Genome sequence of the psychrophilic Cryobacterium sp. LW097 and characterization of its four novel cold-adapted β-galactosidases.

A psychrophilic strain Cryobacterium sp. LW097 was isolated from the subglacial sediments and discovered to show considerable β-galactosidases activity at low temperatures. To provide access to genes predicted to encode cold-adapted glycoside hydrolases with biotechnological relevance, we have sequenced the genome of Cryobacterium sp. LW097. Annotation with CAZy database revealed four β-galactosidase genes, bgal322, bgal435, bgal436, and bgal2567 belonging to the GH-42 family and GH-35 family. All the four β-galactosidases recombinantly expressed retained a high level of relative activity at 5 °C and showed different optimum temperatures ranging from 25 °C to 40 °C. The enzyme kinetics proved that Bgal322, Bgal436, and Bgal2567 had lower Km to both oNPG and lactose at 5 °C, further proving their adaption to low temperature. Substrate specificity analysis showed that these four β-galactosidases owned different preferences. The novel GH-42 β-galactosidases Bgal435 showed β-D-glucosidase activity (33.67 ± 0.28%) in addition to β-D-galactosidase activity. Bgal322 preferred β-D-(1,4)-galactobiose, whereas the other three preferred lactulose. Bgal435 showed the highest kcat value of 68.2 ± 1.7 s -1 at 5 °C toward lactose among these four enzymes. The exquisite substrate specificity of Bgal436 in milk made it a potential candidate for applications in milk lactulose quantification.