Screening and cloning of crystalline chitin degrading chitinase gene

Abstract

Soil samples from Chachengsao, Mahachai, Pattaya, and pp Islands of Thailand were selected for chitinase producing bacteria. Chitinase activities were assayed by using colorimetric method with two substrates, powdered chitin (PC) and colloidal chitin (CC). After calculating the percentage ratio of PC/CC, where PC is chitinase activity when used powdered chitin as substrate and CC is chitinase activity when used colloidal chitin as substrate, chitinase-producing bacteria can be divided into five groups. Group 1 is bacterium, which cannot degrade powdered chitin. Group 2 to group 5 have percentage ratio of PC/CC from less than 10% to over 40%. Bacillus circulans PP8, one of member in group 5, which has percentage ratio of PC/CC over 40% was selected for study. Bacillus circulans PP8 was isolated from pp Islands and produced crystalline chitin degrading enzymes that can digest powdered chitin, colloidal chitin and chitosan (100% deacetylation). During growth in colloidal chitin minimum medium, the highest activity, Chi A, was detected at 12 hours when colloidal chitin was used as substrate and 24 hours, ChiB, when powdered chitin was used as substrate. Chitinase activity slowly dropped and vanished after 48 hours. Chitosanase activity was then detected at 60 hours, peaking at 84 hours. The optimum pH and temperature of ChiA, ChiB, and chitosanase are 7/50℃, 6/30℃, and 7/60℃, respectively. Shot gun cloning experiment was performed by partially digesting genomic DNA of B. circulans PP8 with PstI. Fragments of 2-9 kb in size were ligated to pBluescript/SK and then transformed to E.coli DH5α cells. After screening 1,800 transformants, 2 positive clones with chitinase activity on colloidal chitin agar plate were found, Clone 847 and 1691.