5298
MAHA TAIMOUR HASSAN EMAM
GENETIC IMPROVEMENT OF BACTERIAL
XYLANASE PRODUCTION
Bacillus, E. coli; xylanase; UV mutation; EMS; protoplast
fusion; sequence analysis; gene cloning and expression
Evaluation of the collected bacterial strains revealed that, the maximum xylanase activity of Bacillus pumilus GH and Geobacillus. sterothermophilus 2027 was 80 and 40 U/ml, respectively at 60oC and pH7 with negligible amounts of cellulase activity. Xylanase activity from both strains showed tolerance to high temperature and alkaline conditions. Both strains were subjected to UV and EMS mutagenesis. The results proved that EMS is more effective as a mutagenic agent than UV irradiation for induction of high xylanase production. The best mutants of B. pumilus GH and G. sterothermophilus 2027 were selected for construction of intraspecific and intergeneric protoplast fusion. The highest xylanase activity (294.3 U/ml) was obtained from intraspecific protoplast fusion between B. pumilus mutants which increased by about two folds and 3.67 folds in comparison to parental strains and B. pumilus GH wild type, respectively. The thermostable endo-1,4-beta-xylanase gene of B. pumilus GH strain was isolated from chromosomal DNA using specific primers, then cloned into pET29a (+) vector and transformed into E. coli DH5α. The positive clone was selected, sequenced and submitted to gene bank with the accession number KT757524.1. The open reading frame of the xylanase gene was 687 bp encoding a protein of 228 amino acids with a molecular mass of 23 kDa. The recombinant plasmid containing xylanase gene was transformed to expression host E.coli BL21 (DE3) and the xylanase gene was successfully expressed but xylanase activity is lower than B. pumilus GH wild type strain.
2017
Ph.d
Ain Shams
Agriculture