Cloning, expression and activity analysis of cutinase from Sclerotinia sclerotiorum / 生物工程学报

Cutinase can degrade aliphatic and aromatic polyesters, as well as polyethylene terephthalate. Lack of commercially available cutinase calls for development of cost-effective production of efficient cutinase. In this study, eight cutinase genes were cloned from Sclerotinia sclerotiorum. The most active gene SsCut-52 was obtained by PCR combined with RT-PCR, expressed in Escherichia coli BL21 and purified by Ni-NTA affinity chromatography to study its characteristics and pathogenicity. Sscut-52 had a total length of 768 bp and 17 signal peptides at the N terminals. Phylogenetic analysis showed that its amino acid sequence had the highest homology with Botrytis keratinase cutinase and was closely related to Rutstroemia cutinase. Sscut-52 was highly expressed during the process of infecting plants by Sclerotinia sclerotiorum. Moreover, the expression level of Sscut-52 was higher than those of other cutinase genes in the process of sclerotia formation from mycelium. The heterologously expressed cutinase existed in the form of inclusion body. The renatured SsCut-52 was active at pH 4.0-10.0, and mostly active at pH 6.0, with a specific activity of 3.45 U/mg achieved. The optimum temperature of SsCut-52 was 20-30 ℃, and less than 60% of the activity could be retained at temperatures higher than 50 ℃. Plant leaf infection showed that SsCut-52 may promote the infection of Banlangen leaves by Sclerotinia sclerotiorum.

The application of carbohydrate binding module-Thermobifida fusca cutinase fusion protein in polyethylene terephthalate degradation / 生物工程学报

With the development of global economy, the dramatically increased production of polyethylene terephthalate (PET) plastics has led to a remarkably increased amount of plastic waste. PET waste can be treated by landfill, incineration, or biodegradation. While landfilling and incineration may cause secondary pollution, biodegradation has since received increased attentions due to its environmental friendliness. Recent studies have indicated that the carbohydrate binding module (CBM) can effectively enhance the binding of PET degrading enzymes to PET, and consequently increasing PET degradation rate. Here we constructed a fusion protein BaCBM2-Tfuc containing the BaCBM2 from Bacillus anthraci and the cutinase Tfuc from Thermobifida fusca, by megaprimer PCR of whole plasmids (MEGAWHOP). Notabaly, the PET film degradation efficiency (at 60 ℃) of BaCBM2-Tfuc was 2.8 times that of Tfuc. This study may provide technical support for constructing fusion proteins capable of efficiently degrading PET.

Characterization of Humicola insolens cutinase-tachystatin A2 fusion protein and its application in treatment of recycled paper stickies / 生物工程学报

With the decrease of forest timber resources, the recycling of waste paper has received increasing attention. However, the stickies produced in the process of waste paper recycling may negatively affect the production of recycled paper. The biological decomposition of stickies, which has the advantages of high efficiency, high specificity and pollution-free, is achieved mainly through the enzymatic cleavage of the ester bond in the stickies components to prevent flocculation. Cutinase is a serine esterase that can degrade some components of the stickies. Previous research indicated that the anchor peptide tachystatin A2 (TA2) is able to bind polyurethane. In this study, the cutinase HiC derived from Humicola insolens was used to construct a fusion protein HiC-TA2 by megaprimer PCR of the whole plasmid (MEGAWHOP). The enzymatic properties and the degradation efficiency of the fusion protein on poly(ethyl acrylate) (PEA), a model substrate of stickies component, were determined. The results showed that the degradation efficiency, the size decrease of PEA particle, and the amount of ethanol produced by HiC-TA2 were 1.5 times, 6.8 times, and 1.4 times of that by HiC, respectively. These results demonstrated that TA2 improved the degradation efficiency of HiC on PEA. This study provides a useful reference for biological decomposition of stickies produced in the process of recycled paper production.

Research progress in structure and function of pectin methylesterase / 生物工程学报

Pectin methylesterase (PME) is an important pectinase that hydrolyzes methyl esters in pectin to release methanol and reduce the degree of methylation of pectin. At present, it has broad application prospects in food processing, tea beverage, paper making and other production processes. With the in-depth study of PME, the crystal structures with different sources have been reported. Analysis of these resolved crystal structures reveals that PME belongs to the right-hand parallel β-helix structure, and its catalytic residues are two aspartic acids and a glutamine, which play the role of general acid-base, nucleophile and stable intermediate, in the catalytic process. At the same time, the substrate specificity is analyzed to understand the recognition mechanism of the substrate and active sites. This paper systematically reviews these related aspects.

Enzymatic preparation of fructooligosaccharides-rich burdock syrup with enhanced antioxidative properties

Background: Burdock (Arctium lappa L.) is a fructan-rich plant with prebiotic potential. The aim of this study was to develop an efficient enzymatic route to prepare fructooligosaccharides (FOS)-rich and highly antioxidative syrup using burdock root as a raw material. Results: Endo-inulinase significantly improved the yield of FOS 2.4-fold while tannase pretreatment further increased the yield of FOS 2.8-fold. Other enzymes, including endo-polygalacturonase, endo-glucanase and endo-xylanase, were able to increase the yield of total soluble sugar by 11.1% (w/w). By this process, a new enzymatic process for burdock syrup was developed and the yield of burdock syrup increased by 25% (w/w), whereas with FOS, total soluble sugars, total soluble protein and total soluble polyphenols were enhanced to 28.8%, 53.3%, 8.9% and 3.3% (w/w), respectively. Additionally, the scavenging abilities of DPPH and hydroxyl radicals, and total antioxidant capacity of the syrup were increased by 23.7%, 51.8% and 35.4%, respectively. Conclusions: Our results could be applied to the development of efficient extraction of valuable products from agricultural materials using enzyme-mediated methods.

Investigating the effect of intracameral cefuroxime on oxidative stress and apoptosis in the rat cornea / Investigando o efeito de cefuroxima intracameral sobre o estresse oxidativo e a apoptose na córnea de ratos

ABSTRACT PURPOSE: We examined the effect of intracameral administration of cefuroxime on oxidative stress and endothelial apoptosis in rat corneal tissue. METHODS: In total, 30 rats were divided into 3 groups of 10 rats each (intracameral administration of cefuroxime 0.1 mg/0.01 mL (cefuroxime group); intracameral administration of balanced salt solution 0.01 mL (control group); or absence of intracameral injection (sham group). Corneal endothelial apoptosis was assessed by immunohistochemical analysis using caspase-3 and caspase-8. Total oxidant status, total antioxidant status, oxidative stress index, and paraoxonase and arylesterase levels were examined in corneal endothelial tissue and serum. RESULTS: Paraoxonase levels in the serum were significantly different between the sham and cefuroxime groups (p=0.027). A significant difference was also observed in total oxidant status levels between the cefuroxime and balanced salt solution groups (p=0.023). In addition, there were significant differences in total antioxidant status levels in corneal tissue between the cefuroxime and sham groups (p<0.001) and between the cefuroxime and balanced salt solution groups (p<0.001). Furthermore, significant differences were also observed in oxidative stress index levels between the cefuroxime and balanced salt solution groups (p=0.001) and between the cefuroxime and sham groups (p=0.026). According to the immunohistochemical staining results, a significant association with caspase-3 activity existed between the cefuroxime and balanced salt solution groups (p=0.007), while no significant difference was found with caspase-8 activity (p=0.541). Caspase-3 activity exhibited a significant relationship between the sham and balanced salt solution groups (p=0.018), but no relationship was found with caspase-8 activity (p=0.623). CONCLUSION: Immunohistochemical examination revealed that intracameral cefuroxime increased apoptosis when compared to the sham and balanced salt solution groups. Moreover, intracameral cefuroxime increased oxidative stress in the cornea and simultaneously induced apoptosis.

RESUMO OBJETIVO: Examinamos o efeito da administração intracameral da cefuroxima sobre o estresse oxidativo e a apoptose endotelial no tecido corneano de ratos. MÉTODOS: No total, 30 ratos foram divididos em 3 grupos de 10 ratos cada (administração intracameral de cefuroxima 0,1 mg/0,01 mL (grupo cefuroxima), administração intracameral de solução salina balanceada 0,01 mL (grupo controle) ou ausência de injeção intracameral (grupo sham)). A apoptose endotelial da córnea foi avaliada por análise imuno-histoquimica usando caspase-3 e -8. O status oxidante total, o status antioxidante total, o índice de estresse oxidativo e os níveis de a paraoxonase e arilesterase foram investigados no tecido endotelial da córnea e no soro. RESULTADOS: Os níveis de paraoxonase no soro foram significativamente diferentes entre os grupos sham e cefuroxima (p=0,027). Foi também observada uma diferença significativa nos níveis de estado oxidante total entre os grupos cefuroxima e solução salina balanceada (p=0,023). Além disso, houve diferenças significativas nos níveis de status antioxidante total no tecido da córnea entre os grupos cefuroxima e sham (p<0,001) e entre os grupos cefuroxima e solução salina balanceada (p<0,001). Diferenças significativas também foram observadas nos níveis do índice de estresse oxidativo entre os grupos cefuroxima e solução salina balanceada (p=0,001) e entre os grupos cefuroxima e sham (p=0,026). De acordo com os resultados de coloração imuno-histoquimica, houve associação significativa com a atividade da caspase-3 entre os grupos cefuroxima e solução salina balanceada (p=0,007), enquanto não houve diferença significativa com a atividade da caspase-8 (p=0,541). A atividade da caspase-3 exibiu uma relação significativa entre os grupos sham e solução salina balanceada (p=0,018), mas nenhuma relação foi encontrada com a atividade da caspase-8 (p=0,623). CONCLUSÃO: O exame imuno-histoquímico revelou que a cefuroxima intracameral aumentou a apoptose quando comparada com os grupos sham e solução salina balanceada. Além disso, a cefuroxima intracameral aumentou o estresse oxidativo na córnea e induziu simultaneamente a apoptose.

Oligomerization triggered by foldon to enhance the catalytic efficiency of feruloyl esterase / 生物工程学报

A new method to express oligomerized feruloyl esterase (FAE) in Pichia pastoris GS115 to improve the catalytic efficiency was developed. It was realized by fusing the foldon domain at the C-terminus of FAE, and the fusion protein was purified by histidine tag. Fusion of the feruloyl esterase with the foldon domain resulted spontaneously forming a trimer FAE to improve the catalytic performance. The oligomerized FAE and monomeric FAE were obtained by purification. The apparent molecular weight of the oligomerized FAE was about 110 kDa, while the monomeric FAE about 40 kDa, and the optimum temperature of the oligomerized FAE was 50 °C, which is the same as the monomeric one. The optimal pH of the oligomerized FAE is 5.0, while the optimal pH of the monomer FAE is 6.0. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the oligomerized FAE increased 7.57-folds. The catalytic constant (kcat) of the oligomerized FAE increased 3.42-folds. The oligomerized FAE induced by foldon have advantages in the catalytic performances, which represents a simple and effective enzyme-engineering tool. The method proposed here for improving the catalytic efficiency of FAE would have great potentials for improving the catalytic efficiency of other enzymes.

Cypermethrin induces cell injury in primary cortical neurons of C57BL/6 mice by inhibiting Nrf2/ARE signaling pathway / 南方医科大学学报

OBJECTIVE@#To study the role of Nrf2/ARE signaling pathway in cypermethrin-induced oxidative stress and apoptosis of cerebral cortex neurons in C57BL/6 mice.@*METHODS@#The cortical neurons of C57BL/6 mice were cultured and identified, and a cypermethrin-induced cell injury model was established by treating the cells with 0, 25, 50 and 100 μmol/L of cypermethrin for 48 h. CCK-8 assay was used to analyze the effects of cypermethrin on the cell viability, and the fluorescence probe DCFH-DA was used for detecting intracellular reactive oxygen species (ROS); flow cytometry was performed for determining the apoptosis rate of the cells. The mRNA and protein expression levels of Nrf2 and its downstream genes HO-1 and NQO1 were detected using qPCR and Western blotting.@*RESULTS@#Exposure to cypermethrin at different doses inhibited the viability of the cultured cortical neurons. With the increase of cypermethrin dose, the viability of the neurons decreased progressively, the intracellular ROS and the cell apoptosis rate increased, and the neuronal injury worsened. At the dose of 50 and 100 μmol/L, cypermethrin significantly down-regulated the expressions of HO-1, NQO1 and Nrf2 at both the mRNA and protein levels in the cells ( < 0.01).@*CONCLUSIONS@#Cypermethrin exposure shows a dose-dependent neurotoxicity by inhibiting Nrf2/ARE signaling pathway, down-regulating the expression of Nrf2 and its downstream genes HO-1, NQO1 mRNA and protein, and inducing oxidative damage and apoptosis in primary mouse cortical neurons, .

Type C feruloyl esterase from Aspergillus ochraceus: a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Background: Aspergillus ochraceus was isolated from coffee pulp and selected as an interesting hydroxycinnamoyl esterase strain producer, using an activity microplate high-throughput screening method. In this work, we purified and characterized a new type C A. ochraceus feruloyl esterase (AocFaeC), which synthesized specifically butyl hydroxycinnamates in a ternary solvent system. Results: AocFaeC was produced by solid state fermentation, reaching its maximal activity (1.1 U/g) after 48 h of culture. After purification, the monomeric protein (34 kDa) showed a specific activity of 57.9 U/mg towards methyl ferulate. AocFaeC biochemical characterization confirmed its identity as a type C feruloyl esterase and suggested the presence of a catalytic serine in the active site. Its maximum hydrolytic activity was achieved at 40°C and pH 6.5 and increased by 109 and 77% with Ca2+ and Mg2+, but decreased by 90 and 45% with Hg2+ and Cu2+, respectively. The initial butyl ferulate synthesis rate increased from 0.8 to 23.7 nmol/min after transesterification condition improvement, using an isooctane:butanol:water ternary solvent system, surprisingly the synthesis activity using other alcohols was negligible. At these conditions, the synthesis specific activities for butyl p-coumarate, sinapinate, ferulate, and caffeate were 87.3, 97.6, 168.2, and 234 U/µmol, respectively. Remarkably, AocFaeC showed 5 folds higher butyl caffeate synthesis rate compared to type B Aspergillus niger feruloyl esterase, a well-known enzyme for its elevated activity towards caffeic acid esters. Conclusions: Type C feruloyl esterase from A. ochraceus is a butanol specific biocatalyst for the synthesis of hydroxycinnamates in a ternary solvent system

Esterasa leucocitaria como prueba diagnóstica eficaz, económica y rápida ante un proceso infeccioso articular de rodilla / Leucocyte esterase as a reliable diagnostic, cost-effective and fast in knee infections

Resumen: Antecedentes: La infección articular es un reto ortopédico por la complejidad diagnóstica y sus efectos devastadores al no tratarse oportunamente. Se cuenta con diversos estudios de diagnóstico: cultivo, VSG, PCR, conteo de leucocitos, entre otros, pero ninguno es preciso, tardan más de 30 minutos en realizarse y requieren una infraestructura compleja. En este estudio se determina la sensibilidad y especificidad de la esterasa leucocitaria para la detección de un proceso infeccioso articular en población mexicana. Material y métodos: de Noviembre de 2015 a Abril de 2016 se obtuvo líquido sinovial de pacientes con diagnóstico de infección articular con o sin implante y sin infección con patología degenerativa de rodilla. Se evaluó la muestra mediante el test de esterasa leucocitaria COMBI-SCREEN 11SYS con lectura colorimétrica a los dos minutos, determinando positivo para infección: dos cruces, el resto de la muestra fue enviado a cultivo. Resultados: Se aplicó el test a 64 muestras de líquido sinovial de rodilla, 19 diagnosticadas con infección articular y 45 sin infección. Se obtuvo una sensibilidad 100%, especificidad 88.24% VPP 68.42% y VPN 100%, índice de concordancia kappa 0.753 mediante el programa IBM SPSS Statistics 22, Python versión 2.7. Conclusiones: La esterasa leucocitaria es una prueba rápida, económica y eficaz para detectar un proceso infeccioso contra un proceso inflamatorio con alta probabilidad de acierto. Este estudio presentó un índice de concordancia kappa de 0.753, demostrando ser reproducible, por lo que se recomienda implementarse en los servicios de urgencias a nivel nacional.

Abstract: Background: The articular infection represents a challenge due to its complexity and its devastating effect when not treated promptly. We have various diagnostic studies: cultures, ESR, CRP, count of leukocytes, among others but none is specific, it takes more than 30 minutes to complete and require complex infrastructure. In this study we determine the sensitivity and specificity of the leukocyte esterase for detection of an infectious process joint in Mexican population. Material and methods: From November 2015 to April 2016 was obtained synovial fluid from patients with diagnosis of knee infection with or without implant and without infection with degenerative pathology of the knee. It assessed the sample through the COMBI-SCREEN 11SYS leukocyte esterase with reading colorimetric test at two minutes determining positive for infection: two crosses, the remainder of the sample was sent to culture. Results: We perform the test in 64 samples of synovial fluid of knee joint 19 diagnosed with infection and 45 without infection. Was obtained a sensitivity 100%, specificity of 88.24%, PPV 68.42% and PNV 100%, kappa index 0.753 using the program IBM SPSS Statistics 22, Python ver. 2.7. Conclusions: Leukocyte esterase is a fast, economical and effective to detect an infectious process against one inflammatory with high probability of success. This study showed an index of concordance 0.753 Kappa, proving to be reproducible so recommend be implemented in the emergency department at the national level.

Serum paraoxonase, arylesterase, and glutathione-s-transferase activities and oxidative stress levels in patients with mushroom poisoning

OBJECTIVES: Consumption of toxic species of mushrooms may have detrimental effects and increase oxidative stress. Paraoxonase, arylesterase and glutathione-S-transferase are antioxidants that resist oxidative stress. In this study, we analyzed the changes in these enzymes during intoxication due to mushrooms. METHODS: The study enrolled 49 adult patients with a diagnosis of mushroom poisoning according to clinical findings and 49 healthy volunteers as the control group. The patients with mild clinical findings were hospitalized due to the possibility that the patient had also eaten the mushrooms and due to clinical findings in the late period, which could be fatal. Paraoxonase, arylesterase, and glutathione-S-transferase concentrations, as well as total antioxidant and oxidant status, were determined in the 49 patients and 49 healthy volunteers by taking blood samples in the emergency department. RESULTS: While paraoxonase, arylesterase, and total antioxidant status were significantly decreased in the patient group (p<0.05), glutathione-S-transferase, total oxidant status and the oxidative stress index were significantly higher (p<0.05). There was a positive correlation between the hospitalization time and the oxidative stress index (r=0.752, p<0.001), whereas a negative correlation was found with glutathione-S-transferase (r=-0.420, p=0.003). CONCLUSION: We observed a significant decrease in paraoxonase and arylesterase and an increase in glutathione-S-transferase and oxidative stress indexes in patients with mushroom poisoning, indicating that these patients had an oxidative status. In particular, a low total antioxidant status and high oxidative stress index may gain importance in terms of the assessment of hospitalization duration.

Carboxylesterases in lipid metabolism: from mouse to human

Mammalian carboxylesterases hydrolyze a wide range of xenobiotic and endogenous compounds, including lipid esters. Physiological functions of carboxylesterases in lipid metabolism and energy homeostasis in vivo have been demonstrated by genetic manipulations and chemical inhibition in mice, and in vitro through (over)expression, knockdown of expression, and chemical inhibition in a variety of cells. Recent research advances have revealed the relevance of carboxylesterases to metabolic diseases such as obesity and fatty liver disease, suggesting these enzymes might be potential targets for treatment of metabolic disorders. In order to translate pre-clinical studies in cellular and mouse models to humans, differences and similarities of carboxylesterases between mice and human need to be elucidated. This review presents and discusses the research progress in structure and function of mouse and human carboxylesterases, and the role of these enzymes in lipid metabolism and metabolic disorders.

Tannase application in secondary enzymatic processing of inferior Tieguanyin oolong tea

Background: Inferior Tieguanyin oolong tea leaves were treated with tannase. The content and bioactivity of catechins in extracts from the treated tea leaves were investigated to assess the improvement in the quality of inferior Tieguanyin oolong tea. Results: Analysis showed that after treatment, the esterified catechin content decreased by 23.5%, whereas non-galloylated catechin and gallic acid contents increased by 15.3% and 182%, respectively. The extracts from tannase-treated tea leaves showed reduced ability to bind to BSA and decreased tea cream levels. The extracts also exhibited increased antioxidant ability to scavenge OH and DPPH radicals, increased ferric reducing power, and decreased inhibitory effects on pancreatic α-amylase and lipase activities. Conclusions: These results suggested that tannase treatment could improve the quality of inferior Tieguanyin oolong tea leaves.

Synthesis of structured triacylglycerols enriched in n-3 fatty acids by immobilized microbial lipase

Abstract The search for new biocatalysts has aroused great interest due to the variety of micro-organisms and their role as enzyme producers. Native lipases from Aspergillus niger and Rhizopus javanicus were used to enrich the n-3 long-chain polyunsaturated fatty acids content in the triacylglycerols of soybean oil by acidolysis with free fatty acids from sardine oil in solvent-free media. For the immobilization process, the best lipase/support ratios were 1:3 (w/w) for Aspergillus niger lipase and 1:5 (w/w) for Rhizopus javanicus lipase using Amberlite MB-1. Both lipases maintained constant activity for 6 months at 4 °C. Reaction time, sardine-free fatty acids:soybean oil mole ratio and initial water content of the lipase were investigated to determine their effects on n-3 long-chain polyunsaturated fatty acids incorporation into soybean oil. Structured triacylglycerols with 11.7 and 7.2% of eicosapentaenoic acid + docosahexaenoic acid were obtained using Aspergillus niger lipase and Rhizopus javanicus lipase, decreasing the n-6/n-3 fatty acids ratio of soybean oil (11:1 to 3.5:1 and 4.7:1, respectively). The best reaction conditions were: initial water content of lipase of 0.86% (w/w), sardine-free faty acids:soybean oil mole ratio of 3:1 and reaction time of 36 h, at 40 °C. The significant factors for the acidolysis reaction were the sardine-free fatty acids:soybean oil mole ratio and reaction time. The characterization of structured triacylglycerols was obtained using easy ambient sonic-spray ionization mass spectrometry. The enzymatic reaction led to the formation of many structured triacylglycerols containing eicosapentaenoic acid, docosahexaenoic acid or both polyunsaturated fatty acids.

Esterasa leucocitaria como prueba diagnóstica ante un proceso infeccioso articular de rodilla / Leukocyte esterase as a diagnostic tool for an infectious disease of the knee

Resumen: Antecedentes: La infección articular es un reto ortopédico por su complejidad diagnóstica y efectos devastadores al no tratarse oportunamente. Contamos con diversos estudios de diagnóstico: cultivo, VSG, PCR, conteo de leucocitos, entre otros, pero ninguno es preciso, tardan más de 30 minutos en realizarse y requieren de infraestructura compleja. En este estudio determinamos la sensibilidad y especificidad de la esterasa leucocitaria para la detección de un proceso infeccioso articular en población mexicana. Material y métodos: Durante Noviembre de 2015 a Abril de 2016, se obtuvo líquido sinovial de pacientes con diagnóstico de infección articular con o sin implante, y de otros sin infección, con patología degenerativa de rodilla. Se evaluó la muestra mediante el test de esterasa leucocitaria COMBI-SCREEN 11SYS con lectura colorimétrica a los dos minutos; se determinó positivo para infección con dos cruces; el resto de la muestra fue enviado a cultivo. Resultados: Realizamos el test en 64 muestras de líquido sinovial de rodilla, 19 diagnosticadas con infección articular y 45 sin infección. Se obtuvo una sensibilidad de 100%, especificidad de 88.24%, VPP de 68.42% y VPN de 100%; índice de Kappa de .753. Conclusiones: La esterasa leucocitaria es una prueba eficaz para detectar un proceso infeccioso contra uno inflamatorio con alta probabilidad de acierto. Este estudio presentó un índice de concordancia Kappa de 0.753, con lo que demostró ser reproducible, por lo que recomendamos implementarlo en los servicios de urgencias a nivel nacional.

Abstract: Background: Articular infection is an orthopedic challenge due to its difficult diagnosis and devastating results. Various diagnostic studies exist: culture, ESR, CRP, count of leukocytes, among others, but none is specific, they all take more than 30 minutes to complete, and require complex infrastructure. In this study, we determine the sensitivity and specificity of the leukocyte esterase for detection of an infectious process joint in Mexican population. Material and methods: During November 2015 to April 2016, we obtained synovial fluid from two groups of patients: one with a diagnosis of synovial joint infection with or without implant, and the control group, without infection but with degenerative pathology of the knee. We evaluated the sample using the leukocyte esterase test COMBI-SCREEN 11SYS with colorimetric reading at two minutes; two crosses determined positive for infection; the remainder of the sample was sent for culture. Results: We performed the test in 64 samples of synovial fluid, 19 diagnosed with articular infection and 45 without it. We obtained a sensitivity of 100%, specificity of 88.24%, PPV of 68.42%, and NPV of 100%; Kappa index of .753. Conclusions: Leukocyte esterase is an effective test to detect an infectious process against an inflammatory one with a high probability of success. This study presented an index of agreement Kappa of 0.753, proving to be reproducible.

Effects of vitamin A, C and E, or omega-3 fatty acid supplementation on the level of paraoxonase and arylesterase activity in streptozotocin-induced diabetic rats: an investigation of activities in plasma, and heart and liver homogenates

<p><b>INTRODUCTION</b>This study was designed and conducted to evaluate the effects of vitamin A, C and E supplementation, and omega-3 fatty acid supplementation on the activity of paraoxonase and arylesterase in an experimental model of diabetes mellitus.</p><p><b>METHODS</b>A total of 64 male Sprague Dawley® rats, each weighing 250 g, were randomly distributed into four groups: (a) normal control; (b) diabetic control; (c) diabetic with vitamin A, C and E supplementation; and (d) diabetic with omega-3 fatty acid supplementation. The animals were anaesthetised after four weeks of intervention, and paraoxonase and arylesterase activity in blood plasma, and liver and heart homogenates were measured.</p><p><b>RESULTS</b>Arylesterase activity in the heart and liver homogenates was significantly lower in the diabetic control group than in the normal control group (p < 0.01). Vitamin A, C and E supplementation, and omega-3 fatty acid supplementation significantly increased liver arylesterase activity (p < 0.05). No significant change was observed in paraoxonase activity and other investigated factors.</p><p><b>CONCLUSION</b>Vitamin A, C and E, or omega-3 fatty acid supplementation were found to increase liver arylesterase activity in streptozotocin-induced diabetic rats. These supplements may be potential agents for the treatment of diabetes mellitus complications.</p>

Characterization of a multi-tolerant tannin acyl hydrolase II from Aspergillus carbonarius produced under solid-state fermentation

Background Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20-60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application.

EVALUATION OF LEUKOCYTE ESTERASE REAGENT STRIPS TEST IN THE DIAGNOSIS OF SPONTANEOUS BACTERIAL PERITONITIS IN CHILDREN WITH CIRRHOSIS / Avaliação de tiras reagentes de teste de esterase de leucócitos no diagnóstico de peritonite bacteriana espontânea em crianças com cirrose

BackgroundSpontaneous bacterial peritonitis is defined as an ascetic fluid infection without an evident intra-abdominal surgically treatable source. Spontaneous bacterial peritonitis is one of the severe complications in patients with cirrhosis and ascites. Without early antibiotic treatment, this complication is associated with high mortality rate; therefore, early diagnosis and treatment of spontaneous bacterial peritonitis is necessary for survival. Leukocyte esterase reagent can rapidly diagnose the spontaneous bacterial peritonitis.ObjectiveThis study aimed to find out the diagnostic accuracy of leukocyte esterase dipstick test for the diagnosis of spontaneous bacterial peritonitis.MethodsA single centered hospital-based cross-sectional study was conducted during July 2013 to August 2014 on children with cirrhotic liver disease and ascites who were admitted in the Department of Pediatric Gastroenterology in Nemazee Hospital affiliated to Shiraz University of Medical Sciences (Iran). All patients underwent abdominal paracentesis, and the ascitic fluid was processed for cell count, leukocyte esterase reagent strip test (Combiscreen SL10) and culture. Spontaneous bacterial peritonitis was defined as having a polymorphonuclear count (PMN ≥250/m3) in ascitic fluid. Sensitivity, specificity, positive predictive value and negative predictive value of leukocyte esterase test were calculated according to the formula.ResultsTotally, 150 ascitic fluid sample of cirrhotic male patients (53.2%) and their mean age (4.33±1.88 years) were analyzed. Biliary atresia (n=44, 29.4%) and idiopathic neonatal hepatitis (n=29, 19.3%) were the most frequent etiology of cirrhosis. Also, abdominal pain (68.6%) and distension (64%) were the most common presenting complaint. Of all cases, 41patients (27.35%) were diagnosed to have spontaneous bacterial peritonitis (PMN ≥250/mm3). Sensitivity and specificity of leukocyte esterase reagent test according to PMNs ≥250mm3 were 87.80% and 91.74%, also on ascitic fluid culture results were 88.23% and 77.44%. Positive predictive value and negative predictive value of this test in PMNs ≥250mm3 were 80% and 95.23% and in cases with positive culture 33.33% and 98.09% were obtained, respectively. Efficiency of leukocyte esterase reagent test in diagnosing spontaneous bacterial peritonitis, according to PMNs ≥250mm3 and culture results were 90.66% and 78.66%.ConclusionThe leukocyte esterase strip test may be used as rapid test for diagnosis of spontaneous bacterial peritonitis due to its high diagnostic validity.

ContextoA peritonite bacteriana espontânea é definida como uma infecção do fluido ascítico sem evidente origem intra-abdominal cirurgicamente tratável. A peritonite bacteriana espontânea é uma das complicações graves em pacientes com cirrose e ascite. Sem tratamento antibiótico precoce, esta complicação é associada com alta taxa de mortalidade. Portanto, o diagnóstico precoce e tratamento de peritonite bacteriana espontânea são necessários para a sobrevivência. O reagente de esterase de leucócitos pode rapidamente diagnosticar a peritonite bacteriana espontânea.ObjetivoEste estudo teve como objetivo descobrir a acurácia diagnóstica do teste com tiras de esterase de leucócitos para o diagnóstico de peritonite bacteriana espontânea.MétodosUm estudo transversal hospitalar unicêntrico foi realizado entre julho de 2013 e agosto de 2014 em crianças com cirrose hepática e ascite que foram admitidas no Departamento de Gastroenterologia Pediátrica no Hospital de Nemazee afiliado à Universidade de Ciencias Médicas de Shiraz (Irã). Todos os pacientes foram submetidos a paracentese abdominal, e o líquido ascítico foi processado para contagem de células, teste de tira de reagente de esterase de leucócitos (Combiscreen SL10) e cultura. Peritonite bacteriana espontânea foi definida como tendo uma contagem de polimorfonucleares (PMN ≥250/m3) no líquido ascítico. Sensibilidade, especificidade, valor preditivo positivo negativo do teste de esterase de leucócitos foram calculados de acordo com a fórmula.ResultadosForam analisados um total de 150 amostras de líquido ascítico de pacientes cirróticos; (53,2%) eram do sexo masculino e sua média de idade (4,33±1,88 anos). A atresia biliar (n=44, 29,4%) e hepatite neonatal idiopática (n=29, 19,3%) foram as etiologias mais frequentes de cirrose. Além disso, dor abdominal (68,6%) e distensão (64%) foram as queixas mais comuns de apresentação. De todos os casos, 41 (27,35%) foram diagnosticados com peritonite bacteriana espontânea (PMN ≥250/mm3). A sensibilidade e especificidade do teste de reagente de esterase de leucócitos segundo PMN ≥250mm3 foi de 87,80% e 91,74% e, para os resultados de cultura de líquido ascítico, de 88,23% e 77,44%. Valor preditivo positivo e valor preditivo negativo do teste em PMN ≥250mm3 foi de 80% e 95,23% e em casos com cultura positiva 33,33% e 98,09%, respectivamente. A eficiência do teste de reagente esterase de leucócitos no diagnóstico de peritonite bacteriana espontânea, de acordo com resultados de ≥250mm3 e cultura PMN, foi de 90,66% e 78,66%.ConclusãoO teste de tiras de esterase de leucócitos pode ser usado como um teste rápido para diagnóstico de peritonite bacteriana espontânea, devido a sua alta validade diagnóstica.

Construction and expression of two-copy engineered yeast of feruloyl esterase

Background Aspergillus niger has the ability to secrete feruloyl esterase. However, for economically viable industrial applications, it is necessary to increase their catalytic activities and/or protein yields to satisfy the increasing needs for feruloyl esterases. Results The gene AnFaeA that encodes a type A feruloyl esterase was successfully expressed in Pichia pastoris by a two-copy engineered yeast. After a screen in shaker flask, a one-copy strain GSKFA3 having the highest feruloyl esterase activity of 2.4 U/mL was obtained. Then, the pPICZaA-AnFaeA plasmid was transformed into GSKFA3 and the transformants were grown on YPDS plates with antibiotic Zeocin. After cultivation, a two-copy strain GSKZaFA20 with the highest feruloyl esterase activity of 15.49 U/mL was obtained. The expressed protein (recombinant AnFaeA) may be a glycoprotein with an apparent molecular weight of 40 kDa. It displayed the maximum activity at pH 6.0 and 50°C, and was stable at a pH range of 4.0-6.5 and at below 45°C. Its activity was not significantly affected by K+, Ca2 +, Mg2 +, Cu2 +, Zn2 +, Mn2 +, Na+ and EDTA, but activated by Fe2 +. The Km and Vmax toward 4-nitrophenyl ferulate were 5.5 mM and 69.0 U/mg, respectively. Conclusions The two-copy strain GSKZaFA20 showed a 4.4-fold increase in extracellular enzyme activity compared with the one-copy strain GSKFA3. Construction of two-copy strain improved secretion of recombinant AnFaeA in P. pastoris.

Statistical optimization for tannase production by Aspergillus tubingensis in solid-state fermentation using tea stalks

Background A sequential statistical strategy was used to optimize tannase production from Aspergillus tubingensis using tea stalks by solid-state fermentation. Results First, using a Plackett-Burman design, inoculum size and incubation time (among seven tested variables) were identified as the most significant factors for tannase yield. The effects of significant variables were further evaluated through a single steepest ascent experiment and central composite design with response surface analysis. Under optimal conditions, the experimental value of 84.24 units per gram of dry substrate (U/gds) closely matched the predicted value of 87.26 U/gds. Conclusions The result of the statistical approach was 2.09 times higher than the basal medium (40.22 U/gds). The results were fitted onto a second-order polynomial model with a correlation coefficient (R²) of 0.9340, which implied an adequate credibility of the model.