99A-23 |
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S. H. YI, Dept. of Food Science & Agricultural Chemistry, McGill Univ. Macdonald Campus, 21111 Lakeshore Rd, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada and B. Lee, Dept. of Food Science & Agricultural Chemistry, McGill Univ., Macdonald Campus, 21111 Lakeshore Rd., Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada. Effective utilization of lactose in dairy fluid wastes such as whey and whey permeate continues to be a serious problem in the dairy industry. Bioconversion of lactose in whey through an enzymatic process into useful products such as whey sweeteners and oligosaccharides, etc. is an attractive avenue for whey utilization. However the enzyme costs are a major impediment of lactose products. The objectives were to clone, overexpress, and characterize a relatively smaller size, b-galactosidase from Bifidobacterium breve. A b-galactosidase gene was screened from Bifidobacterium breve genomic DNA library constructed in pBR322 with three different restriction enzymes and cloned into pDrive vector for the sequencing. The cloned b-galactosidase gene contained 2076 bp nucleotides and had a high homology with other Bifidobacterium spp. One primer set (5'-Nde I- and 3'-BamH-) was designed and synthesized for the PCR amplification, and the amplified fragments were ligated into pET-36(B)+ overexpression vector. pET-36(B)+ plasmid containing a b-galactosidase gene was transformed into E. coli ER2566, successfully induced and overexpressed. The overexpressed b-galactosidase was purified using a FPLC system equipped with Mono Q, Superose 12, and Superdex 200 columns. Molecular mass of the purified enzyme was estimated to be 75 kDa on SDS-PAGE, but the enzyme was composed of dimer that was confirmed by activity staining on NATIVE-PAGE. Relatively smaller size of this enzyme than others should be useful as they would be better suited for industrial applications.
Session 99A, Biotechnology: General
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