Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Reatividade fotoquímica da atmosfera de Cubatão e a influência de fontes exógenas / Photochemical reactivity of the atmosphere of Cubatão and the influence of exogenous pollutants

Objetivo. Demonstrar que a ocorrência de episódios críticos de poluição por ozônio na região de Cubatão-Centro é influenciada pelo aporte externo de poluentes, que participam dos processos físico-químicos de formação e remoção de reagentes fotoquímicos na atmosfera. Métodos. O estudo envolveu a obtenção e o tratamento de dados meteorológicos e de concentrações de poluentes fornecidos pela rede de monitoramento da qualidade do ar, bem como a realização de campanhas de amostragem passiva de ozônio (O ) e ativa de COVs, aldeídos e etanol na atmosfera da região. A amostragem de O VI 3 foi realizada com amostradores tipo Ogawa® e as concentrações foram determinadas por cromatografia iônica, com detecção por condutividade elétrica. Na amostragem de COVs foram utilizados tubos de aço contendo o adsorvente Tenax-GR, e a quantificação das amostras foi realizada por cromatografia gasosa e espectrometria de massas. A amostragem de aldeídos foi realizada com cartuchos de sílica gel revestido com 2,4 DNPH, e a quantificação realizada por cromatografia liquida de alta performance, com detector ultravioleta (UV/VIS). A amostragem de etanol foi realizada com cartuchos revestidos de florisil, e a quantificação realizada por flame ionization detector. O transporte de massas de ar foi analisado com base em backward trajectories, calculadas pelo modelo STILT. A interpretação dos dados foi realizada por estatística descritiva, boxplot, análise de correlação e análise multivariada (clusters). Resultados. A maioria dos episódios de ultrapassagem do PQAr de ozônio ocorreu no verão, entre 15h e 16h, com ventos predominantes de S/SE e velocidades superiores a 2,5m/s, típicos de períodos diurnos (brisa marítima). As espécies orgânicas precursoras de ozônio mais importantes foram: formaldeído, acetaldeído, tolueno, 1,2,3-trimetilbenzeno, m-xileno, etanol, oxileno, etilbenzeno, 1,3,5-trimetilbenzeno e benzeno. A retrotrajetória das massas de ar que chegam à Cubatão-Centro comprovou a influência de emissões exógenas. Os resultados obtidos na circulação de mesoescala, simulada pelo modelo BRAMS, concordam com os resultados obtidos por JAESCHKE (1997), onde apenas uma parcela da concentração média de poluentes de Cubatão-Centro era oriunda de fontes locais, sendo o restante decorrente do transporte de massas, com ocorrência de elevadas concentrações de poluentes com fluxo de direção norte-noedeste (brisa terrestre) e sul-sudeste (brisa marítima)

Surface detail reproduction and dimensional accuracy of stone models: influence of disinfectant solutions and alginate impression materials

This study compared the surface detail reproduction and dimensional accuracy of stone models obtained from molds disinfected with 2% sodium hypochlorite, 2% chlorhexidine digluconate or 0.2% peracetic acid to models produced using molds which were not disinfected, with 3 alginate materials (Cavex ColorChange, Hydrogum 5 and Jeltrate Plus). The molds were prepared over matrix containing 20-, 50-, and 75-µm lines, performed under pressure with perforated metal tray. The molds were removed following gelation and either disinfected (using one of the solutions by spraying followed by storage in closed jars for 15 min) or not disinfected. The samples were divided into 12 groups (n=5). Molds were filled with dental gypsum Durone IV and 1 h after the start of the stone mixing the models were separated from the tray. Surface detail reproduction and dimensional accuracy were evaluated using optical microscopy on the 50-µm line with 25 mm in length, in accordance with the ISO 1563 standard. The dimensional accuracy results (%) were subjected to ANOVA. The 50 µm-line was completely reproduced by all alginate impression materials regardless of the disinfection procedure. There was no statistically significant difference in the mean values of dimensional accuracy in combinations between disinfectant procedure and alginate impression material (p=0.2130) or for independent factors. The disinfectant solutions and alginate materials used in this study are no factors of choice regarding the surface detail reproduction and dimensional accuracy of stone models.

Este estudo comparou a reprodução de detalhes da superfície e alteração dimensional de modelos de gesso obtidos a partir de moldes desinfetados com hipoclorito de sódio 2%, digluconato de clorexidina 2%, ou ácido peracético 0,2% a modelos confeccionados utilizando moldes que não foram desinfetados com três alginatos (Cavex ColorChange, Hydrogum 5, Jeltrate Plus). Os moldes foram preparados sobre matriz contendo linhas de 20, 50 e 75 µm realizado sob pressão com moldeira de metal perfurada. Os moldes foram removidos após a geleificação e desinfetados (utilizando uma das soluções por pulverização, armazenados em frascos fechados durante 15 min) ou não desinfetados. Assim, as amostras foram divididas em 12 grupos (n=5). Os moldes foram preenchidos com gesso dental Durone IV e uma hora após a manipulação do gesso os modelos foram separados da moldeira. A reprodução de detalhes da superfície e a precisão dimensional foram avaliadas usando microscopia óptica na linha 50 µm com 25 mm de comprimento, de acordo com a norma ISO 1563. Os resultados de precisão dimensional (%) foram submetidos à ANOVA. A linha de 50 µm foi completamente reproduzida por todos os alginatos, independentemente do processo de desinfecção. Não houve diferença estatisticamente significativa nos valores médios de precisão dimensional nas combinações entre procedimento de desinfecção e alginato (p=0,2130), ou para fatores independentes. Soluções desinfetantes e alginatos utilizados neste estudo não são fatores de escolha em relação à reprodução de detalhes da superfície e alteração dimensional de modelos de gesso.

Quantitative Determination of Plasmodium Parasitemia by Flow Cytometry and Microscopy

The traditional light microscopy has limitations for precise growth assays of malaria parasites in culture or for assessment of new compounds for antimalarial activity; the speed and high reproducibility of flow cytometry can overcome these limitations. A flow cytometric method using PicoGreen, a DNA-binding fluorochrome, was developed with optimal precision suitable for performing growth assays of low-parasitemia field isolates. In addition, intra- and inter-person reproducibility of the flow cytometric and the microscopic method were compared in order to quantitatively demonstrate the improved precision. RNase treatment contributed to the precision of the flow cytometric measurements by enhancing the signal-to-noise ratios. Coefficients of variation of the method were smaller than 10% for 0.1% or higher parasitemia samples. The intra- and inter-person coefficients of variation of the flow cytometric method were three to six times smaller than those of the microscopic method. The flow cytometric method developed in this study yielded substantially more precise results than the microscopic method, allowing determination of parasitemia levels of 0.1% or higher, with coefficients of variation smaller than 10%. Thus, the PicoGreen method could be a reliable high sensitivity assay for analysis of low parasitemia samples and might be applied to a high throughput system testing antimalarial drug activity.

Detection of alpha-thalassemia-1 Southeast Asian and Thai Type Deletions and beta-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis / 대한진단검사의학회지

BACKGROUND: Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. METHODS: Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of alpha-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and beta-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with alpha-thalassemia-1 SEA type deletion, 2 with alpha-thalassemia-1 Thai type deletion, and 2 with heterozygous beta-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from alpha-thalassemia-1 SEA type deletion, alpha-thalassemia-1 Thai type deletion, beta-thalassemia 3.5-kb gene deletion, and the wild-type beta-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89degrees C, 85.66degrees C, 77.24degrees C, and 74.92degrees C, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. CONCLUSIONS: Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of alpha- and beta-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.

Detection of alpha-thalassemia-1 Southeast Asian and Thai Type Deletions and beta-thalassemia 3.5-kb Deletion by Single-tube Multiplex Real-time PCR with SYBR Green1 and High-resolution Melting Analysis / 대한진단검사의학회지

BACKGROUND: Prevention and control of thalassemia requires simple, rapid, and accurate screening tests for carrier couples who are at risk of conceiving fetuses with severe thalassemia. METHODS: Single-tube multiplex real-time PCR with SYBR Green1 and high-resolution melting (HRM) analysis were used for the identification of alpha-thalassemia-1 Southeast Asian (SEA) and Thai type deletions and beta-thalassemia 3.5-kb gene deletion. The results were compared with those obtained using conventional gap-PCR. DNA samples were derived from 28 normal individuals, 11 individuals with alpha-thalassemia-1 SEA type deletion, 2 with alpha-thalassemia-1 Thai type deletion, and 2 with heterozygous beta-thalassemia 3.5-kb gene deletion. RESULTS: HRM analysis indicated that the amplified fragments from alpha-thalassemia-1 SEA type deletion, alpha-thalassemia-1 Thai type deletion, beta-thalassemia 3.5-kb gene deletion, and the wild-type beta-globin gene had specific peak heights at mean melting temperature (Tm) values of 86.89degrees C, 85.66degrees C, 77.24degrees C, and 74.92degrees C, respectively. The results obtained using single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis showed 100% consistency with those obtained using conventional gap-PCR. CONCLUSIONS: Single-tube multiplex real-time PCR with SYBR Green1 and HRM analysis is a potential alternative for routine clinical screening of the common types of alpha- and beta-thalassemia large gene deletions, since it is simple, cost-effective, and highly accurate.

Evaluation of Two Commercial HLA-B27 Real-Time PCR Kits / 대한진단검사의학회지

BACKGROUND: The standard PCR with sequence-specific primers (SSP) is a widely used method of HLA-B27 typing in clinical practice. The aim of our study was to evaluate 2 Korean HLA-B27 kits with different real-time PCR chemistries. METHODS: To validate the accuracy of real-time PCR kits, we selected 28 HLA-B27-positive samples and 33 HLA-B27-negative samples with a wide range of different HLA-B specificities typed by standard PCR-SSP. The 2 real-time PCR kits used were the AccuPower(R) HLA-B27 real-time PCR kit (Bioneer, Korea) with TaqMan probes and the Real-Q(TM) HLA-B*27 detection kit (BioSewoom, Korea) with SYBR Green I dye for melting curve analysis. RESULTS: All 61 samples typed by PCR-SSP demonstrated a perfect concordance with the 2 real-time PCR assays. It was possible to clearly discriminate between HLA-B27-positive and -negative samples in both real-time assays. CONCLUSIONS: In summary, both real-time PCR assays for HLA-B27 were fast, reliable, well-adapted for routine laboratory testing, and attractive alternatives to the conventional PCR-SSP method.

Polaron hopping in some biomolecular solids and their charge transfer complexes.

The solid state spectroscopy of charge transfer complexes of biomolecules such as fatty acids, tripalmitin, lysozyme. folic acid, beta-carotene, cytochrome c, valinomycin and gramicidin has been carried out. The absorption coefficient is related with electronic conductivity. A half-power beta density is found common among these macromolecular solids, indicating photon-induced polaron hopping or hopping of a charge carrier between two branches of a polariton. Band gap vs full width at half-maximum of the mid-IR peak also reveals a linear relation.

Comparison of the effectiveness of four organic chemoattractants towards zoospores of Ulva pertusa and macrofouling.

Algal spores respond to many environmental variables, especially to chemical "cues". This chemotactic response can be utilized to attract spores, thereby colonization of a new substrata is possible to be influenced. In this attempt, four chemoattractant candidates were screened against spores of Ulva pertusa to reveal their efficiencies. Attachment and subsequent germination of Ulva spores were effectively influenced by these chemoattractant candidates. In particular 100 microg cm2 of D-glucose coating was found to enhance spore attachment by > 150%. Furthermore, field investigations carried out with test panels, clearly indicate the chemoattractive properties of test coatings. In recent years, various anthropogenic activities and natural hazards cause detrimental impacts on the benthic algae and other fishery resources. Artificial reefs have been laid on many coastal regions to increase or restore marine resources. Chemoattractant coatings can be applied on artificial surfaces to increase the colonization of benthic forms. It also can be used in the mariculture devices. Influence of chemoattractants on Ulva spores and fouling biomass estimated on test panels are discussed.

Biomanagement of sago-sludge using an earthworm, Eudrilus eugeniae.

Sago, the tapioca starch is manufactured by over 800 small-scale units located in the Salem district, Tamil Nadu, India. During the processing of sago it generates huge quantities of biodegradable solid waste, as crushed tubers. In present study an attempt was made to convert these biodegradable solid sago tubers into value added compost using an exotic earthworm, Eudrilus eugeniae. The experiments were carried out in a plastic tray at various concentrations of sago-sludge (50% 75% and 100%) for a period of 90 days. During the vermicomposting, data were collected on life form (cocoon, non clitellates, clitellates) of earthworm and it was found to be high in 50% followed by 75% and 100% concentrations. Chemical analysis of worked substrates showed a step wise increase of nitrogen and phosphorus. The fold increase of phosphorus and nitrogen were found to be high for sago-sludge undergoing vermicomposting than the control. During the composting period the organic carbon decreased from its initial value of 58, 76 and 107 mg/kg to 21, 24 and 65 mg/kg for 50, 70 and 100%, respectively The microbial analysis showed that after 75 days of composting, their population stabilized and further increase in composting period did not increase their population size. The results indicate that 50% and 75% concentration of sludge mixed with bedding material was ideal for the vermicomposting.

Risco ambiental da aplicação de éteres de difenilas polibromadas como retardantes de chama / Environmental risks involved with the application of polybrominated diphenyl ethers as flame retardants

Flame retardants are additives of combustible materials, such as plastics, textile, electronic circuitry, wood and paper providing resistance to the combustion process when exposed to fire and high temperature. The main flame retardants used are inorganic chemicals (such as antimony oxides), organic phosphate esters with or without halogens, and chlorinated and brominated organic compounds. The brominated flame retardants (BFRs) are largely used due to its efficiency and low cost. The most used flame retardants are the polybrominated diphenyl ethers (PBDEs), produced inlarge-scale whose degradation is very difficult. Thus, they have been found in many different environmental samples. These observations suggest the current destination of these substances is still devoid of recycling or specific treatment. Despite the increased application in oil polymers, little is known about its impact upon the ecosystem. In this review, we provide an overview about the use and risks related to PBDEs as a recognized toxicants found in industries.

Os retardantes de chama, dentre os quais se destacam os éteres de difenilas polibromadas, são aditivos de materiais destinados a torná-los resistentes ao fogo ou a altas temperaturas, inibindo ou suprimindo o processo de combustão, dentre os quais se destacam os éteres de difenilas polibromadas (PBDEs, do inglês, polibromated diphenyl ethers). Devido a sua produção em grande escala e sua difícil degradação, os PBDEs têm sido um contaminante emergente frequentemente encontrados em diferentes amostras ambientais, demonstrando que o processo produtivo, em especial o destino desse material, requer medidas estratégicas que racionalize seu uso. Apesar da ampla utilização desses aditivos em polímeros (na maioria derivados de petróleo) e tecidos inflamáveis comumente utilizados, pouco se sabe a respeito do impacto dessas substâncias sobre o ecossistema. Nessa revisão, uma relação consistente a respeito do risco ambiental resultante do uso indiscriminado desses aditivos, ainda carentes de regulamentação específica, foi estabelecida com base na ação desses compostos bem como a prevalência de algumas classes, reconhecidamente tóxicas em alguns ambientes.

Docking mode of delvardine and its analogues into the p66 domain of HIV-1 reverse transcriptase: screening using molecular mechanics-generalized born/surface area and absorption, distribution, metabolism and excretion properties.

Delvardine and its structural derivatives are important non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs). In this work,15 delvardine analogues were studied. A free energy-of-binding (FEB)expression was developed in the form of an optimized linear combination of van der Waal (vdW), electrostatic, solvation and solvent-accessible surface area (SASA) energy terms. The solvation energy terms estimated by generalized born/surface area (GB/SA) play an important role in predicting the binding affinity of delvardine analogues. Out of 15 derivatives, substitution of CH3 with H at the Y and R positions, as well as substitution of SO2CH3 with only CH2 at the Z position in S2, S8 and S12 analogues, were found to be the most potent (glide score = -7.60, -8.06 and -7.44; pIC50 =7.28, 7.37 and 7.64) in comparison with the template delvardine (which is used currently as the drug candidate). All the three analogues also passed the absorption, distribution,metabolism and excretion (ADME) screening and Lipinski's rule of 5, and have the potential to be used for second-generation drug development. The work demonstrates that dock molecular mechanics-generalized born/surface area (MM-GB/SA-ADME) is a promising approach to predict the binding activity of ligands to the receptor and further screen for a successful candidate drug in a computer-aided rational drug design.

Using respirometer in biodegradation phenomenon of natural organic matters.

This study conducted in monitoring respirometer oxygen consumption of aerobic microorganism during biodegradation processes of ozonated organic matters, which can estimate both biodegraded efficiency and coefficient of natural organic matters (NOMs) in water source. It can be proposed that different ozone dosage might change biodegradation characteristics of organic matters. The result reveals that higher ozone dosage may cause higher biomass yield coefficient of microorganism, and cultured microorganism may easily utilize biodegradation organic matters (BOMs) produced by ozonation, finally increasing overall removal efficiency. Therefore, using respirometer to evaluate the production of BOMs by ozonation before the biological treatment is effective for controling ozone dosage and enhancement of NOMs removal by biological processes.

Effects of Air Pollutants on Childhood Asthma

Epidemiologic studies have suggested the association between environmental exposure to volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) and the increased risk of incurring asthma. Yet there is little data regarding the relationship between personal exposure to air pollution and the incidence of asthma in children. This study was designed to evaluate the effect of exposure to air pollution on children with asthma by using exposure biomarkers. We assessed the exposure level to VOCs by measuring urinary concentrations of hippuric acid and muconic acid, and PAHs by 1-OH pyrene and 2-naphthol in 30 children with asthma and 30 children without asthma (control). The mean level of hippuric acid was 0.158+/-0.169micromol/mol creatinine in the asthma group and 0.148+/-0.249micromol/mol creatinine in the control group, with no statistical significance noted (p=0.30). The mean concentration of muconic acid was higher in the asthma group than in the control group (7.630+/-8.915micromol/mol creatinine vs. 3.390+/-4.526micromol/mol creatinine p=0.01). The mean level of urinary 1-OHP was higher in the asthma group (0.430+/-0.343micromol/mol creatinine) than the control group (0.239+/-0.175micromol/mol creatinine), which was statistically significant (p=0.03). There was no difference in the mean concentration of 2-NAP between the two groups (9.864+/-10.037micromol/mol in the asthma group vs. 9.157+/-9.640micromol/mol in the control group, p=0.96). In conclusion, this study suggests that VOCs and PAHs have some role in asthma.

The effects of cyclophosphamide treatment on the pathogenesis of subgroup J avian leukosis virus (ALV-J) infection in broiler chickens with Marek's disease virus exposure

Studies were performed to determine the effects of Bcell suppression on the pathogenesis of Subgroup J avian leukosis virus (ALV-J) in broiler chickens. Neonatal chickens were treated with cyclophosphamide (CY) or PBS, and then infected with ALV-J (ADOL-7501) at 2 weeks of age. CY treatment induced B cell specific immunosuppression throughout the experiment confirmed by decreased bursal weight, intact lymphocyte mitogenetic activity stimulated by Con A and increased relative subpopulation of CD3-positive cells as measured by flow cytometry. Chickens in this experiment had Mareks disease virus exposure prior to three weeks of age as determined by the presence of lymphocytic infiltration and antibody. Virus neutralizing antibody against ALV-J was first observed at 6 weeks post-infection in some of the infected chickens in the PBS group. As expected, none of the chickens from the CY group and uninfected chickens developed virus-neutralizing antibody. The viremic status was measured by real time RT-PCR using SYBR green I dye. The percentage of viremic chickens was significantly higher, and more chickens had high titered viremia, in the CY treated group. No neoplastic foci consistent with ALVJ infection were observed in any of the experimental chickens. The frequency and intensity of viral antigen expression determined by immunohistochemistry was significantly higher in tissues from CY treated birds than those of PBS treated chickens at 3 weeks post-infection. This study showed that B cell specific immunosuppression with CY treatment in chickens resulted in increase in viremia and viral antigen load in tissues.

Self-Association of some organic acids in aqueous solutions

The self-association of both phenyl acetic acid [PAA] and hydrocinnamic acid [HCA] in aqueous solutions has been studied at 25 degree. Vapor pressure osmometry was used to count the number of particles presented in dilute aqueous solutions of non-volatile compounds. The data obtained were best fitted by assuming the formation of 1: 1 dimers. The association is most likely to occur through stacking of the phenyl groups with the side chain oriented on the opposite sides of the stacked pair. The dimerization equilibrium constants have been computed for both acids. From the results, it is possible to discuss the effect of the side chain length on the dimerization constant