Subcloning of cyclodextrin glycosyltransferase gene from Bacillus sp. A11 into Bacillus Vectors

Abstract

The aim of this study was to subclone CGTase gene from Bacillus sp. A11 in E. coli recombinant plasmids CSBC5 or pCSBC8, into an expression system of Bacillus subtilis host using pUB110, Bacillus sp. DNA vector and HV33, as the DNA vector However, subcloning of CGTase gene into pHV33 at HindIII, SalI ,EcoRl sites and subcloning of CGTase gene into pUB110 at EcoRl .PvuII sites were unsuccessful. Since, the vector pHV33 and CGTase gene containing fragment from pCSBC5 could not be prepared for ligation by cleaving at the designing of restriction sites, and when CGTase gene from pCSBC5 or pCSBC8 was subcloned into pHV33, it appeared that there were no transformant at all. It was later found out that the CGTase activity in pCSBC5 and pCSBC8 could not be detected. Therefore, recloning of the CGTase gene from Bacillus sp. A11 into the plasmid vector pUC18 was performed. The CGTase gene fragments were isolated from Hindlll-digested chromosomal DNA of Bacillus sp. A11 via DNA-DNA hybridization using pCSBC5 as a DNA probe, then ligated with Hindlll digested pUC18, and transformed into E. coli JM101. More than 1,000 transformants containing recombinant plasmids were screened for dextrinizing activity by iodine test. It appeared that only one transformant, namely no.17, showed dextrinizing activity by hydrolyzing starch resulting in clear zone formation. To ascertain if the plasmid isolated from ransformant no.17 contained the CGTase gene, it was hybridized with 2 CGTase gene probes (1.7 and 3kb) prepared from the CGTase gene in pCSBC5 The DNA fragment 3.54 kb from the HindIII-digested recombinant plasmid no 17 could hybridize with both the labeled 1.7 and 3 kb CGTase probes from pCSBC5. Upon subculture, however, the transformant no.17 lost its dextrinizing activity.