Traditional Uses of Animals in the Himalayan Region of Azad Jammu and Kashmir.

Background: The use of animals and animal-derived products in ethnopharmacological applications is an ancient human practice that continues in many regions today. The local people of the Himalayan region harbor rich traditional knowledge used to treat a variety of human ailments. The present study was intended with the aim of examining animal-based traditional medicine utilized by the population of the Himalayan region of Azad Jammu and Kashmir. Methods: Data were collected from 2017 to 2019 through individual and group interviews. Data on traditional uses of animal products were analyzed, utilizing following indices such as the frequency of citation, use value, relative importance, similarity index, principal component analysis, and cluster analysis to find the highly preferred species in the area. Results: Ethnomedicinal uses of 62 species of vertebrates and invertebrates were documented. Flesh, fat, bone, whole body, milk, skin, egg, head, feathers, bile, blood, and honey were all used in these applications. The uses of 25 animals are reported here for the first time from the study area (mainly insects and birds, including iconic species like the kalij pheasant, Lophura leucomelanos; Himalayan monal, L. impejanus; and western tragopon, Tragopan melanocephalus). The diversity and range of animal-based medicines utilized in these communities are indications of their strong connections with local ecosystems. Conclusion: Our results provide baseline data valuable for the conservation of vertebrate and invertebrate diversity in the region of Himalayan of Azad Jammu and Kashmir. It is possible that screening this fauna for medicinally active chemicals could contribute to the development of new animal-based drugs.

Production, partial purification and characterization of ligninolytic enzymes from selected basidiomycetes mushroom fungi.

In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 × 10-6 IU/ml) and manganese peroxidase (24.11 × 10-6 IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 × -6 IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.

Carbapenemases producing Klebsiella pneumoniae from the pus of hospitalized patients: In-vitro antibiotic properties of Streptomyces against multidrug resistant infectious bacteria.

BACKGROUND: Klebsiella pneumoniae is predominantly exists in the pus of the human wounds and it creates massive infections in the skin and causes serious health associated infections. Modern antibiotics are highly active in the treatment of wound infections. In this study was aimed to determine resistance of K. pneumoniae screened from wound specimens of patients. Sample was collected from the pus of the patients associated with secondary infection. METHODS: Samples were serially diluted and the isolated bacterial strains were characterized by biochemical tests, colony morphology and Gram's staining methods. Resistance of K. pneumoniae was tested using antibiotics such as, Gentamycin, Ampicillin, Tetracycline, Cefurooxime, Oxacillin, Ofloxacin, Erythromycin, Nalidic acid, Cefepine, Piperacillin, Norfloxacin, Imipenem, Nitrofurantoin, Amikacin, Ciprofloxacin, Vancomycin, Meropeneum and Cefotaxime with Kirby-Bauer disc diffusion method. RESULTS: Among the 73 K. pneumoniae strains, four strains produced AmpC and ESBLs, 42 strains produced ESBLs and 7 bacterial strains synthesized only AmpC enzyme. Four stains produced ESBLs and showed multidrug resistance against various antibiotics. Most of the strains synthesized extracellular polysaccharides and mediated biofilm formation. Among the K. pneumoniae strains, K. pneumoniae PS02 showed multidrug resistant against most of the tested antibiotics. It produced ESBLs and AmpC enzyme. To produce secondary metabolites, actinomycetes were isolated and characterized as Streptomyces sp. AC14. The secondary metabolite was effective against Klebsiella strains. CONCLUSIONS: To conclude, secondary metabolites extracted from Streptomyces sp. AC14 was found to be effective against multidrug resistant bacterium. Further studies are warranted to analyze the drug hydrolyzing pathways of bacteria and to identify the mechanism of action of secondary metabolites from Streptomyces sp. AC14.

A novel biogenic Allium cepa leaf mediated silver nanoparticles for antimicrobial, antioxidant, and anticancer effects on MCF-7 cell line.

In the present study, Allium cepa leaf extract was utilized to reduce the silver nitrate into the nanoscale range of silver ions (Ag NPs). The biosynthesized Ag NPs were extensively characterized by X-ray diffraction analysis (XRD), Dynamic light scattering analysis (DLS), UV-Visible spectroscopy (UV-vis), Transmission electron microscopy (TEM), Energy dispersive X-ray analysis (EDX) and Fourier transform infrared spectroscopy (FTIR). The antioxidant activity of synthesized Ag NPs was verified by DPPH assay. From the results obtained from XRD and DLS studies, the size of Ag NPs was determined to be around 54.3 nm. The measured zeta potential value of -19.1 mV confirms the excellent stability of biosynthesized Ag NPs. TEM analyses reveal that the biosynthesized Ag NPs have a spherical structure of 13 nm in size. The presence of various functional groups was confirmed through FTIR studies and EDAX verifies the weight percentage of silver content in biosynthesized nanoparticles to be 30.33%. In the present study, anti-cancer activity was carried out by using breast cancer cell line MCF-7. Further, silver nanoparticles exhibited antimicrobial effectiveness against gram-positive Bacillus cereus and gram-negative Escherichia coli. The MTT assay also showed better cytotoxic activity against the MCF- 7 cell line.

Partial purification and characterization of 2, 4-diacetylphloroglucinol producing Pseudomonas fluorescens VSMKU3054 against bacterial wilt disease of tomato.

We find out the antimicrobial potential of partially purified 2,4-diacetylphloroglucinol (DAPG) against Ralstonia solanacearum and fungal plant pathogens isolated from tomato rhizobacterium Pseudomonas fluorescens VSMKU3054. The present study is mainly focused on the control of wilt disease of tomato by our isolate VSMKU3054 and DAPG. The cell free culture filtrate of P. fluorescens VSMKU3054 was significantly arrested the growth of R. solanacearum and fungal pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Macrophomina phaseolina and Fusarium oxysporum compared to control. The existence of DAPG from the crude metabolites of P. fluorescens VSMKU3054 was confirmed on TLC with Rf value 0.34, which is coincide with that of authentic phloroglucinol. The partially purified DAPG exhibited much higher activity against R. solanacearum at 30 µg/ml than the fungal plant pathogens compared to control. The antimicrobial partially purified compound was identified as DAPG by UV, FT-IR and GC-MS analysis. The percentage of live cells of R. solanacearum when supplemented with DAPG at 30 µg/ml, significantly controlled the living nature of R. solanacearum up to 68% compared to tetracycline and universal control observed under high content screening analysis. The selected isolate P. fluorescens VSMKU3054 and DAPG significantly controlled wilt disease of tomato up to 59.5% and 42.12% on 3rd and 7th days compared to positive and negative control by detached leaf assay. Further, in silico analysis revealed that high interaction of DAPG encoding protease with lectin which is associated with R. solanacearum. Based on our findings, we confirmed that P. fluorescens VSMKU3054 and DAPG could be used a potential bio inoculants for the management of bacterial wilt disease of tomato.

Stabilization and improved properties of Salipaludibacillus agaradhaerens alkaline protease by immobilization onto double mesoporous core-shell nanospheres.

In this study, serine alkaline protease from halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R was purified and immobilized onto double mesoporous core-shell silica (DMCSS) nanospheres. Covalent immobilization of AK-R protease onto activated DMCSS-NH2 nanospheres was more efficient than physical adsorption and was applied in further studies. DMCSS-NH2 nanospheres showed high loading capacity of 103.8 µg protein/mg nanospheres. Relative to free AK-R protease, the immobilized enzyme exhibited shifts in the optimal temperature and pH from 60 to 65 °C and pH 10.0 to 10.5, respectively. While the soluble enzyme retained 47.2% and 9.1% of its activity after treatment for 1 h at 50 and 60 °C, the immobilized protease maintained 87.7% and 48.3%, respectively. After treatment for 2 h at pH 5 and 13, the immobilized protease maintained 73.6% and 53.4% of its activity, whereas the soluble enzyme retained 32.9% and 1.4%, respectively. Furthermore, the immobilized AK-R protease showed significant improvement of enzyme stability in high concentration of NaCl, organic solvents, surfactants, and commercial detergents. In addition, the immobilized protease exhibited a very good operational stability, retaining 79.8% of its activity after ten cycles. The results clearly suggest that the developed immobilized protease system is a promising nanobiocatalyst for various protease applications.

Alkaline serine protease from the new halotolerant alkaliphilic Salipaludibacillus agaradhaerens strain AK-R: purification and properties.

Herein, we report the purification and characterization of an alkaline protease from the alkaliphilic Salipaludibacillus agaradhaerens (formerly Bacillus agaradhaerens) strain AK-R, which was previously isolated from Egyptian soda lakes. The purification procedures resulted in enzyme purification up to 13.3-fold, with a recovery yield of 16.3% and a specific activity of 3488 U/mg protein. AK-R protease was a monomeric protein with an estimated molecular weight of 33.0 kDa. The optimum pH and temperature for AK-R protease were pH 10 and 60 °C, respectively. The enzyme thermostability was significantly enhanced in the presence of CaCl2 by approximately 1.3-fold. Moreover, under optimal conditions, the K m and V max values of the enzyme were 2.63 mg/ml and 4166.7 U/mg, respectively. PMSF caused complete inhibition of the enzyme activity, suggesting that AK-R belongs to the serine protease family. In addition, the enzyme was completely inhibited by EDTA, revealing the requirement of metal ions for AK-R protease activity; hence, it can be classified as a metalloprotease. AK-R protease is a mostly thiol-independent enzyme, since thiol reductants such as ß-mercaptoethanol and dithiothreitol had no effect on the enzyme activity. AK-R protease exhibited high stability in several organic solvents, including butanol, amyl alcohol, dimethyl ether, toluene, diethyl ether and methanol. Moreover, AK-R protease showed significant stability to a variety of surfactants and commercial detergents. The features and properties of AK-R alkaline protease are favourable and suggest its potential applications in various industries, particularly in the laundry detergent industry.

Prevalence, serotyping and antimicrobials resistance mechanism of Salmonella enterica isolated from clinical and environmental samples in Saudi Arabia

Abstract Salmonella is recognized as a common foodborne pathogen, causing major health problems in Saudi Arabia. Herein, we report epidemiology, antimicrobial susceptibility and the genetic basis of resistance among S. enterica strains isolated in Saudi Arabia. Isolation of Salmonella spp. from clinical and environmental samples resulted in isolation of 33 strains identified as S. enterica based on their biochemical characteristics and 16S-rDNA sequences. S. enterica serovar Enteritidis showed highest prevalence (39.4%), followed by S. Paratyphi (21.2%), S. Typhimurium (15.2%), S. Typhi and S. Arizona (12.1%), respectively. Most isolates were resistant to 1st and 2nd generation cephalosporin; and aminoglycosides. Moreover, several S. enterica isolates exhibited resistance to the first-line antibiotics used for Salmonellosis treatment including ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. In addition, the results revealed the emergence of two S. enterica isolates showing resistance to third-generation cephalosporin. Analysis of resistance determinants in S. enterica strains (n = 33) revealed that the resistance to β-lactam antibiotics, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline, was attributed to the presence of carb-like, dfrA1, floR, tetA gene, respectively. On the other hand, fluoroquinolone resistance was related to the presence of mutations in gyrA and parC genes. These findings improve the information about foodborne Salmonella in Saudi Arabia, alarming the emergence of multi-drug resistant S. enterica strains, and provide useful data about the resistance mechanisms.

Prevalence, serotyping and antimicrobials resistance mechanism of Salmonella enterica isolated from clinical and environmental samples in Saudi Arabia.

Salmonella is recognized as a common foodborne pathogen, causing major health problems in Saudi Arabia. Herein, we report epidemiology, antimicrobial susceptibility and the genetic basis of resistance among S. enterica strains isolated in Saudi Arabia. Isolation of Salmonella spp. from clinical and environmental samples resulted in isolation of 33 strains identified as S. enterica based on their biochemical characteristics and 16S-rDNA sequences. S. enterica serovar Enteritidis showed highest prevalence (39.4%), followed by S. Paratyphi (21.2%), S. Typhimurium (15.2%), S. Typhi and S. Arizona (12.1%), respectively. Most isolates were resistant to 1st and 2nd generation cephalosporin; and aminoglycosides. Moreover, several S. enterica isolates exhibited resistance to the first-line antibiotics used for Salmonellosis treatment including ampicillin, trimethoprim-sulfamethoxazole and chloramphenicol. In addition, the results revealed the emergence of two S. enterica isolates showing resistance to third-generation cephalosporin. Analysis of resistance determinants in S. enterica strains (n=33) revealed that the resistance to ß-lactam antibiotics, trimethoprim-sulfamethoxazole, chloramphenicol, and tetracycline, was attributed to the presence of carb-like, dfrA1, floR, tetA gene, respectively. On the other hand, fluoroquinolone resistance was related to the presence of mutations in gyrA and parC genes. These findings improve the information about foodborne Salmonella in Saudi Arabia, alarming the emergence of multi-drug resistant S. enterica strains, and provide useful data about the resistance mechanisms.

Alkaline protease from a new halotolerant alkaliphilic Bacillus agaradhaerens strain ak-r isolated from egyptian soda lakes / Protease alcalina de uma nova estirpe ak-r halotolerante e alcalófila de Bacillus agaradhaerens isolada a partir dos lagos alcalinos egípcios

Alkaline proteases are hydrolytic enzymes that cleave peptide bonds in proteins and peptides in alkaline conditions, which occupy a pivotal importance with respect to their industrial applications. This study aimed to isolate new alkaline protease producing alkaliphilic bacteria from Egyptian soda lakes and optimize the fermentation process to enhance the enzyme production. The extensive screening process of the samples collected from Egyptian soda lakes resulted in isolation of a potent alkaline protease producing alkaliphilic strain AK-R. The isolate was identified as Bacillus agaradhaerens strain AK-R based on 16S rRNA gene analysis (99%). Wheat bran and gelatin supported maximum alkaline protease production as carbon and nitrogen sources, respectively. Strain AK-R is halo-tolerant thermotolerant alkaliphilic bacterium in nature, as it can grow over a wide range of NaCl concentrations (up to 25%) and up to 55 °C, with maximal growth and enzyme production at 2.5-5%, and pH 11 at 35 °C. Among the tested cations, only Mg2+ and Ca2+ ions significantly enhanced the enzyme production by about 1.2, and 1.3 fold compared to control, respectively. Alkaline protease secretion was coherent with the growth pattern, reaching maximal yield after about 32 h (mid stationary phase). In conclusion a new halo-tolerant thermo-tolerant alkaliphilic alkaline protease producing Bacillus agaradhaerens strain AK-R was isolated from Egyptian soda lakes. Optimization of the nutritional and cultivation conditions resulted in increase of enzyme yield by 20 fold. Strain AK-R and its extracellular alkaline protease with salt, pH and temperature, tolerance signify their potential application in laundry and pharmaceuticals industries.

Proteases alcalinas são enzimas hidrolíticas que quebram ligações peptídicas em proteínas e peptídeos em condições alcalinas, o que ocupa uma importância fundamental em relação às suas aplicações industriais. Este estudo teve como objetivo isolar novas proteases alcalinas e produzir bactérias alcalófilas a partir dos lagos salgados alcalinos egípcios e otimizar o processo de fermentação para aumentar a produção de enzimas. O extensivo processo de triagem das amostras coletadas dos lagos salgados alcalinos egípcios resultou no isolamento de uma protease alcalina potente produzindo uma estirpe alcalófila AK-R. O isolado foi identificado como sendo a estirpe AK-R de Bacillus agaradhaerens baseado na análise de genes 16S rRNA (99%). O farelo de trigo e a gelatina suportaram a produção máxima de protease alcalina como fontes de carbono e nitrogênio, respectivamente. A estirpe AK-R é uma bactéria alcalófila halotolerante e termotolerante, pois pode crescer dentro de uma vasta gama de concentrações de NaCl (até 25%) e até 55ºC, com crescimento e produção de enzimas máximos a 2.5-5% e pH 11 a 35ºC. Dentre os cátions testados, somente os íons Mg2+ e Ca2+ aumentaram significativamente a produção de enzimas em cerca de 1.2 e 1.3 em comparação ao controle, respectivamente. A secreção de protease alcalina foi coerente com o padrão de crescimento, atingindo o rendimento máximo após 32h (fase estacionária média). Pode-se concluir que uma nova estirpe AK-R de Bacillus agaradhaerens halotolerante, termotolerante e alcalófila produtora de protease alcalina foi isolada a partir dos lagos salgados alcalinos egípcios. A otimização das condições de nutrição e cultivo resultou num aumento da produção de enzima em 20 vezes. A estirpe AK-R e a sua protease alcalina extracelular com tolerância ao sal, pH e temperatura tornam significantes as suas potenciais aplicações nas indústrias farmacêutica e de lavanderia.

Development of novel robust nanobiocatalyst for detergents formulations and the other applications of alkaline protease.

Alkaline protease from alkaliphilic Bacillus sp. NPST-AK15 was immobilized onto functionalized and non-functionalized rattle-type magnetic core@mesoporous shell silica (RT-MCMSS) nanoparticles by physical adsorption and covalent attachment. However, the covalent attachment approach was superior for NPST-AK15 protease immobilization onto the activated RT-MCMSS-NH2nanoparticles and was used for further studies. In comparison to free protease, the immobilized enzyme exhibited a shift in the optimal temperature and pH from 60 to 65 °C and pH 10.5-11.0, respectively. While free protease was completely inactivated after treatment for 1 h at 60 °C, the immobilized enzyme maintained 66.5% of its initial activity at similar conditions. The immobilized protease showed higher k cat and K m , than the soluble enzyme by about 1.3-, and 1.2-fold, respectively. In addition, the results revealed significant improvement of NPST-AK15 protease stability in variety of organic solvents, surfactants, and commercial laundry detergents, upon immobilization onto activated RT-MCMSS-NH2nanoparticles. Importantly, the immobilized protease maintained significant catalytic efficiency for ten consecutive reaction cycles, and was separated easily from the reaction mixture using an external magnetic field. To the best of our knowledge this is the first report about protease immobilization onto rattle-type magnetic core@mesoporous shell silica nanoparticles that also defied activity-stability tradeoff. The results clearly suggest that the developed immobilized enzyme system is a promising nanobiocatalyst for various bioprocess applications requiring a protease.

Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres.

The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS-NH2 nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 µg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher V(max), k(cat) and k(cat)/K(m), than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

Production of extracellular alkaline protease by new halotolerant alkaliphilic Bacillus sp. NPST-AK15 isolated from hyper saline soda lakes

Background Alkaline proteases are among the most important classes of industrial hydrolytic enzymes. The industrial demand for alkaline proteases with favorable properties continues to enhance the search for new enzymes. The present study focused on isolation of new alkaline producing alkaliphilic bacteria from hyper saline soda lakes and optimization of the enzyme production. Results A new potent alkaline protease producing halotolerant alkaliphilic isolate NPST-AK15 was isolated from hyper saline soda lakes, which affiliated to Bacillus sp. based on 16S rRNA gene analysis. Organic nitrogen supported enzyme production showing maximum yield using yeast extract, and as a carbon source, fructose gave maximum protease production. NPST-AK15 can grow over a broad range of NaCl concentrations (0-20%), showing maximal growth and enzyme production at 0-5%, indicated the halotolerant nature of this bacterium. Ba and Ca enhanced enzyme production by 1.6 and 1.3 fold respectively. The optimum temperature and pH for both enzyme production and cell growth were at 40°C and pH 11, respectively. Alkaline protease secretion was coherent with the growth pattern, started at beginning of the exponential phase and reached maximal in mid stationary phase (36 h). Conclusions A new halotolerant alkaliphilic alkaline protease producing Bacillus sp. NPST-AK15 was isolated from soda lakes. Optimization of various fermentation parameters resulted in an increase of enzyme yield by 22.8 fold, indicating the significance of optimization of the fermentation parameters to obtain commercial yield of the enzyme. NPST-AK15 and its extracellular alkaline protease with salt tolerance signify their potential applicability in the laundry industry and other applications.

Cyclodextrin glucanotransferase immobilization onto functionalized magnetic double mesoporous core-shell silica nanospheres

Background Cyclodextrin glucanotransferase (CGTase) from Amphibacillus sp. NPST-10 was covalently immobilized onto amino-functionalized magnetic double mesoporous core-shell silica nanospheres (mag@d-SiO2@m-SiO2-NH2), and the properties of the immobilized enzyme were investigated. The synthesis process of the nanospheres included preparing core magnetic magnetite (Fe3O4) nanoparticles, coating the Fe3O4 with a dense silica layer, followed by further coating with functionalized or non-functionalized mesoporous silica shell. The structure of the synthesized nanospheres was characterized using TEM, XRD, and FT-IR analyses. CGTase was immobilized onto the functionalized and non-functionalized nanospheres by covalent attachment and physical adsorption. Results The results indicated that the enzyme immobilization by covalent attachment onto the activated mag@d-SiO2@m-SiO2-NH2, prepared using anionic surfactant, showed highest immobilization yield (98.1%), loading efficiency (96.2%), and loading capacity 58 µg protein [CGTase]/mg [nanoparticles]) which were among the highest yields reported so far for CGTase. Compared with the free enzyme, the immobilized CGTase demonstrated a shift in the optimal temperature from 50°C to 50-55°C, and showed a significant enhancement in the enzyme thermal stability. The optimum pH values for the activity of the free and immobilized CGTase were pH 8 and pH 8.5, respectively, and there was a significant improvement in pH stability of the immobilized enzyme. Moreover, the immobilized CGTase exhibited good operational stability, retaining 56% of the initial activity after reutilizations of ten successive cycles. Conclusion The enhancement of CGTase properties upon immobilization suggested that the applied nano-structured carriers and immobilization protocol are promising approach for industrial bioprocess for production of cyclodextrins using immobilized CGTase.

Immobilization of cyclodextrin glucanotransferase on aminopropyl-functionalized silica-coated superparamagnetic nanoparticles

Background: Cyclodextrin glycosyltransferase (CGTase) from Amphibacillus sp. NPST-10 was successfully covalently immobilized on aminopropyl-functionalized silica coated superparamagnetic nanoparticles; and the properties of immobilized enzyme were investigated. The synthesis process included preparing of core magnetic magnetite (Fe3O4) nanoparticles using solvothermal synthesis; followed by coating of Fe3O4 nanoparticles with dense amino-functionalized silica (NH2-SiO2) layer using in situ functionalization method. The structure of synthesized Fe3O4@NH2-SiO2 nanoparticles was characterized using TEM, XRD, and FT-IR analysis. Fe3O4@NH2-SiO2 nanoparticles were further activated by gluteraaldehyde as bifunctional cross linker, and the activated nanoparticles were used for CGTase immobilization by covalent attachment. Results: Magnetite nanoparticles was successfully synthesized and coated with and amino functionalized silica layer (Fe3O4/NH2-SiO2), with particle size of 50-70 nm. The silica coated magnetite nanoparticles showed with saturation magnetization of 65 emug-1, and can be quickly recovered from the bulk solution using an external magnet within 10 sec. The activated support was effective for CGTase immobilization, which was confirmed by comparison of FT-IR spectra of free and immobilized enzyme. The applied approach for support preparation, activation, and optimization of immobilization conditions, led to high yields of CGTase immobilization (92.3%), activity recovery (73%), and loading efficiency (95.2%); which is one of the highest so far reported for CGTase. Immobilized enzyme showed shift in the optimal temperature from 50 to 55ºC, and significant enhancement in the thermal stability compared with free enzyme. The optimum pH for enzyme activity was pH 8 and pH 7.5 for free and immobilized CGTase, respectively, with slight improvement of pH stability of immobilized enzyme. Furthermore, kinetic studies revealed that immobilized CGTase had higher affinity toward substrate; with k m values of 1.18 ± 0.05 mg/ml and 1.75 ± 0.07 mg/ml for immobilized and free CGTase, respectively. Immobilized CGTase retained 87% and 67 of its initial activity after 5 and 10 repeated batches reaction, indicating that immobilized CGTase on Fe3O4/NH2-SiO2 had good durability and magnetic recovery. Conclusion: The improvement in kinetic and stability parameters of immobilized CGTase makes the proposed method a suitable candidate for industrial applications of CGTase. To best of our knowledge, this is the first report about CGTase immobilization on silica coated magnetite nanoparticles.

Hexavalent chromate reduction by alkaliphilic Amphibacillus sp. KSUCr3 is mediated by copper-dependent membrane-associated Cr(VI) reductase.

The present study was aimed to localize and characterize hexavalent chromate [Cr(VI)] reductase activity of the extreme alkaliphilic Amphibacillus sp. KSUCr3 (optimal growth pH 10.5). The resting cells were able to reduce about 62 % of the toxic heavy metal Cr(VI) at initial concentration of 200 µM within 30 min. Cell permeabilization resulted in decrease of Cr(VI) reduction in comparison to untreated cells. Enzymatic assays of different sub-cellular fractions of Amphibacillus sp. KSUCr3 demonstrated that the Cr(VI) reductase was mainly associated with the membranous fraction and expressed constitutively. In vitro studies of the crude enzyme indicated that copper ion was essential for Cr(VI) reductase activity. In addition, Ca²âº and Mn²âº slightly stimulated the chromate reductase activity. Glucose was the best external electron donor, showing enhancement of the enzyme activity by about 3.5-fold. The K (m) and V (max) determined for chromate reductase activity in the membranous fraction were 23.8 µM Cr(VI) and 72 µmol/min/mg of protein, respectively. Cr(VI) reductase activity was maximum at 40 °C and pH 7.0 and it was significantly inhibited in the presence of disulfide reducers (2-mercaptoethanol), ion chelating agent (EDTA), and respiratory inhibitors (CN and Azide). Complete reduction of 100 and 200 µM of Cr(VI) by membrane associated enzyme were observed within 40 and 180 min, respectively. However, it should be noted that biochemical characterization has been done with crude enzyme only, and that final conclusion can only be drawn with the purified enzyme.

Effects of substrates and reaction conditions on production of cyclodextrins using cyclodextrin glucanotransferase from newly isolated Bacillus agaradhaerens KSU-A11

The effects of reaction conditions on cyclodextrins (CDs) production by CGTase from newly isolated Bacillus agaradhaerens KSU-A11 is reported. Among six types of starch tested, potato starch gave highest starch conversion into CDs. In addition, CDs yield was about three fold higher when using gelatinized potato starch in comparison to raw starch. The total CDs production was increased with increasing pH, showing maximum starch conversion at pH 10. Furthermore, the proportion of gamma-CD was relatively higher under slightly acidic-neutral conditions than at alkaline pH with a maximum proportion of 35.6 percent at pH 7 compared to 7.6 percent at pH 10. Maximum starch conversion into CDs was seen at reaction temperature of 55ºC. Lower reaction temperature led to higher proportion of gamma-CD with maximum percentage at 35ºC. Cyclization reaction was significantly promoted in the presence CaCl2 (10 mM), while in the presence of ethyl alcohol there was significant decrease in CD production particularly at high concentration. beta-CD was the major product up to 1 hr reaction period with traces of alpha-CD and no detectable gamma-CD. However, as the reaction proceed, gamma-CD started to be synthesised and alpha-CD concentration increased up to 4 hrs, where the CDs ratios were 0.27:0.65:0.07 for alpha-CD:beta-CD:gamma-CD, respectively. In addition, optimum CGTase/starch ratio was obtained at 80 U/g starch, showing highest starch conversion into CDs. All the parameters involved have been shown to affect the products yield and/or specificity of B. agaradhaerens KSU-A11 CGTase.

Isolation and characterization of novel potent Cr(VI) reducing alkaliphilic amphibacillus sp. ksuCr3 from hypersaline soda lakes

A strain KSUCr3 with extremely high Cr(VI)-reducing ability under alkaline conditions was isolated from hypersaline soda lakes and identified as Amphibacillus sp. on the basis of 16S rRNA gene sequence analysis. The results showed that Amphibacillus sp. strain KSUCr3 was tolerance to very high Cr(VI) concentration (75 mM) in addition to high tolerance to other heavy metals including Ni2+ (100 mM), Mo2+ (75 mM), Co2+ (5 mM), Mn2+ (100 mM), Zn2+ (2 mM), Cu2+ (2 mM) and Pb (75 mM). Strain KSUCr3 was shown to be of a high efficiency in detoxifying chromate, as it could rapidly reduce 5 mM of Cr(VI) to a non detectable level over 24 hrs. In addition, strain KSUCr3 could reduce Cr(VI) efficiently over a wide range of initial Cr(VI) concentrations (1-10 mM) in alkaline medium under aerobic conditions without significant effect on the bacterial growth. Addition of glucose, NaCl and Na2CO3 to the culture medium caused a dramatic increase in Cr(VI)-reduction by Amphibacillus sp. strain KSUCr3. The maximum chromate removal was exhibited in alkaline medium containing 1.5 percent Na2CO3, 0.8 percent glucose, and 1.2 percent NaCl, at incubation temperature of 40ºC and shaking of 100 rpm. Under optimum Cr(VI) reduction conditions, Cr(VI) reduction rate reached 237 uMh¹ which is one of the highest Cr(VI) reduction rate, under alkaline conditions and high salt concentration, compared to other microorganisms that has been reported so far. Furthermore, the presence of other metals, such as Ni2+, Co2+, Cu2+ and Mn2+ slightly stimulated Cr(VI)-reduction ability by the strain KSUCr3.The isolate, Amphibacillus sp. strain KSUCr3, exhibited an ability to repeatedly reduce hexavalent chromium without any amendment of nutrients, suggesting its potential application in continuous bioremediation of Cr(VI). The results also revealed the possible isolation of potent heavy metals resistant bacteria from extreme environment such as hypersaline soda lakes.