Molecular cloning of β-glucosidase gene from alcaligenes faecalis to E. coli

Abstract

A gene encoding $\beta$-glucosidase was transferred form Alcaligenea faecalis ATCC 21400 to Escherichia coli HB 101 by molecular cloning using pBR 322 as a vector. Chromosomal DNA of A. faecalis was isolated from an overnight culture and digested with Eco RI. DNA fragments above 3.2Kb. were collected by fractionation with sucrose density gradient ultracentrifugation. The pBR 322 DNA was also digested with Eco and dephosphorylated by treating with bacterial alkaline phosphatase. Both digested DNA preparations were mixed and ligated with T4 DNA ligase. With the ligation mixture, E. coli HB101 cells we tried to transform. To screen for $\beta$-glucosidase positive colonies, the p-nitro-phenyl-$\beta$-D glucopyranoside -filter paper-blotting method was used. Colonies were lyzed with lysozyme and Triton X-100 and PNPG was used as the substrate for $\beta$-glucosidase. Two colonies showed distinct yellow color on the filter paper indicating hydrolysis of PNPG by $\beta$-glucosidase. From the two $\beta$-glucosidase positive colonies, two different plasmids were isolated and named pBAF 1200 and pBAF 1400, respectively. With the two plasmids, E. coli HB 101 was netrans-formed and the PNPG hydrolysis in the transformants was reconfirmed. Restriction analysis indicated that the two plasmids contain identical chromosomal DNA inserts except that the pBAF 1400 had extra 2-KB. chromosomal fragment which was not related to the $\beta$-glucosidase production. The E. coli HB 101 harboring pBAF 1200 produced $\beta$-glucosidase three times higher than the gene donor A. faecalis.