Abdelnasser S. S. Ibrahim

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.

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.

Brown macroalgae as bio-indicators for heavy metals pollution of Al-Jubail coastal area of Saudi Arabia

Wastes from both industrial and domestic sources, as well as habitat destruction have a substantial impact on the coastal environments. It causes serious problems in many countries and for several seas and oceans which leads to the extinction of several plant and animal species. Many water resources are no longer suitable for drinking or for agriculture as a result of pollution. The main aim of this study was to investigate the efficiency of four brown macroalage as bioindicators for toxic heavy metals (manganese (Mn), copper (Cu), zinc (Zn), arsenite (As), cadmium (Cd), and lead (Pb)) along Al-jubail industrial city coast at Persian Gulf (Saudi Arabia). Brown macroalage samples were collected from three different sites in three time points, January, March and May, 2010. The four collected brown macroalgae were identified as Sargassum angustifolium, Sargassum boveanum, Sargassum latifolium, and Padina gymnospora. The algal samples were cleaned using sea water and distilled water, dried, and the concentrations of various toxic metals were determined. The average concentrations of Mn, Co, Ni and Cd were within the expected limits of un-contaminated areas. However, the results indicate the high level of Zn ion accumulation in all tested brown algae, showing highest concentration in S. angustifolium > P. gymnospora > S. latifolium > S. boveanum with highest Zn concentration of 991 ± 49.1, 988 ± 47.5, 980 ± 44.2, and 911 ± 39.7 µg g -1 dry weights, respectively. In addition, Cu was detected at high concentration of 92.1 ± 3.7 ig g -1 dry weight in S. boveanum . These results clearly indicate the high pollution levels of Al-jubail industrial city coast with Zn and Cu toxic heavy metals, which is mostly due to uncontrolled disposal of industrial waste into coastal area. Furthermore, the consistency of Zn concentrations in all tested brown algae indicated the efficiency of the tested algae, including P. gymnospora, S. angustifolium, S. latifolium, and S. boveanum, for bioaccumulation and bio-monitoring studies of Zn. Key words : Brown algae, heavy metals, bio-indicators, Sargassum sp., Padina sp.

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.

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 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.