A new strategy for the treatment of middle ear infection using ciprofloxacin/amoxicillin-loaded ethyl cellulose/polyhydroxybutyrate nanofibers.

A middle ear infection occurs due to the presence of several microorganisms behind the eardrum (tympanic membrane) and is very challenging to treat due to its unique location and requires a well-designed treatment. If not treated properly, the infection can result in severe symptoms and unavoidable side effects. In this study, excellent biocompatible ethyl cellulose (EC) and biodegradable polyhydroxybutyrate (PHB) biopolymer were used to fabricate drug-loaded nanofiber scaffolds using an electrospinning technique to overcome antibiotic overdose and insufficient efficacy of drug release during treatment. PHB polymer was produced from Halomonas sp., and the purity of PHB was found to around be 90 %. Additionally, ciprofloxacin (CIP) and amoxicillin (AMX) are highly preferable since both drugs are highly effective against gram-negative and gram-positive bacteria to treat several infections. Obtained smooth nanofibers were between 116.24 and 171.82 nm in diameter and the addition of PHB polymer and antibiotics improved the morphology of the nanofiber scaffolds. Thermal properties of the nanofiber scaffolds were tested and the highest Tg temperature resulted at 229 °C. The mechanical properties of the scaffolds were tested, and the highest tensile strength resulted in 4.65 ± 6.33 MPa. Also, drug-loaded scaffolds were treated against the most common microorganisms that cause the infection, such as S.aureus, E.coli, and P.aeruginosa, and resulted in inhibition zones between 10 and 21 mm. MTT assay was performed by culturing human adipose-derived mesenchymal stem cells (hAD MSCs) on the scaffolds. The morphology of the hAD MSCs' attachment was tested with SEM analysis and hAD MSCs were able to attach, spread, and live on each scaffold even on the day of 7. The cumulative drug release kinetics of CIP and AMX from drug-loaded scaffolds were analysed in phosphate-buffered saline (pH: 7.4) within different time intervals of up to 14 days using a UV spectrophotometer. Furthermore, the drug release showed that the First-Order and Korsmeyer-Peppas models were the most suitable kinetic models. Animal testing was performed on SD rats, matrix and collagen deposition occurred on days 5 and 10, which were observed using Hematoxylin-eosin and Masson's trichrome staining. At the highest drug concentration, a better repair effect was observed. Results were promising and showed potential for novel treatment.

A two-step purification platform for efficient removal of Fab-related impurities: A case study for Ranibizumab.

Antibodies (mAbs) and antibody fragments (Fabs) constitute one of the largest and most rapidly expanding groups of protein pharmaceuticals. In particular, antibody fragments have certain advantages over mAbs in some therapeutic settings. However, due to their greater chemical diversity, they are more challenging to purify for large-scale production using a standard purification platform. Besides, the removal of Fab-related byproducts poses a difficult purification challenge. Alternative Fab purification platforms could expedite their commercialization and reduce the cost and time invested. Accordingly, we employed a strong cation exchanger using a pH-based, highly linear gradient elution mode following Protein L affinity purification and developed a robust two-step purification platform for an antibody fragment. The optimized pH gradient elution conditions were determined on the basis of purity level, yield, and the abundance of Fab-related impurities, particularly free light chain. The purified Fab molecule Ranibizumab possessed a high degree of similarity to its originator Lucentis. The developed purification platform highly intensified the process and provided successful clearance of formulated Fab- and process-related impurities (∼98 %) with an overall process recovery of 50 % and, thus, might be a new option for Fab purification for both academic and industrial purposes.

Investigation of the Physiology of the Obligate Alkaliphilic Bacillus marmarensis GMBE 72T Considering Its Alkaline Adaptation Mechanism for Poly(3-hydroxybutyrate) Synthesis.

The novel extreme obligate alkaliphilic Bacillus marmarensis DSM 21297 is known to produce polyhydroxybutyrate (PHB). However, the detailed mechanism of PHB synthesis in B. marmarensis is still unknown. Here, we investigated which metabolic pathways and metabolic enzymes are responsible for PHB synthesis in order to understand the regulatory pathway and optimize PHB synthesis in B. marmarensis. In accordance with the fact that beta-galactosidase, 3-hydroxyacyl-CoA dehydrogenase, and Enoyl-CoA hydratase together with acyl-CoA dehydrogenase and lipase were annotated in B. marmarensis according to the RAST server, we used glucose, lactose, and olive oil to understand the preferred metabolic pathway for the PHB synthesis. It was found that B. marmarensis produces PHB from glucose, lactose, and olive oil. However, the highest PHB titer and the highest amount of PHB synthesized per dry cell mass (YP/X) were achieved in the presence of lactose, as compared to glucose and olive oil. Additionally, in the absence of peptone, the amount of PHB synthesized is reduced for each carbon source. Interestingly, none of the carbon sources studied yielded an efficient PHB synthesis, and supplementation of the medium with potassium ions did not enhance PHB synthesis. According to these experimental results and the presence of annotated metabolic enzymes based on the RAST server, PHB accumulation in the cells of B. marmarensis could be improved by the level of the expression of 3-hydroxybutyryl-CoA dehydrogenase (1.1.1.157), which increases the production of NADPH. Additionally, the accumulation of 3-hydroxyacyl-CoA could enhance the production of PHB in B. marmarensis in the presence of fatty acids. To our knowledge, this is the first report investigating the regulatory system involved in the control of PHB metabolism of B. marmarensis.

Glioblastoma cell adhesion properties through bacterial cellulose nanocrystals in polycaprolactone/gelatin electrospun nanofibers.

Glioblastoma (GBM), the most common and extremely lethal type of brain tumor, is resistant to treatment and shows high recurrence rates. In the last decades, it is indicated that standard two-dimensional (2D) cell culture is inadequate to improve new therapeutic strategies and drug development. Hence, well-mimicked three-dimensional (3D) tumor platforms are needed to bridge the gap between in vitro and in vivo cancer models. In this study, bacterial cellulose nano-crystal (BCNC) containing polycaprolactone (PCL) /gelatin (Gel) nanofibrous composite scaffolds were successfully fabricated by electrospinning for mimicking the extracellular matrix of GBM tumor. The fiber diameters in the nanofibrous matrix were increased with an increased concentration of BCNC. Moreover, fiber morphology changed from the smooth formation to the beaded formation by increasing the concentration of the BCNC suspension. In-vitro biocompatibilities of nanofibrous scaffolds were tested with U251 MG glioblastoma cells and improved cell adhesion and proliferation was compared with PCL/Gel. PCL/Gel/BCNC were found suitable for enhancing axon growth and elongation supporting communication between tumor cells and the microenvironment, triggering the process of tumor recurrence. Based on these results, PCL/Gel/BCNC composite scaffolds are a good candidate for biomimetic GBM tumor platform.

What Are the Multi-Omics Mechanisms for Adaptation by Microorganisms to High Alkalinity? A Transcriptomic and Proteomic Study of a Bacillus Strain with Industrial Potential.

Alkaliphilic organisms are among an industrially important class of extremophile microorganisms with the ability to thrive at pH 10-11.5. Microorganisms that exhibit alkaliphilic characteristics are sources of alkali-tolerant enzymes such as proteases, starch degrading enzymes, cellulases, and metabolites such as antibiotics, enzyme inhibitors, siderophores, organic acids, and cholic acid derivatives, which have found various applications in industry for human and environmental health. Yet, multi-omics mechanisms governing adaptation to high alkalinity have been poorly studied. We undertook the present work to understand, as a case study, the alkaliphilic adaptation strategy of the novel microorganism, Bacillus marmarensis DSM 21297, to alkaline conditions using a multi-omics approach that employed transcriptomics and proteomics. As alkalinity increased, bacteria remodeled the peptidoglycan layer by changing peptide moieties along with the peptidoglycan constituents and altered the cell membrane to reduce lipid motility and proton leakiness to adjust intracellular pH. Different transporters also contributed to the maintenance of this pH homeostasis. However, unlike in most well-known alkaliphiles, not only sodium ions but also potassium ions were involved in this process. Interestingly, increased pH has triggered the expression of neither general stress proteins nor gene encoding proteins associated with heat, salt, and nutrient stresses. Only an increase in the expression of oxidative stress related genes was evident. Endospore formation, also a phenomenon closely linked to stress, was unclear. This questioned if high pH was a real stress for B. marmarensis. These new findings, corroborated using the multi-omics approach of the present case study, broaden the knowledge on the mechanisms of alkaliphilic adaptation and might also potentially offer useful departure points for further industrial applications with other microorganisms.

Assessment of poly(3-hydroxybutyrate) synthesis from a novel obligate alkaliphilic Bacillus marmarensis and generation of its composite scaffold via electrospinning.

In this study, poly(3-hydroxybutyrate) (PHB) production from a newly isolated obligate alkaliphilic Bacillus marmarensis DSM 21297 was investigated to evaluate the ability of obligate alkaliphilic strain to produce a biopolymer. Additionally, electrospun nanofibers from B. marmarensis PHB (Bm-PHB) were generated using Bm-PHB/polycaprolactone (PCL) blend to evaluate the applicability of Bm-PHB. According to the experimental results, the metabolic activity of B. marmarensis decreased the pH of the medium by generating H+ ions to initiate Bm-PHB production, which was achieved at pH below 9.0. Regarding medium components, the addition of MgSO4.7H2O and KH2PO4 to the medium containing 1% glucose enhanced the amount of Bm-PHB synthesis, and an approximately 60% increase in PHB concentration was obtained in the presence of mineral salts. Based on FTIR analysis, the chemical structures of Bm-PHB and commercial PHB were found to be highly similar. Additionally, the Tg and Tm values of Bm-PHB were determined to be 17.77 °C and 165.17 °C, respectively. Moreover, Bm-PHB/PCL composite scaffold was generated by electrospinning method that produced nanofibers between 150 and 400 nm in diameter, with an average of 250 nm. To our knowledge, this is the first report to produce PHB from an obligate alkaliphilic Bacillus strain and PHB scaffold.

Assessment of hazelnut husk as a lignocellulosic feedstock for the production of fermentable sugars and lignocellulolytic enzymes.

The present work focuses firstly on the evaluation of the effect of laccase on enzymatic hydrolysis of hazelnut husk which is one of the most abundant lignocellulosic agricultural residues generated in Turkey. In this respect, the co-enzymatic treatment of hazelnut husk by cellulase and laccase, without a conventional pretreatment step is evaluated. Using 2.75 FPU/g substrate (40 g/L substrate) and a ratio of 131 laccase U/FPU achieved the highest reducing sugars concentration. Gas chromatography mass spectrometry confirmed that the hydrolysate was composed of glucose, xylose, mannose, arabinose and galactose. The inclusion of laccase in the enzyme mixture [carboxymethyl cellulase (CMCase) and ß-glucosidase] increased the final glucose content of the reducing sugars from 20 to 50%. Therefore, a very significant increase in glucose content of the final reducing sugars concentration was obtained by laccase addition. Furthermore, the production of cellulases and laccase by Pycnoporus sanguineus DSM 3024 using hazelnut husk as substrate was also investigated. Among the hazelnut husk concentrations tested (1.5, 6, 12, 18 g/L), the highest CMCase concentration was obtained using 12 g/L husk concentration on the 10th day of fermentation. Besides CMCase, P. sanguineus DSM 3024 produced ß-glucosidase and laccase using hazelnut husk as carbon source. In addition to CMCase and ß-glucosidase, the highest laccase activity measured was 2240 ± 98 U/L (8.89 ± 0.39 U/mg). To the best of our knowledge, this is the first study to report hazelnut husk hydrolysis in the absence of pretreatment procedures.

Transcriptomic analysis displays the effect of (-)-roemerine on the motility and nutrient uptake in Escherichia coli.

Among the different families of plant alkaloids, (-)-roemerine, an aporphine type, was recently shown to possess significant antibacterial activity in Escherichia coli. Based on the increasing demand for antibacterials with novel mechanisms of action, the present work investigates the potential of the plant-derived alkaloid (-)-roemerine as an antibacterial in E. coli cells using microarray technology. Analysis of the genome-wide transcriptional reprogramming in cells after 60 min treatment with 100 µg/mL (-)-roemerine showed significant changes in the expression of 241 genes (p value <0.05 and fold change >2). Expression of selected genes was confirmed by qPCR. Differentially expressed genes were classified into functional categories to map biological processes and molecular pathways involved. Cellular activities with roles in carbohydrate transport and metabolism, energy production and conversion, lipid transport and metabolism, amino acid transport and metabolism, two-component signaling systems, and cell motility (in particular, the flagellar organization and motility) were among metabolic processes altered in the presence of (-)-roemerine. The down-regulation of the outer membrane proteins probably led to a decrease in carbohydrate uptake rate, which in turn results in nutrient limitation. Consequently, energy metabolism is slowed down. Interestingly, the majority of the expressional alterations were found in the flagellar system. This suggested reduction in motility and loss in the ability to form biofilms, thus affecting protection of E. coli against host cell defense mechanisms. In summary, our findings suggest that the antimicrobial action of (-)-roemerine in E. coli is linked to disturbances in motility and nutrient uptake.

Response of Escherichia coli to Prolonged Berberine Exposure.

Berberine is a plant-derived alkaloid possessing antimicrobial activity; unfortunately, its efflux through multidrug resistance pumps reduces its efficacy. Cellular life span of Escherichia coli is generally shorter with prolonged berberine exposure; nevertheless, about 30% of the cells still remain robust following this treatment. To elucidate its mechanism of action and to identify proteins that could be involved in development of antimicrobial resistance, protein profiles of E. coli cells treated with berberine for 4.5 and 8 hours were compared with control cells. A total of 42 proteins were differentially expressed in cells treated with berberine for 8 hours when compared to control cells. In both 4.5 and 8 hours of berberine-treated cells, carbohydrate and peptide uptake regimens remained unchanged, although amino acid maintenance regimen switched from transport to synthesis. Defect in cell division persisted and this condition was confirmed by images obtained from scanning electron microscopy. Universal stress proteins were not involved in stress response. The significant increase in the abundance of elongation factors could suggest the involvement of these proteins in protection by exhibiting chaperone activities. Furthermore, the involvement of the outer membrane protein OmpW could receive special attention as a protein involved in response to antimicrobial agents, since the expression of only this porin protein was upregulated after 8 hours of exposure.

Proteomic Analysis of Kidney Preservation Solutions Prior to Renal Transplantation.

One of the main issues in kidney transplantation is the optimal functional preservation of the organ until its transplantation into the appropriate recipient. Despite intensive efforts, the functional preservation period remains limited to hours. During this time, as a result of cellular injury, various proteins, peptides, and other molecules are released by the organ into the preservation medium. In this study, we used proteomic techniques to analyze the protein profiles of preservation solutions in which organs had been preserved prior to their transplantation. Samples were obtained from the preservation solutions of 25 deceased donor kidneys scheduled for transplantation. The protein profiles of the solutions were analyzed using 2D gel electrophoresis/MALDI-TOF and LC-MS/MS. We identified and quantified 206 proteins and peptides belonging to 139 different groups. Of these, 111 proteins groups were belonging to kidney tissues. This study used proteomic techniques to analyze the protein profiles of organ preservation solutions. These findings will contribute to the development of improved preservation solutions to effectively protect organs for transplantation.

Metabolic Biomarkers and Neurodegeneration: A Pathway Enrichment Analysis of Alzheimer's Disease, Parkinson's Disease, and Amyotrophic Lateral Sclerosis.

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS) lack robust diagnostics and prognostic biomarkers. Metabolomics is a postgenomics field that offers fresh insights for biomarkers of common complex as well as rare diseases. Using data on metabolite-disease associations published in the previous decade (2006-2016) in PubMed, ScienceDirect, Scopus, and Web of Science, we identified 101 metabolites as putative biomarkers for these three neurodegenerative diseases. Notably, uric acid, choline, creatine, L-glutamine, alanine, creatinine, and N-acetyl-L-aspartate were the shared metabolite signatures among the three diseases. The disease-metabolite-pathway associations pointed out the importance of membrane transport (through ATP binding cassette transporters), particularly of arginine and proline amino acids in all three neurodegenerative diseases. When disease-specific and common metabolic pathways were queried by using the pathway enrichment analyses, we found that alanine, aspartate, glutamate, and purine metabolism might act as alternative pathways to overcome inadequate glucose supply and energy crisis in neurodegeneration. These observations underscore the importance of metabolite-based biomarker research in deciphering the elusive pathophysiology of neurodegenerative diseases. Future research investments in metabolomics of complex diseases might provide new insights on AD, PD, and ALS that continue to place a significant burden on global health.

Pyrolysis kinetics and thermal characteristics of microalgae Nannochloropsis oculata and Tetraselmis sp.

In this study non-isothermal thermogravimetric analysis were used to investigate pyrolysis behavior and kinetics of microalgae Nannochloropsis oculata (NO) and Tetraselmis sp. (TS). TG/DTG experiments at different heating rates were carried out. Heating rates had slight effect on the decomposition trend, however the maximum temperature and peak of weight loss rate in the DTG curves shifted towards higher temperature with the increase in heating rate. The average activation energy and pre-exponential factor for pyrolysis of NO and TS were estimated by distributed activation energy model. The highest activation energies were observed as 152.20 and 334kJ/mol for NO and TS, respectively, at various conversions. The pre-exponential factors for the corresponding activation energies were observed to be in the order of 10(8)-10(13) and 10(12)-10(25)s(-1) for NO and TS, respectively. Calculated kinetic parameters were used to predict devolatilization curves and results were in well agreement with experimental data.

Schizosaccharomyces pombe and its Ni(II)-insensitive mutant GA1 in Ni(II) uptake from aqueous solutions: a biodynamic model.

In the present study, Ni(II) uptake from aqueous solution by living cells of the Schizosaccharomyces pombe haploid 972 with h (-) mating type and a Ni(II)-insensitive mutant GA1 derived from 972 was investigated at various initial glucose and Ni(II) concentrations. A biodynamic model was developed to predict the unsteady and steady-state phases of the uptake process. Gompertz growth and uptake process parameters were optimized to predict the maximum growth rate µ m and the process metric C r, the remaining Ni(II) content in the aqueous solution. The simulated overall metal uptake values were found to be in acceptable agreement with experimental results. The model validation was done through regression statistics and uncertainty and sensitivity analyses. To gain insight into the phenomenon of Ni(II) uptake by wild-type and mutant S. pombe, probable active and passive metal transport mechanisms in yeast cells were discussed in view of the simulation results. The present work revealed the potential of mutant GA1 to remove Ni(II) cations from aqueous media. The results obtained provided new insights for understanding the combined effect of biosorption and bioaccumulation processes for metal removal and offered a possibility for the use of growing mutant S. pombe cell in bioremediation.

Water miscible mono alcohols' effect on the proteolytic performance of Bacillus clausii serine alkaline protease.

In this study, our investigations showed that the increasing concentrations of all examined mono alcohols caused a decrease in the Vm, kcat and kcat/Km values of Bacillus clausii GMBE 42 serine alkaline protease for casein hydrolysis. However, the Km value of the enzyme remained almost the same, which was an indicator of non-competitive inhibition. Whereas inhibition by methanol was partial non-competitive, inhibition by the rest of the alcohols tested was simple non-competitive. The inhibition constants (KI) were in the range of 1.32-3.10 M, and the order of the inhibitory effect was 1-propanol>2-propanol>methanol>ethanol. The ΔG(≠) and ΔG(≠)E-T values of the enzyme increased at increasing concentrations of all alcohols examined, but the ΔG(≠)ES value of the enzyme remained almost the same. The constant Km and ΔG(≠)ES values in the presence and absence of mono alcohols indicated the existence of different binding sites for mono alcohols and casein on enzyme the molecule. The kcat of the enzyme decreased linearly by increasing log P and decreasing dielectric constant (D) values, but the ΔG(≠) and ΔG(≠)E-T values of the enzyme increased by increasing log P and decreasing D values of the reaction medium containing mono alcohols.

Halomonas smyrnensis sp. nov., a moderately halophilic, exopolysaccharide-producing bacterium.

Four Gram-negative, moderately halophilic, exopolysaccharide-producing strains, designated AAD6(T), AAD4, AAD17 and AAD21, were isolated from Çamalti Saltern Area, a wildlife reserve in Sasali, Izmir province located in the Aegean Region of Turkey. The isolates grew at an optimum NaCl concentration of 10% (w/v). The major cellular fatty acids were C(16:0), C(18:1)ω7c, C(16:1)ω7c and C(12:0) 3OH, respectively and the predominant lipoquinone was ubiquinone Q-9. The G+C content of the genomic DNA of strains AAD6(T), AAD4, AAD17 and AAD21 was 63.0, 63.3, 62.8 and 62.6 mol %, respectively. Comparative 16S rRNA gene sequence studies showed that the isolates belonged to the genus Halomonas. The DNA-DNA hybridization mean values between the representative strain AAD6(T) and the closely related species Halomonas salina DSM 5928(T), Halomonas halophila DSM 4770(T), Halomonas maura DSM 13445(T), Halomonas organivorans DSM 16226(T), Halomonas elongata DSM 2581(T), Halomonas koreensis JCM 12237(T) and Halomonas nitroreducens LMG 24185, were 40.8, 39.6, 24.2, 23.3, 12.6, 14.5 and 12.2%, respectively. Based on these data the strains represent a novel species of the genus Halomonas for which the name Halomonas smyrnensis sp. nov. is proposed. The type strain is AAD6(T) (= DSM 21644(T) = JCM 15723(T)).

Interplay of adaptive capabilities of Halomonas sp. AAD12 under salt stress.

In the present study, osmoadaptive mechanism of Halomonas sp. AAD12 was studied through analysis of changes in its proteome maps and osmolyte accumulation strategy to understand how this euryhaline microorganism masters osmotic stress of saline environments. Under salt stress, there were significant variations in the expression of proteins involved in osmoregulation, stress response, energy generation and transport. This was accompanied by an increase in proline and hydroxyectoine but a decrease in ectoine accumulation. The major osmolyte at high salinity was proline. Unexpectedly the size of the total ectoines' pool was smaller at elevated salinity. Experimental findings were then integrated with a metabolic model to get insight into carbon trafficking during osmoadaptation. Simulations predicted that the total flux through energy generating pathways, namely gluconeogenesis and the pentose phosphate pathway, was significantly lower and carbon source that entered the system as citrate was mainly diverted to osmolyte synthesis at high salinity. Overall these results suggested that the moderately halophilic Halomonas sp. AAD12 pursued a different osmoregulatory strategy than the two well known moderate halophiles, Chromohalobacter salexigens and Halobacillus halophilus. The climbing value of osmolytes such as ectoine in health care and skin care products places significant attention to halophilic microorganisms hence an understanding of the osmoadaptive mechanism and osmolyte accumulation strategy of this isolate is very valuable to be able to manipulate its metabolism towards desired goals.

Proteomic insight into phenolic adaptation of a moderately halophilic Halomonas sp. strain AAD12.

A gram-negative, moderately halophilic bacterium was isolated from Çamalti Saltern area, located in the Aegean Region of Turkey. Analysis of its 16S rRNA gene sequence and physiological characteristics showed that this strain belonged to the genus Halomonas ; hence, it was designated as Halomonas sp. strain AAD12. The isolate tolerated up to 800 mg⋅L(-1) phenol; however, at elevated concentrations, phenol severely retarded cell growth. The increase in lag phase with increasing phenol concentrations indicated that the microorganism was undergoing serious adaptative changes. To understand the physiological responses of Halomonas sp. strain AAD12 to phenol, a 2-dimensional electrophoresis approach combined with mass spectrometric analysis was used. This approach showed that the expression of 14 protein spots were altered as phenol concentration increased from 200 to 800 mg⋅L(-1). Among the identified proteins were those involved in protein biosynthesis, energy, transport, and stress metabolism. So far, this is the first study on phenolic adaptation of a gram-negative, moderately halophilic bacteria using proteomic tools. The results provided new insights for understanding the general mechanism used by moderately halophilic bacteria to tolerate phenol and suggested the potential for using these microorganisms in bioremediation.

Bacillus marmarensis sp. nov., an alkaliphilic, protease-producing bacterium isolated from mushroom compost.

A Gram-stain-positive, obligately alkaliphilic bacterium designated strain GMBE 72(T) was isolated from mushroom compost from Yalova, located in the Marmara region of Turkey. Cells were aerobic, straight rods and they formed subterminal to terminal ellipsoidal endospores. The isolate was catalase-positive, oxidase-negative and motile and contained a type A1gamma peptidoglycan based on meso-diaminopimelic acid. The strain grew at pH 8.0-12.5. The major cellular fatty acid was anteiso-C(15 : 0). The genomic DNA G+C content was 40.2 mol%. Phylogenetic analyses based on 16S rRNA gene sequencing showed that strain GMBE 72(T) belonged to the genus Bacillus and exhibited 98.2 % sequence similarity to Bacillus pseudofirmus DSM 8715(T). DNA-DNA reassociation was 56 % between GMBE 72(T) and B. pseudofirmus DSM 8715(T). According to our polyphasic characterization, strain GMBE 72(T) represents a novel species of the genus Bacillus, for which the name Bacillus marmarensis sp. nov. is proposed. The type strain is GMBE 72(T) (=DSM 21297(T) =JCM 15719(T)).

Studies on alkaline serine protease produced by Bacillus clausii GMBE 22.

An alkali tolerant Bacillus strain having extracellular serine alkaline protease activity was newly isolated from compost and identified as Bacillus clausii GMBE 22. An alkaline protease (AP22) was 4.66-fold purified in 51.5% yield from Bacillus clausii GMBE 22 by ethanol precipitation and DEAE-cellulose anion exchange chromatography. The purified enzyme was identified as serine protease by LC-ESI-MS analysis. Its complete inhibition by phenylmethanesulfonylfluoride (PMSF) also justified that it is a serine alkaline protease. The molecular weight of the enzyme is 25.4 kDa. Optimal temperature and pH values are 60 degrees C and 12.0, respectively. The enzyme showed highest specificity to N-Suc-Ala-Ala-Pro-Phe-pNA. The K(m) and k(cat) values for hydrolysis of this substrate are 0.347 mM and 1141 min(-1) respectively. The enzyme was affected by surface active agents to varying extents. The enzyme is stable for 2 h at 30 degrees C and pH 10.5. AP22 is also stable for 5 days over the pH range 9.0-11.0 at room temperature. AP22 has good pH stability compared with the alkaline proteases belonging to other strains of Bacillus clausii reported in the literature.

Preliminary phenotypic characterization of newly isolated halophilic microorganisms by footprinting: a rapid metabolome analysis.

The emerging need for rapid screening and identification methods for microbiological purposes necessitates the combined uses of high-tech instruments. In this work, electrospray ionization mass spectrometry was used to visualize the relation of ten newly isolated moderately halophilic microorganisms, to Halomonas salina DSMZ 5,928 and Halomonas halophila DSMZ 4,770. The method was based on the global analysis of the metabolites in culture media and is termed as metabolic footprinting. Since it was not possible to gain insight into the similarities solely based on the visual inspection of the chromatograms, principal component (PC) analysis was applied on the data. Three PCs alone were able to explain 99% of the information in the data set. The score plots revealed the relation of the new isolates to the two type strains whereas the loading plots gave important clues on the significant ions responsible for the observed clustering. Loading plots also indicated inversely correlated ions that give clues on differing metabolic pathways. The work described here offers a potentially useful way for preliminary rapid phenotypic characterization of new and closely related isolates and a method for screening of similar microorganisms for different and valuable secondary metabolites.