Cloning of chitinase gene from Burkholderia cepasia

[Synopsis] In this study, we isolated a bacterium producing a large quantity of chitinase and identified it as Burkholderia cepasia. The chitinases isolated from the bacterium were detected by SDS-PAGE and at least two chitinases (40 and 50 kDa) were identified in the zymogram. The genomic DNA extracted from the bacterium was used for the construction of genomic library by the use of λEMBL3 phage vector and phage clones producing chitinase were selected by using 4-MU chitotrioside as a substrate and exposing to UV light. DNA fragment inserted in λEMBL3 phage vector was subcloned into pBluescript vector with restriction enzymes and pBlueCAK12 ( 4.4 kb ) clone including chitinase gene was selected. The inserted DNA fragment was deleted by exonuclease III and chitinase-coding region was specified by chitinase activity detected by the use of 4-MU chitotrioside and UV light exposure. The chitinase of B. cepasia was found to be coded in 2.3 kb of pBlueCAK12