores of Bacillus subtilis using CotE as an anchor protein.

Tyrosinases, copper-containing monooxygenases, are widely used enzymes for industrial, medical, and environmental applications. We report the first functional surface display of Bacillus megaterium tyrosinase on Bacillus subtilis spores using CotE as an anchor protein. Flow Cytometry was used to verify surface expression of tyrosinase on the purified spores. Moreover, tyrosinase activity of the displayed enzyme on B. subtilis spores was monitored in the presence of L-tyrosine (substrate) and CuSO4 (inducer). The stability of the spore-displayed tyrosinase was then evaluated after 15 days maintenance of the spores at room temperature, and no significant decrease in the enzyme activity was observed. In addition, the tyrosinase-expressing spores could be repeatedly used with 62% retained enzymatic activity after six times washing with Tris-HCl buffer. This genetically immobilized tyrosinase on the spores would make a new advance in industrial, medical, and environmental applications.