Basic Informations

C.V

Master Title

A Study on Microbial Pectinase Activity: Characterization of the Enzyme(S) Involved In Pectin Degradation and the Technology Used For Optimizing Enzyme Activity and Productivity

Master Abstract

Pectins are the most structurally complex polysaccharides; mainly occur in plant cell walls; especially in the middle lamella of the primary cell wall of higher plants. Pectins contribute to many biological and physiological functions; especially during abscission and growth as they determine the size, shape of cells and consequently the integrity and rigidity of plant tissues. Pectins are subjected to many pectinolytic enzymes of plant and microbial origin. These enzymes are composed of large number of different enzymes collectively called pectinases. One hundred isolates of different bacteria and fungi had a pectinolytic activity. These isolates were used as representatives of the microbial flora of rotten fruits, vegetables and seeds. Pectinolytic activity was followed up resulted in selection of 21 isolates with high hydrolyses activity. Characterization of pectinases secreted by 21 isolates was carried out through determination of the following enzymes: polygalacturonase (PG); polygalacturonase lyases (PGL); polymethylgalacturonase (PMG); polymethylgalacturonase lyase (PMGL) and pectinoesterase (PE). The obtained results showed that isolates No. 100, 98, and 94 were higher for both PG and PMG producers while isolates No. 97, 60 and 19, 38 were best producers of PG and PMG ;respectively. These isolates were species of Aspergillus and were identified as follows: • Three isolates (No. 19, 97 and 98) belong to Aspergillus fumigatus. • Three isolates (No. 38, 94 and 100) belong to Aspergillus niger. • One isolate (No. 60) belongs to Aspergillus carbonarius. In this concern, different factors affecting production of PG and PMG were studied including: carbon, nitrogen sources, and initial pH of the enzyme production medium, agitation rate, and fermentation period and temperature. In case of the effect of carbon source on production, data showed that soluble starch and sucrose resulted in highest production of PG in all the tested isolates while high PMG production was attained upon utilizing carboxymethylcellulose, and mixture of pectin and oxalic acid by Asp. fumigatus No.98. The source of nitrogen incorporated in enzyme production medium greatly affected the production of both PG and PMG. The obtained results showed that gelatin, yeast extract, ammonium nitrate, ammonium sulphate and peptone promoted the highest production of PG by certain isolates, while alanine, glycine, sodium nitrate and ammonium sulphate were fairly good in production of PMG by certain isolates. Concerning the influence of initial pH of the enzyme production medium, the obtained results showed that pH values of 4.0 and 7.0 seemed to be optimal for production of both PG and PMG enzymes; respectively . In case of agitation, the data showed that there is a direct relation between the rate of agitation and the production of both enzymes. As productivity of both enzymes were increased as the rate of agitation reached 150rpm. As regards the effect of fermentation period on the production of the two enzymes by Asp. fumigatus No.98, it was found that optimum production of PG was between 4-8 days while that of PMG was at 7 days. The effect of fermentation temperature on the production of PG and PMG by the selected isolates was investigated. The highest level of PG was achieved at 30oC in case of Asp. fumigatus No. 97 and No. 98 and Asp. niger No. 100. While other isolates of PG were at 25oC except isolate No.100. However, PMG produced by Asp. niger isolates No.38 and 94 showed high levels at 25oC. While Asp. fumigatus No. 19 and No. 98 produced highest levels at 35oC.on the other hand production by Asp. niger No. 100 was optimum at 30oC. Some factors affecting the activity of extracellular crude PG and PMG enzymes including pH, temperature and the presence of cations have been studied: On studying the influence of various pH values of the reaction mixture on the enzyme activity, the obtained results showed that the optimum pH of the activity of PG produced by Asp. fumigatus No.98 and Asp. carbonarius No.60 was 4.6, while the optimum pH of produced by Asp. niger No.100 was 7.0. A pH range of 4-8 was optimal for the activity of PMG produced by Asp. fumigatus No.98. However, a pH 6.6 was optimal for the activity of PMG produced by Asp. niger No. 94 and No.100 at pH 6.6. Incubation temperature of reaction mixture affected greatly PG and PMG activities. It was found that a temperature of 45 oC was optimum for the activity of PG produced by Asp. fumigatus No.98 while the optimum temperature for the activity of PG produced by Asp. carbonarius No.60 and Asp. niger No. 100 was at 50 oC and 55oC; respectively. However 45oC was the optimum temperature for the activity of PMG produced by Asp. fumigatus No.98 and Asp. niger No. 100. While in case of PMG produced by Asp. niger No. 94 was 50oC. On studying the influence of different cations on the activities of PG and PMG crude enzymes, the obtained results showed that Fe+2 and Na+ ions caused activation in both enzymes in all the tested strains, while Ca+2, Cu+2, Hg+2, Zn+2 had an inhibitory effect in case of all tested enzyme preparations, however Mg+2, Mn+2, Ni+2, Al+3, Li+ and Cr+2 showed activation for some isolates and inhibition for others. The obtained data showed that Asp. fumigatus isolate No. 98 was the best isolate for the production of polygalacturonases so it was selected for further studies. This isolate produced two exo-polygalacturonases (PGI, PGII).purification of these two enzymes was carried out using chromatography on Sephadex G-100 and diethyl amino ethyl cellulose (DEAE)-cellulose. The exo-PGI and exo-PGII were purified 19.3 and 21.5 folds; respectively. The two enzymes have the same molecular weight of 78.75 K.D. They have similar bimodal pH profiles with two pH optima at 5.6 and 8.3. Their optimum temperatures were 45°C and 55°C; respectively. Complete inactivation of the two enzymes was observed with (NH4)+, Zn2+, Hg2+, Cu2+, Cr2+, Ca2+ and Al3+. Variable degrees of inactivation were observed with other metallic ions: Ni +, Ba2+, Mg2+, Fe3+, Na+, Li+, K+ and Mn2+. On the other hand these enzymes were stimulated by Fe2+. PG-I and PG-II hydrolyzed polygalacturonic acid and to a lesser extent citrus pectin. The thin layer chromatography (TLC) of the reaction products supported the exo-activity for PGI and PGII since galacturonic acid was identified as sole product of polygalacturonic acid breakdown by the enzyme.

PHD Title

Characterization of ß-Lactamase Isolated from Salmonella Spp. of Different Samples.

PHD Abstract

Salmonella serovar Typhimurium is a leading cause of foodborne diseases worldwide. Its ability to acquire new antibiotic resistance mechanisms is an increasing therapeutic concern. We report acquisition of the CTX-M-14 ESBL by 6 pediatric isolates of Salmonella serovar Typhimurium from Egypt. To our data, this is the first report of CTX-M-producing Salmonella in Egypt. Antibiotic susceptibility and ESBL detection were investigated by CLSI agar dilution methodology. ß-lactamase production was investigated by isoelectric focusing (IEF) incorporating clavulanate, cloxacillin, and cefotaxime overlays, CTX-M, OXA, and SHV-specific PCR, sequencing, and conjugation. Strain relatedness was investigated by PFGE. Two clusters CI and CII were resulted, however the clinical isolates in cluster CI have 85% related by PFGE suggesting they may be subtypes of a common strain, but CI was unrelated to cluster CII. The clinical isolates were highly cefotaxime-resistant, but exhibited only reduced susceptibility to ceftazidime, and were ESBL-positive by the CLSI criteria for E. coli, Klebsiella, and P. mirabilis. All clinical isolates produced multiple clavulanate-sensitive ß-lactamase bands on IEF with pIs of 7.9 and 5.4 (all-clinical isolates), the clinical isolates 68, 79, 93, 108, 115, 126, and 128, showed possibly CTX-M-14 without OXA-1-Like or SHV-12-Like activity. The clinical isolates 92, 96, 99, 100, 102, 111, 120, and 132 showed a resistance pattern possibly OXA-1-Like activity. Eventually, the clinical isolates 59, 65, 76, 78, 94, 95, 113, 119, and 127, showed a resistance pattern possibly SHV-12-Like activity. The pI 7.9 enzyme was transferrable by conjugation and conferred a CTX-M ESBL phenotype in clinical isolates Sal.68, 94, and 111. PCR and sequencing identified the CTX-M gene as CTX-M-14 in clinical isolates Sal.68, 94, 111, 113, 119, and 127. In addition, genes for SHV-12 and OXA-1 were also identified by sequencing in clinical isolates number Sal.94, 113, 119, 127, and Sal.102, 111, 120, 132, respectively. This report of clinical isolates of Salmonella from Egypt producing CTX-M-14 are of epidemiological significance. The successful detection of this ESBL with CLSI methodology suggests that CLSI methodology should be further evaluated for ESBL detection in this genus.