Ali A. Al-Salamah

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.

Synthesis and characterization of novel organotin monomers and copolymers and their antibacterial activity

Two novel organotin monomers, (N-tri-n-butyltin) maleimide and m-acryloylamino-(tri-n-butyltin benzoate), were synthesized. Copolymerization of these two monomers with styrene was carried out in the bulk at 65°C using asobisisobutyronitrile as the free radical initiator. The monomers and copolymers were characterized by elemental analysis; the molecular weights of the copolymers were determined by GPC, solubility, IR, and 1H-NMR spectral studies. The antibacterial activities of the synthesized organotin monomers and copolymers toward various types of bacteria were also reported.

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, K m and V max , values for free and immobilized enzymes were estimated and while there was no change in the V max value (83.3 μmol/min. mg) for both free and immobilized CGTase, the K m 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.

Hexavalent chromium reduction by novel chromate resistant alkaliphilic Bacillus sp. strain KSUCr9a

Alkaliphilic bacterial strain termed KSUCr9a was isolated from soil and water samples collected from various soda lakes located in northern Egypt. KSUCr9a was tolerance up to 75 mM Cr (VI), with minimum inhibition concentration (MIC) value of 80 mM, in alkaline medium (pH 10.5) containing 10% NaCl. Analysis of 16S rDNA of strain KSUCr9a identified this bacterial strain as Bacillus sp., with sequence similarity of 99%, and was referred to as Bacillus sp. strain KSUCr9a. In addition to its tolerance to Cr(VI), Bacillus sp. KSUCr9a showed high resistance to other heavy metals including Cd 2+ (50 mM), Mo 2+ (75 mM), Mn 2+ (100 mM), Cu 2+ (2 mM), Ni 2+ (100 mM), Pb 2+ (75 mM), Co 2+ (5 mM) and Zn 2+ (2 mM). Bacillus sp. KSUCr9a demonstrated good chromate bio-reduction ability, as it could rapidly reduce up to 100 μM within 24 h. In addition, at initial Cr(VI) concentration of 200 μM, complete chromate reduction was achieved within 48 h. Furthermore, at initial Cr(VI) concentration of 300, 400 and 500 μM, 92.8, 75.5 and 39.8% of chromate reduction was achieved within 72 h. Bacillus sp. KSUCr9a was able to reduce Cr(VI) in a wide range of NaCl (0 to 20%), indicating the halotolerance nature of this alkaliphilic bacterial strain. Addition of glucose as an electron donor to the culture medium led to significant increase of both growth and chromate reduction by Bacillus sp. KSUCr9a. Maximum Cr(VI) reduction was exhibited in alkaline medium (pH 9) containing 0.8% glucose at incubation temperature of 35°C and under static culture condition. Under optimum Cr (VI) bioreduction conditions, 169.2 μM of Cr(VI) was completely reduced within 24 h, indicating a good ability of Bacillus sp. KSUCr9a of Cr(VI) detoxification under alkaline condition. Furthermore, Cr(VI)-reduction by Bacillus sp. KSUCr9a was slightly induced in the presence of other heavy metals, such as Mn 2+ , Co 2+ , Mo 2+ and Cu 2+ at concentration of 50 mg/L along with Cr(VI) in the culture medium. Moreover, Bacillus sp. KSUCr9a showed the ability of repeated bioreduction of chromate without any addition of exogenous nutrients, indicating its possible application in chromate detoxification. Key words : Chromate reduction, bioremediation, heavy metals, Bacillus sp., soda lakes.

Comparison of phenotypic and PCR methods for detection of carbapenemases production by Enterobacteriaceae.

Dissemination of carbapenem resistance via Enterobacteriaceae, particularly among Klebsiella pneumoniae and Escherichia coli, is a major public health concern. Rapid methods for determining antimicrobial susceptibility are important to ensure adequate and appropriate use of antimicrobial agents and to limit the spread of these bacteria. In the current study, we compared the rapidity, sensitivity and specificity of traditional methods and molecular-based Xpert Carba-R PCR assay to identify sixty isolates, (26 E. coli and 34 K. pneumoniae). The specificity of MicroScan was 100% while sensitivity to ertapenem (ERT), imipenem (IMI), and meropenem (MER) was 93%, 68.9%, and 55.17%, respectively. For the modified Hodge test, the specificity was 96.77% and sensitivity was 89.65%. Although some results of phenotypic assays matched with the definite PCR identification, some results were misleading. Out of the 29 positive PCR samples, three samples of K. pneumoniae were negative for the MHT and one E. coli sample was MHT positive but negative for the PCR. Nine samples were positive for the PCR but were determined as carbapenem sensitive by MicroScan. While MicroScan and MHT requires several hours and multi-steps to obtain results, Xpert Carba-R PCR assay takes less than an hour. Therefore, we recommend using Gene xpert Carba-R assay for the optimal carbapenemnase detection with reducing material, manpower and cost. Also it is important to know the type of carbapenemase is present.

Detoxification of hexavalent chromate by Amphibacillus sp. KSUCr3 cells immobilised in silica-coated magnetic alginate beads

Recently isolated Cr(VI)-reducing Amphibacillus KSUCr3 whole cells were immobilised in magnetic gels. Magnetic magnetite (Fe3O4) nanoparticles were synthesised with an average particle size of 47 nm and 80 electromagnetic unit (emu)/g saturation magnetisation. Whole cells were immobilised by entrapment in agar, agarose, alginate, or gelatin in the presence or absence of Fe3O4 nanoparticles for the preparation of both magnetic and nonmagnetic immobilised cells. Of the gels tested, alginate was selected as the best immobilisation matrix, and following optimisation of the entrapment process, the immobilisation yield reached 92.5%. In addition to the ease of separation and reuse of the magnetic cell-containing alginate beads using an external magnet, the magnetically immobilised cells showed approximately 16% higher Cr(VI) reduction activity compared with nonmagnetic immobilised cells. To improve their physical and mechanical properties, the magnetic alginate beads were successfully coated with a dense silica layer using sol-gel chemistry and Ca(OH)2, an alkaline catalyst for tetraethyl orthosilicate, to avoid leaching of Ca2+ ions. Amphibacillus KSUCr3 cells immobilised in silica-coated magnetic alginate beads showed approximately 1.4- to 3.9-fold enhancement of thermal stability compared with free cells. Furthermore, after seven batch cycles, the Cr(VI) reduction activity of free cells decreased to 48%, whereas immobilised cells still retained 81.1% of their original activity. In addition, the Cr(VI)-reduction rate of immobilised cells was higher relative to free cells, especially at higher Cr(VI) concentrations. These results supported the development of a novel, efficient biocatalysts for Cr(VI) detoxification using a combination of whole cell immobilisation, sol-gel chemistry, and nanotechnology.

Prevalence of Antibiotic Resistance and Minimum Inhibitory Concentrations of Helicobacter pylori Isolates from Clinical Gastritis Patients, Saudi Arabia

Background : Establishing effective and acceptable treatment regimens for patients infected with Helicobacter pylori continues to pose problems for physician and patient alike in Saudi Arabia. The aim of the study was to assess the antibiotics resistance and to find out the MICs of the drugs which are commonly used against H. Pylori. Methods : A total of one hundred antral biopsy specimens were collected and processed from which seventy-six strains of H. pylori were isolated. All the isolates were performed antibiotics sensitivity and E-test. Results : The antibiotics result revealed that of H. pylori isolates 72% strains were resistant to metronidazole and 8% of isolates were resistant to clarithromycin. On the basis of antibiotics resistant a five different antibiotics phenotypes were found among H. pylori isolates. The distribution of metronidazole MICs was high in the range between 32 to128 mg/L. However all other antibiotics showed the effective MICs range was below 1 mg/L. Conclusions : Our findings suggested that resistance to metronidazole has been increasing in the Saudi population. The resistance may be linked to the frequentuse of these antimicrobial agents for treating parasitic infections. The E test results revealed that the most effective antibiotic MICs were amoxicillin and clarithromycin against H. Pylori infection. Keywords: Peptic ulcer, Helicobacter pylori , Antral biopsy, Antibiotics, Resistance.