Genomics And β-Ggalactosidase Gene Function Analysis On A Marinomonas Strain Isolated From Sea Ice, Canada Basin, The Arctic Ocean

A fundamental genomics analysis was carried out on a Marinomonas strain BSi20584 isolated from an ice core sample collected from Canada Basin, the Arctic Ocean. And two P-D-galactosidase genes bgal584-1 and bga1584-2 were cloned, expressed and characterized afterwards.Genomics analysis shows that Marinomonas sp. BSi20584 contains 4462 genes. Through KEGG database analysis, BGAL584-1 was found out to be able to hydrolyze both lactose and galactan, and could work in extracellular polysaccharide degradation pathway and sphingolipid metabolic pathway at the same time, however, BGAL584-2 could only work in galactan degradation pathway. Homology modeling implied that the BGAL584-1 was a monomer, while BGAL584-2 was a homotrimer. The predicted catalytic residues of BGAL584-1 and BGAL584-2 were Glu453/Glu557/Tyr526 and Glul42/Glu314/Trp183, respectively.The bga.1584-1 gene obtained by hiTAIL-PCR was inserted into pET-30 Ek/LIC vector, while bgal584-2 was combined with pET-28a(+) vector, and then both transformed into E.coli BL21 (DE3). The recombinant enzymes were purified to apparent electrophoretic homogeneity by the way of one step Ni2+ affinity chromatography. The results of enzymatic analysis showed that enzyme BGAL584-1 was cold adaptive with an optimal tempertature at 30℃, and enzyme BGAL584-2 was a mesophilic enzyme with an optimal temperature at 50℃. Mg2+ was identified as an activator to BGAL584-1, but showed no significant influence on the activity of BGAL584-2. Fe2+, Mn2+ were identified to be activators to BGAL584-2. Zn2+ and L-glutathione strongly inhibited the activity of both enzymes. HPLC analysis showed that BGAL584-1 can hydrolyze both Gal β-1,4 GlcNAc and Gal β1-3 GlcNAc, however, BGAL584-2 can only hydrolyze Gal P-1,4 GlcNAc.