Introduction: Campylobacter spp are the major cause of enteritis in humans and more than 90% of reported infections are caused by Campylobacter jejuni. Fluoroquinolones such as ciprofloxacin are the antibiotics of choice for treatment. An increase in the frequency of ciprofloxacin-resistant Campylobacter has been reported globally due to a single base mutation (C-257 to T) in codon 86 of the quinolone resistance determining region (QRDR) of the gyrA gene altering the amino acid sequence from threonine at position 86 to isoleucine (Thr-86 to Ile). Methodology: Campylobacter spp (n = 118) were selected from a collection of Egyptian isolates spanning 1998 to 2005. The presence of C. jejuni gyrA gene was confirmed in each isolate by a PCR assay amplifying 368bp portion of the gyrA gene. C to T alteration was detected by the mismatch amplification mutation assay MAMA PCR. The MIC of nalidixic acid (NA) and ciprofloxacin (CIP) was determined by E-test. Results: C. jejuni gyrA gene was detected in 100 of the Campylobacter spp studied; the other 18 isolates were found to be Campylobacter coli by lpxA PCR. The mutation was detected in 89 C. jejuni resistant isolates with MIC values (NA; 8 ->256μg/ml) and (CIP; 4 ->32μg/ml). The other 11 sensitive C. jejuni isolates with MIC values (NA; 0.38 -3μg/ml) and (CIP; 0.03 -0.125μg/ml) were not amplified by the MAMA primers. There was 100% congruence with MAMA PCR, MIC results and gyrA gene sequence analysis. Conclusions: In Egypt the main mechanism for resistance to fluoroquinolones is an alteration in the gyrA QRDR. MAMA PCR provides an economical and rapid means for screening fluoroquinolone resistance.
Background: Amylases are among the most important enzymes which are of great significance for biotechnology and have almost completely replaced chemical hydrolysis of starch in the starch processing industry. The present study was concerned with the production and optimization of extracellular α-amylase from Bacillus sp. NRC22017. Results: The effect of various fermentation conditions on α-amylase production through shake-flask culture was investigated. Bacterial strain produces α-amylase was isolated from water in Wadi El-Natron. Based on microbiological, biochemical tests, and 16S rRNA gene sequences, the isolate was identified as Bacillus sp. NRC22017 and was later used for further studies. Maximum yield of α-amylase is 15.15 ± 0.47 U/ml from Bacillus sp. NRC22017; this strain is characterized with high temperature and high salinity in cultivated culture, and achieved maximum yield of α-amylase at pH 6.0 with inoculum size of 500 μl at 45°C and aerobically incubation period of 72 h. The optimum volume of the fermentation medium was found to be 20 ml in 100 ml Erlenmeyer flask; the best starch and meat extract plus peptone concentration that provided the highest enzyme production from Bacillus sp. NRC22017 were found to be 2% and 1.05% (w/v) respectively. Conclusion: Enzyme production was higher after optimizing the production conditions as compared to the basal medium.
Polygalacturonase (PGI) from Aspergillus niger NRRL3 was purified about 12.0-fold from the cell-free broth using diethylaminoethyl-Sepharose and Sephacryl S-200 columns. The molecular weight of the PGI was 32,000 Da as estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PGI had an isoelectric point of 7.6 and an optimum pH of 5.0. PGI was active on polygalacturonic acid and esterified pectins, but the activity on pectin decreased with an increase in degree of esterification. PGI had higher affinity (low Km) and turnover number (Vmax/Km and Kcat/Km) toward polygalacturonic acid. PGI was found to have a temperature optimum at 40 degrees C and was approximately stable up to 30 degrees C. All the examined metal cations had partial inhibitory effects on PGI, while Mn+2 at 5 mM caused a complete inhibition for the enzyme. Comparison of viscosity reduction rates with release of reducing sugars indicated that the enzyme from A. niger is exoacting. The storage stability study of PGI showed that the enzyme in powder form retained 56% of its activity after 9 months of storage at 4 degrees C. The above properties of PGI may be suitable for food processing.
A novel extreme halophilic exochitinase enzyme was produced by honey isolate Aspergillus awamori EM66. The enzyme was immobilized successfully on k-carrageenan-alginate gel carrier (CA) with 93 % immobilization yield. The immobilization process significantly improved the enzyme specific activity 2.6-fold compared to the free form. The significant factors influencing the immobilization process such as enzyme protein concentration and loading time were studied. Distinguishable characteristics of optimum pH and temperature, stability at different temperatures and NaCl tolerance for free and immobilized enzyme were studied. The immobilization process improved optimum temperature from 35 to 45 °C. The immobilized enzyme retained 76.70 % of its activity after 2 h at 75 °C compared to complete loss of activity for the free enzyme. The reusability test proved the durability of the CA gel beads for 28 cycles without losing its activity.
A b s t r a c tGamma irradiation is used on Penicillium cyclopium in order to obtain mutant cells of high L-asparaginase productivity. Using gamma irradiation dose of 4 KGy, P. cyclopium cells yielded L-asparaginase with extracellular enzyme activity of 210.8 ± 3 U/ml, and specific activity of 752.5 ± 1.5 U/mg protein, which are 1.75 and 1.53 times, respectively, the activity of the wild strain. The enzyme was partially purified by 40-60% acetone precipitation. L-asparaginase was immobilized onto Amberlite IR-120 by ionic binding. Both free and immobilized enzymes exhibited maximum activity at pH 8 and 40°C. The immobilization process improved the enzyme thermal stability significantly. The immobilized enzyme remained 100% active at temperatures up to 60°C, while the free asparaginase was less tolerant to high temperatures. The immobilized enzyme was more stable at pH 9.0 for 50 min, retaining 70% of its relative activity. The maximum reaction rate (V max ) and Michaelis-Menten constant (K m ) of the free form were significantly changed after immobilization. The K m value for immobilized L-asparaginase was about 1.3 times higher than that of free enzyme. The ions K + , Ba 2+ and Na + showed stimulatory effect on enzyme activity with percentages of 110%, 109% and 106% respectively. K e y w o r d s: Penicillium cyclopium, Amberlite IR-120, gamma irradiation, ionic binding immobilization, L-asparaginase
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