Enzyme assay, cloning and sequencing of novel β-glucosidase gene from Aspergillus niger f321 (unidentified Nigerian strain)

β-glucosidase is a cellulase enzyme under intense investigation for its potential role in cellulose degradation for the generation of fermentable sugars used in biofuels production. Ten catalytic sites have been identified that are conserved in β- glucosidases from a range of prokaryotes and eukaryotes. NCBI Primer BLAST was used in this study to design primers that successfully clone a partial β-glucosidase gene from an uncharacterised Nigerian strain of the filamentous fungi Aspergillus niger F321 (A. niger F321). Two β-glucosidase genes from A. niger F321 denoted as ANRA12.6 and ANRA12.9 were amplified from genomic DNA using PCR techniques and the amplicons gave estimated PCR products of 1,190 bp and 1,950 bp respectively. Subsequent cloning into E. coli produced positive results for blue/white screening of transformed colonies while the colony PCR of their pDNA gave estimated sizes of 860 bp and 1,600 bp respectively. DNA sequencing confirmed that the chosen A. niger F321 partial β-glucosidase sequences had been successfully cloned. Bioinformatics studies also suggested that the cloned β-glucosidases share some characteristics with their bacterial counterparts. The findings in this study highlight the increasing need for more information on β-glucosidase structure and function.Keywords: Aspergillus niger, β-glucosidase, cellulase, PCR, sequencing, Bioinformatic

Expression and characterization of recombinant β-glucosidases from Aspergillus nidulans AN2227

Recombinant β-glucosidase (EC 3.2.1.21) from Aspergillus nidulans AN2227 was expressed using Buffered Methanol Complex Medium (BMMY). Purification was conducted using ammonium sulphate precipitation and anion exchange chromatography on DEAE-Sephadex A-50 column. The enzyme was purified 2.58 fold from the crude extract. β-glucosidase was purified to electrophoretic homogeneity and had a relative molecular weight of 100 kDa as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH and temperature of 6.0 and 40 oC respectively. The most striking characteristics of this enzyme are the dramatically broad pH and temperature profile. The enzyme also had a Km of 0.42 mM for 4-Nitrophenyl-β-D-glucopyranoside (pNPG). The activity of the enzyme was inhibited by HgCl2 and slightly activated by CoCl2, FeCl3, CaCl2, FeCl2 and ZnCl2 suggesting that the enzyme may not be a metalloprotein and therefore does not require metal ions for optimum activity.Keywords: Aspergillus nidulans, Cellulase, β-glucosidase, p-nitrophenol-β-glucopyranoside

Improving survival of probiotic bacteria using bacterial poly-γ-glutamic acid

A major hurdle in producing a useful probiotic food product is bacterial survival during storage and ingestion. The aim of this study was to test the effect of γ-PGA immobilisation on the survival of probiotic bacteria when stored in acidic fruit juice. Fruit juices provide an alternative means of probiotic delivery, especially to lactose intolerant individuals. In addition, the survival of γ-PGA-immobilised cells in simulated gastric juice was also assessed. Bifidobacteria strains (B. longum, B. breve), immobilised on 2.5 % γ-PGA, survived significantly better (P < 0.05) in orange and pomegranate juice for 39 and 11 days respectively, compared to free cells. However, cells survived significantly better (P < 0.05) when stored in orange juice compared to pomegranate juice. Moreover, both strains, when protected with 2.5 % γ-PGA, survived in simulated gastric juice (pH 2.0) with a marginal reduction (<0.47 log CFU/ml) or no significant reduction in viable cells after four hours, whereas free cells died within two hours. In conclusion, this research indicates that γ-PGA can be used to protect Bifidobacteria cells in fruit juice, and could also help improve the survival of cells as they pass through the harsh conditions of the gastrointestinal tract (GIT). Following our previous report on the use of γ-PGA as a cryoprotectant for probiotic bacteria, this research further suggests that γ-PGA could be used to improve probiotic survival during the various stages of preparation, storage and ingestion of probiotic cells

Synthesis of Silver Nanoparticles Using Curcumin-Cyclodextrins Loaded into Bacterial Cellulose-Based Hydrogels for Wound Dressing Applications.

Chronic wounds are often recalcitrant to treatment because of high microbial bioburden and the problem of microbial resistance. Silver is a broad-spectrum natural antimicrobial agent with wide applications extending to proprietary wound dressings. Recently, silver nanoparticles have attracted attention in wound management. In the current study, the green synthesis of nanoparticles was accomplished using a natural reducing agent, curcumin, which is a natural polyphenolic compound that is well-known as a wound-healing agent. The hydrophobicity of curcumin was overcome by its microencapsulation in cyclodextrins. This study demonstrates the production, characterization of silver nanoparticles using aqueous curcumin:hydroxypropyl-β-cyclodextrin complex and loading them into bacterial cellulose hydrogel with moist wound-healing properties. These silver nanoparticle-loaded bacterial cellulose hydrogels were characterized for wound-management applications. In addition to high cytocompatibility, these novel dressings exhibited antimicrobial activity against three common wound-infecting pathogenic microbes , , and

Characterisation and In Vitro Antimicrobial Activity of Biosynthetic Silver-loaded Bacterial Cellulose Hydrogels

Wounds that remain in the inflammatory phase for a prolonged period of time are likely to be colonised and infected by a range of commensal and pathogenic microorganisms. Treatment associated with these types of wounds mainly focuses on controlling infection and providing an optimum environment capable of facilitating re-epithelialisation, thus promoting wound healing. Hydrogels have attracted vast interest as moist wound-responsive dressing materials. In the current study, biosynthetic bacterial cellulose hydrogels synthesised by Gluconacetobacter xylinus and subsequently loaded with silver were characterised and investigated for their antimicrobial activity against two representative wound infecting pathogens, namely S. aureus and P. aeruginosa. Silver nitrate and silver zeolite provided the source of silver and loading parameters were optimised based on experimental findings. The results indicate that both AgNO3 and AgZ loaded biosynthetic hydrogels possess antimicrobial activity (p < .05) against both S. aureus and P. aeruginosa and may therefore be suitable for wound management applications

ДЕЯКІ КЛІНІЧНІ АСПЕКТИ ЛІПІТЕНЗІЇ

The purpose of the study is to evaluate the effectiveness of modern antihypertensive therapy in combination with statin therapy and ursodeoxycholic acid (UDCA) in patients with lipentia (arterial hypertension (hypertension) and hypercholesterolemia).Material and Methods. 36 patients with hypertonic disease (HD) of stage II, grade 2-3 and hypercholesterolemia were examined, which were performed a comprehensive clinical study, monitoring blood pressure (BP), and lipid metabolism on the basis of therapy by Difors, Atorvacor and Ursochol.Results. The results of monitoring blood pressure indicate the beneficial effect of this treatment on the daily blood pressure profile: after 2 months of treatment was recorded a significant reduction in average daily systolic and diastolic blood pressure. Under the influence of atorvastatin and ursochol, lipid metabolism rates improved, which was manifested by a significant decrease in the level of total cholesterol and low density lipoprotein cholesterol, a tendency to increase the content of high density lipoprotein.Conclusions. In this article the combined use of antihypertensive therapy, statin therapy and ursochol in patients with lipentia, especially in clinical cases in which it is necessary to limit the dosage of statins in terms of improving the prognosis of patients with HD due to the synergism of antihypertensive and lipid-lowering potential and pleiotropic effects of these drugs, the effect which is aimed at slowing the progression of HD and atherosclerosis, was justified.Целью исследования является оценка эффективности современной антигипертензивной терапии в сочетании со статинотерапией и урсодезоксихолевой кислотой (УДХК) у больных с липитензией (артериальная гипертензия (АГ) и гиперхолестеринемия).Материал и методы. Обследовано 36 пациентов с гипертонической болезнью (ГБ) ІІ стадии, 2–3 степени, и гиперхолестеринемией, которым на фоне терапии дифорсом, аторвакором, урсохолом проводили комплексное клиническое исследование, мониторинг артериального давления (АД) и липидного метаболизма.Результаты. Результаты мониторинга свидетельствуют о благоприятном влиянии лечения на суточный профиль АД: через 2 месяца терапии зарегистрировано достоверное снижение среднесуточного систолического и диастолического АД. Под воздействием аторвакора и урсохола улучшились показатели липидного обмена, что проявлялось достоверным снижением уровня общего холестерина и холестерина липопротеидов низкой плотности, тенденцией к росту липопротеидов высокой плотности.Выводы. В статье обосновано использование комбинированной антигипертензивной терапии, статинотерапии и урсохола у больных с липитензией, особенно в клинических случаях, при которых необходимо ограничить дозирование статинов, с точки зрения улучшения прогноза больных с ГБ за счет синергизма антигипертензивного и липидоснижающего потенциала и плейотропних эффектов этих препаратов, действие которых направлено на замедление прогрессирования ГБ и атеросклероза.Метою дослідження є оцінка ефективності сучасної антигіпертензивної терапії у поєднанні зі статинотерапією та урсодезоксихолевою кислотою (УДХК) у хворих з ліпітензією (артеріальна гіпертензія (АГ) та гіперхолестеринемія).Матеріал і методи. Обстежено 36 пацієнтів із гіпертонічною хворобою (ГХ) ІІ стадії, 2–3 ступенів, та гіперхолестеринемією, яким на фоні терапії діфорсом, аторвакором, урсохолом проводили комплексне клінічне дослідження, моніторування артеріального тиску (АТ) та ліпідного метаболізму.Результати. Результати моніторування свідчать про сприятливий вплив вказаного лікування на добовий профіль АТ: через 2 місяці терапії зареєстровано достовірне зниження середньодобового систолічного та діастолічного АТ. Під впливом аторвастатину та урсохолу поліпшилися показники ліпідного обміну, що проявля­лось достовірним зниженням рівня загального холестерину та холестерину ліпопротеїдів низької щільності, тенденцією до зростання вмісту ліпопротеїдів високої щільності.Висновки. У статті обґрунтовано поєднане використання антигіпертензивної терапії, статинотерапії та урсохолу у хворих з ліпітензією, особливо у клінічних випадках, при яких необхідно обмежити дозування статинів, з точки зору поліпшення прогнозу хворих на ГХ за рахунок синергізму антигіпертензивного та ліпідознижувального потенціалу і плейотропних ефектів цих препаратів, дія яких направлена на сповільнення прогресування ГХ і атеросклерозу

Production and Characterisation of Bacterial Cellulose Hydrogels Loaded with Curcumin Encapsulated in Cyclodextrins as Wound Dressings

Natural bioactive materials with wound healing properties such as curcumin are attracting interest due to the emergence of resistant bacterial strains. The hydrophobicity of curcumin has been counteracted by using solubility enhancing cyclodextrins. Hydrogels facilitate wound healing due to unique properties and 3D network structures which allows encapsulation of healing agents. In this study, biosynthetic cellulose produced by Gluconacetobacter xylinus (ATCC 23770) was loaded with water soluble curcumin:hydroxypropyl-β-cyclodextrin supramolecular inclusion complex produced by a solvent evaporation method to synthesise hydrogel dressings. The ratios of solvents to solubilise curcumin and hydroxypropyl-β-cyclodextrin were tested for the production of the inclusion complex with optimum encapsulation efficacy. The results confirmed that hydroxypropyl-β-cyclodextrin enhanced the aqueous solubility of curcumin and allowed loading into bacterial cellulose hydrogels. These hydrogels were characterised for wound management applications and exhibited haemocompatability, cytocompatability, anti-staphylococcal and antioxidant abilities and therefore support the potential use of the curcumin:hydroxypropyl-β-cyclodextrin-loaded-bacterial cellulose as hydrogel dressings

Transesterification of PHA to Oligomers Covalently Bonded with (Bio)Active Compounds Containing Either Carboxyl or Hydroxyl Functionalities

© 2015 The Authors. Published by Public Library of Science. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1371/journal.pone.0120149This manuscript presents the synthesis and structural characterisation of novel biodegradable polymeric controlled-release systems of pesticides with potentially higher resistance to weather conditions in comparison to conventional forms of pesticides. Two methods for the preparation of pesticide-oligomer conjugates using the transesterification reaction were developed. The first method of obtaining conjugates, which consist of bioactive compounds with the carboxyl group and polyhydroxyalkanoates (PHAs) oligomers, is "one-pot" transesterification. In the second method, conjugates of bioactive compounds with hydroxyl group and polyhydroxyalkanoates oligomers were obtained in two-step method, through cyclic poly(3-hydroxybutyrate) oligomers. The obtained pesticide-PHA conjugates were comprehensively characterised using GPC, 1H NMR and mass spectrometry techniques. The structural characterisation of the obtained products at the molecular level with the aid of mass spectrometry confirmed that both of the synthetic strategies employed led to the formation of conjugates in which selected pesticides were covalently bonded to PHA oligomers via a hydrolysable ester bond