Production, Characterization and Genetic Study of Pantoeadispersa Producing Cellulase and pectinase

      Soil samples were collected from fields cultivated with wheat and barley and from spoiled orange, apple fruits and carrot roots to enzyme  isolate bacteria producing lytic. Bacterial isolates were screened for enzymes production on selective media containing carboxymethyl cellulose (CMC) or pectin as a sole source for carbon and energy. Results of semi-quantitative screening showed that nine isolates from fifty isolates were able to produce cellulase and pectinase with variable degrees depending on the formation of  halo zones around colonies of these isolates. The most efficient isolates in producing cellulase and pectinase were selected and subjected to quantitative screening by determining cellulase and pectinase specific activities in the culture filtrates. Results showed that the isolate symboled  A3 was the most efficient among the other isolates in cellulase and pectinase  production since it produced the highest yield of these enzymes.The specific activity of cellulase and pectinase  reached 3.9 and 1.31U/mg
protein respectively.The selected isolate (A3) was identified according to its morphological and biochemical characteristics. Results of identification showed that this isolate was belong to Pantoea sp., and identification further confirmed by using Api 20-E and VITEK 2, and identified as Pantoea dispersa. Optimum conditions for cellulase production by local isolate Pantoea dispersa A3 was studied. Results showed that the optimum conditions include the supplementation of  the production medium with  CMC as  a  sole  source  for carbon   and   energy   at   a  concentration  of  1.5%, ammonium   sulfate   as   a nitrogen   source  at  a concentration  of  0.1%; potassium dihydrogen phosphate at a concentration of,  0.1%, in the a medium adjusted to the pH 7, and incubation  at 30 ºC for 72 hrs with shaking at 140 rpm. Under these conditions, specific  activity  of  cellulase  was  increased  to  24 U/mg  protein. Cellulase produced  by  P. dispersa  A3  under   the   optimum   conditions   was   purified throughout four purification steps includes ammonium sulfate precipitation step with a saturation ratio of 70% then dialysis step followed by purification with ion exchange chromatography by using DEAE-cellulose, and then gel filtration step throughout sephadex-G200 which allows larger ability of separation with high degree of purification. Results of purification showed that the specific activity of the purified enzyme was 190.9 U/mg protein with a purification fold and yield of 113.6 and 26.2% respectively. Purified cellulase from P. dispersa A3 was well characterized by studying some enzyme characteristics. Results
showed that the molecular weight of cellulase was 15148 dalton;  pH 7.0 was the optimum for enzyme activity and stability, while 30 ºC was also the optimum for enzyme activity and stability. In attempt to increase cellulase productivity from P. dispersa A3 by physical mutagenesis, bacterial cells from fresh culture of this isolate was subjected to random mutagenesis by UV-ray to induce random mutations affects positively enzyme production. Results of mutagenesis showed that there were nine of overproducer mutants characterized with their higher cellulase productivity were obtained after irradiation with UV ray. Specific activity of cellulase produced by these mutants was ranged between 12.48  and  55.65  U/mg protein in comparison with the productivity of wild-type (3.9 U/mg protein). Genomic  DNA  of  P. dispersa  A3  was extracted  by  using  favorgen  extraction  kit  in  order  to  amplify  cellulase gene  after designing  oligoprimers including genetic elements upstream and downstream sequences of cellulase gene for different species of Pantoea spp.according   to  database   found  in  the  website   of   national center for for biotechnology information (NCBI).  After amplification, PCR products were
analyzed on agarose gel (0.8%) to identify DNA bands in presence of  DNA ladder marker. Results of electrophoresis showed that there   were no any PCR products   identified  for  each  pair  of  oligonucleotids   primers,  which  may which may refer that these primers were not specific for amplification of cellulase gene of  P. dispersa A3.