الفهرس 
Microbial enzymesOnly 14 pages are availabe for public view
 |  | from | 214 |  |  |
| | | from | 214 | | |
Abstract
Two hundred soil samples collected from different Egyptian governorates were used for recovery of seven hundred and twenty two isolates. The recovered isolates were screened for L-asparaginase production on modified M9 medium supplemented with phenol red as an indicator and 1% w/v asparagine. A total of 81 isolates were proved to be L-asparaginase producers and they were categorized into Gram positive (70 isolates) and Gram negative (11 isolates) according to their Gram reaction. Then, the eighty one producing isolates were subsequently quantified for their L-asparaginase productivity using Nesslerization reaction. The L-asparaginase producing isolates were further categorized into high producers (isolates productivity ≥ 3 IU/ml/h), intermediate producers (isolates productivity ≥ 1 < 3 IU/ml/h) and low producers (isolates productivity < 1 IU/ml/h). The highest Gram positive and Gram negative producing isolates were identified using Biolog® and Vitek® system, respectively then their identification were confirmed by 16S rRNA gene sequencing to be Bacillus licheniformis and Stenotrophomonas maltophilia. The assembled sequences of the two isolates were deposited in GenBank database with assigned accession codes of MG665995 for Bacillus licheniformis and MG665996 for Stenotrophomonas maltophilia. L-asparaginase produced from Bacillus licheniformis was relatively thermostable compared to that of Stenotrophomonas maltophilia where the activity of the former retained about 70% of its activity at 70°C/30 min while that of the later preparation retained only 55% of its activity. L-asparaginase activity was assessed at different temperatures where the two isolates showed optimum activities at 37-40°C. The catalytic activity of both enzyme preparations decreased at 45°C by 27.7% in case of Bacillus licheniformis and 21% in case of Stenotrophomonas maltophilia. Optimum pH of L-asparaginase activity for both isolates was recorded to be 8.6 and with considerable activity at pH 9. L-asparaginase activity at different salinities for the both enzyme preparations from both isolates didn’t adversely affected up to 500 mM sodium chloride but on the other hand slight increase could be observed. The optimum substrate concentration for both isolates was found to be 40 mM. Different metal ions were tested, zinc sulphate was the only metal salt that showed significant increase in Bacillus licheniformis enzyme activity. On the other hand all tested metal salts significantly inhibited the activity of Stenotrophomonas maltophilia enzyme preparation. Mutation of both isolates was induced by gamma radiation for improvement of their enzymatic productivity. In case of Bacillus licheniformis a 1.4 fold increase in L-asparaginase production was achieved compared to the wild organism, while in case of Stenotrophomonas maltophilia 1.6 fold increase in enzyme production was obtained compared to its parent strain. Optimization of L-asparaginase production for both mutant strains was performed using one-Factor-at-a-time then by response surface methodology Box-Behnken central composite design. The first step was optimization of process parameters for four variables; incubation temperature, pH, incubation time and agitation rate. The optimal ranges from the preliminary experiments were applied in RSM model. The model consists of 27 experiments, the optimum conditions for maximum L-asparaginase production from Bacillus licheniformis were obtained at pH 7.39, Temperature 39.5°C, time 20.74 h, and agitation rate 196.40 rpm. While that from Stenotrophomonas maltophilia were obtained at pH 6.89, Temperature 38.0°C, time 19.85 h, and agitation rate 179.15 rpm.
Optimization of media components was carried out using the one-Factor-at-a-time experiments which included carbon sources, nitrogen sources and metal ions. Glucose was found to be the best carbon source for both test mutants while ammonium chloride and yeast extract were found to be the optimum nitrogen sources for Bacillus licheniformis and Stenotrophomonas maltophilia, respectively. Magnesium sulphate was found to be the best metal ion for the highest L-asparaginase production for both test mutants. The best medium component was further tested at different concentrations to determine the optimum one. Maximum enzyme production was observed at 0.5% w/v glucose, 0.1% w/v ammonium chloride and 10 mM magnesium sulphate for Bacillus licheniformis and 0.4% w/v glucose, 0.1% w/v yeast extract and 10 mM magnesium sulphate for Stenotrophomonas maltophilia.