Cold Active B-Galactosidase of Hafnia Alvei KNOUC302 Isolated from Raw Milk: Identification of the Bacteria, Cloning Partial Gene of B-Galactosidase in Escherichia Coli

Psychrophilic bacteria producing lactose hydrolyzing enzyme were isolated from the samples of raw milk collected from dairy farms of Kyung-gi province in Korea. Among isolated strains, KNOUC302 showing the best activity of lactose hydrolysis was identified, and its B-galactosidase gene was cloned. This strain was aerobic, asporogenic bacilli, immobile, Gram negative, catalase positive and oxidase positive. It grew optimally at 20 ?C and pH=7.0-7.2. The composition of major fatty acids in the cell membrane of KNOUC 302, were C12:0 (10.2 %), C14:0 3OH (25.41 %), C16:0 (9.94 %) and C16:1 ?7c (29.75 %). Based on morphological and biochemical properties, the strain could be identified as Hafnia sp. and finally as Hafnia alvei by phylogenetic analysis based on 16S rDNA sequence. A part of B-galactosidase gene, 779 bp, coding 296 a.a from N-terminal of the B-galactosidase was isolated from Hafnia alvei KNOUC302 by partial digestion of chromosomal DNA with Sau3AI and cloned in E. coli Top 10F’. The incomplete gene of 779 bp was supplemented, from vector during cloning, with 109 bp nucleotides providing terminal codon to a complete ORF (KNOUC302 B-gal). The gene (KNOUC302 B-gal) consists of 918 bp encoding the protein of 306 amino acids and 34,806 Da of deduced Mw.