Characterization of immobilized alkaline cyclodextringlycosyltransferase from a newly isolated Bacillus agaradhaerens KSU-A11

Alkaliphilic bacteria were isolated from soil and water samples obtained from Egyptian soda lakes (Wadi Natrun area, Egypt). Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria resulted in isolation of 10 positive strains. Strain KSU-A11 was selected as the best CGTase producer (2.1 U/ml). 16S rDNA sequence analysis identified the KSU-A11strain as Bacillus agaradhaerens. CGTase was partially purified using starch adsorption technique. The partially purified CGTase was immobilized on chitin by covalent binding tecnique using cross linking reaction with high immobilization yield (85%). The properties of the free and immobilized CGTase were determined. The optimum pH of the immobilized enzyme was slightly higher than that of the free enzyme at pH 10 and 10.5, respectively. In addition, both free and immobilized enzyme retained 94 to 100% of its initial activity over a wide pH range (pH 6.0 to 11.0). The enzymatic activity of both free and immobilized CGTase was highest at temperature 50°C; however, the relative activities of the immobilized CGTase were slightly higher than those of the free enzyme. Furthermore, investigation of thermostability of the enzyme indicated that the immobilization process of CGTase on chitin significantly protected the enzyme against thermo-inactivation. Kinetic parameters, Km and Vmax, values for free and immobilized enzymes were estimated and while there was no change in the Vmax value (83.3 μmol/min. mg) for both free and immobilized CGTase, the Km of the enzyme increased from 14.28 to 20 mg/ml upon immobilization. The immobilization of the enzyme showed high operational stability by retaining almost 50% of the initial activity after nine uses.Key words: Cyclodextrin glycosyltransferase, Bacillus agaradhaerens, immobilization, chitin, alkaliphiles

Tapping uncultured microorganisms through metagenomics for drug discovery

Natural products have been an important historical source of therapeutic agents. Microorganisms are major source of bioactive natural products, and several microbial products including antibiotics, anti-inflammatory, anti-tumour, immunosuppressants and others are currently used as therapeutic agents for human and  domestic animals. Most of these products were obtained from cultured environmental microorganisms. However, it is widely accepted that a very large majority of the microorganisms present in natural  environments are not readily cultured under laboratory conditions, and therefore are not accessible for drug  discovery. Metagenomics is a recent culture-independent approach that has been developed to access the  collective genomes of natural bacterial populations. It enables discovery of the diverse biosynthetic pathways encoded by diverse microbial assemblages that are known to be present in the environment but not-yet  cultured. Recently, several new bioactive molecules and proteins have been discovered using a metagenomic approach. This review highlights the recent methodologies, limitations, and applications of metagenomics for the discovery of new drugs. Moreover, it shows how a multidisciplinary approach combining metagenomics with other technologies can expedite and revolutionize drug discovery from diverse environmental microorganisms.Key words: Microbial diversity, metagenomics, natural products, drug discovery, microbial ecology

Islamism and the state after the Arab uprisings: Between people power and state power

The authors would like to thank the Arts and Humanities Research Council for facilitating the research for this article through their support of the research network People Power versus State Power of the Centre for the Advanced Study of the Arab World.This paper examines the trajectories of different Islamist trends in the light of the Arab uprisings. It proposes a distinction between statist and non-statist Islamism to help understand the multiplicity of interactions between Islamists and the state, particularly after 2011. It is outlined how statist Islamists (Islamist parties principally) can contribute to the stabilization and democratization of the state when their interactions with other social and political actors facilitate consensus building in national politics. By contrast when these interactions are conflictual, it has a detrimental impact on both the statist Islamists, and the possibility of democratic politics at the national level. Non statist-Islamists (from quietist salafi to armed jihadi) who prioritize the religious community over national politics are directly impacted by the interactions between statist Islamists and the state, and generally tend to benefit from the failure to build a consensus over democratic national politics. Far more than nationally-grounded statist Islamists, non-statist Islamists shape and are shaped by the regional dynamics on the Arab uprisings and the international and transnational relations between the different countries and conflict areas of the Middle East. The Arab uprisings and their aftermath reshaped pre-existing national and international dynamics of confrontation and collaboration between Islamists and the state, and between statist and non-statists Islamists, for better (Tunisia) and for worse (Egypt).PostprintPeer reviewe

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg−1 protein, 20.0 U mg−1 protein and 11.0 U mg−1 protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl2. Km and Vmax values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co2+, Zn2+, Cu2+, Hg2+, Ba2+, Cd2+, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry

Isolation and identification of Alkaline protease Producing Alkaliphilic bacteria from an Egyptian soda lake

Abstract: Screening of water and soil samples collected from Wadi El-Natrun, an Egyptian soda lake, for alkaline proteases producing bacteria, resulted in isolation of 15 alkaline proteases producing alkaliphilic strains. WN-SK5 showed the highest enzyme production (61.0 U/ml) after 48h. This isolate was gram positive and was able to grow in the presence of NaCl up to 15 %. Growth was observed at 25 ºC, 37 ºC, 45 ºC and 55 ºC but no growth was seen at 60 ºC. It could grow at pH value from 8 to 11. No growth was detected at pH 7 after 48 h incubation at 55 ºC, which indicted this strain to be moderate halophilic thermophilic alkaliphiles. It was found that 16S-rDNA sequence of strain WN-SK5 had 97.35 % identity with the corresponding sequence of Bacillus halodurans (Acc. Nr. gb10172612). The crude alkaline protease showed reasonable activity at temperature range of 65 to 75 °C with maximum activity at 70 ºC and had a relatively wide pH range of activity between pH 8 to 11, with maximum enzyme activity at pH 10 in 50 mM Tris -HCl buffer maximum activity at 70 °C and pH 10.0, indicating the enzyme to be thermo-alkaline proteases

Clonal diversity and antimicrobial resistance of Enterococcus faecalis isolated from endodontic infections

Background: Enterococcus faecalis is considered to be one of most prevalent species in the oral cavity, particularly in endodontic infections. The aim of the present study was to investigate the prevalence of E. faecalis in dental root canals, clonal diversity by restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) analysis, and the antibiotic susceptibility of E. faecalis isolates. Results: Among the bacterial strains isolated from dental root canal specimens (n = 82), E. faecalis was determined to have the highest prevalence followed by Streptococcus viridians, Leuconostoc mesenteroides, Staphylococcus aureus, Streptococcus mitis, and Pediococcus pentosaceus. Cluster analysis of RAPD-PCR and RFLP patterns of the E. faecalis isolates discriminated five and six different genotypes, respectively. Among the tested strains, 43%, 52% and 5% were susceptible, intermediate resistant, and resistant to erythromycin, respectively. In addition, one strain (E-12) was intermediate resistant to linezolid, and one isolate (E-16) was resistant to tetracycline. Interestingly, many of the intermediate resistant/resistant strains were grouped in clusters 5 and 6, according RAPD and to RFLP, respectively. Conclusions: E. faecalis demonstrated the highest prevalence in the tested dental root canal specimens collected from Saudi patients and were grouped into five to six different genotypes. Different levels of antimicrobial susceptibility were observed in the tested E. faecalis strains, which clearly indicated that although bacterial strains may be similar, point mutations can result in extreme susceptibility or resistance to various antibiotics. This phenomenon is a cause for concern for clinicians in the treatment of dental infections caused by E. faecalis

A novel cyclodextrin glycosyltransferase from alkaliphilic amphibacillus sp. NPST-10 : purification and properties

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg−1 protein, 20.0 U mg−1 protein and 11.0 U mg−1 protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl2. Km and Vmax values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co2+, Zn2+, Cu2+, Hg2+, Ba2+, Cd2+, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry.Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg−1 protein, 20.0 U mg−1 protein and 11.0 U mg−1 protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl2. Km and Vmax values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co2+, Zn2+, Cu2+, Hg2+, Ba2+, Cd2+, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry

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 Vmax, kcat and kcat/Km, 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.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 Vmax, kcat and kcat/Km, 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