A novel cold-active β-d-galactosidase with transglycosylation activity from the Antarctic Arthrobacter sp. 32cB – Gene cloning, purification and characterization

Abstract

a b s t r a c tA gene encoding a novel ␤-d-galactosidase from the psychrotolerant Antarctic bacterium Arthrobacter sp. 32cB was isolated, cloned and expressed in Escherichia coli. The active form of recombinant ␤-dgalactosidase consists of two subunits with a combined molecular weight of approximately 257 kDa. The enzyme's maximum activity towards o-nitrophenyl-␤-d-galactopyranoside was determined as occurring at 28 • C and pH 8.0. However, it exhibited 42% of maximum activity at 10 • C and was capable of hydrolyzing both lactose and o-nitrophenyl-␤-d-galactopyranoside at that temperature, with K m values of 1.52 and 16.56 mM, and k cat values 30.55 and 31.84 s −1 , respectively. Two units of the enzyme hydrolyzed 90% of the lactose in 1 mL of milk at 10 • C in 24 h. The transglycosylation activity of Arthrobacter sp. 32cB ␤-d-galactosidase was also examined. It synthesized galactooligosaccharides in a temperature range from 10 to 30 • C. Moreover, it catalyzed the synthesis of heterooligosaccharides such as lactulose, galactosyl-xylose and galactosyl-arabinose, alkyl glycosides, and glycosylated salicin from lactose and the appropriate acceptor at 30 • C. The properties of Arthrobacter sp. 32cB ␤-d-galactosidase make it a candidate for use in the industrial removal of lactose from milk and a promising tool for the glycosylation of various acceptors, especially those which are thermosensitive.

Discovery, Molecular Mechanisms, and Industrial Applications of Cold-Active Enzymes

“…One last example of a β-galactosidase with potential applications in milk and dairy product industry is the enzyme from Antarctic Arthrobacter sp. 32cB, which has the capacity to hydrolyzed 90% of the lactose in 1 mL of milk at 10°C in 24 h (Pawlak-Szukalska et al, 2014 ).…”

A Novel Cold-Adapted Leucine Dehydrogenase from Antarctic Sea-Ice Bacterium Pseudoalteromonas sp. ANT178

“…Thermodynamic parameters such as Δ H , Δ S and Δ G at different temperature (0–30 °C) were calculated and listed in Table 4 . At 0, 10, 20, and 30 °C, the k cat value of rPsLeuDH were 12.25, 14.96, 20.20 and 30.13/s, respectively, indicating that the k cat value increased with increasing temperature, which was similar to the k cat change trend of cold-adapted β- d -galactosidase at different temperatures [ 26 ]. rPsLeuDH also exhibited lower Δ H , Δ S and Δ G and higher k cat at low temperature, as compared to mesophilic enzyme, which may be mainly related to the conformation of cold adapted protein [ 27 ].…”

In Situ Random Microseeding and Streak Seeding Used for Growth of Crystals of Cold-Adapted β-d-Galactosidases: Crystal Structure of βDG from Arthrobacter sp. 32cB

“…Overexpression was induced by addition of 20% L-arabinose solution to the final concentration of 0.02%. The culture was further cultivated for 15 h to OD 600 of 3.8 ± 0.2 and harvested by centrifugation (6000 × g, 15 min, 4 • C) [37]. The extraction of intracellular protein was carried out by two separate methods.…”

“…Method 1: The cells were resuspended in buffer A containing: 20 mM K 2 HPO 4 /KH 2 PO 4 (pH 6.0), 50 mM KCl and the cell suspension was disrupted by sonication on an ice bath using 20 repetitions of 15 s impulses with 60 s pauses to avoid sample overheating. The lysate was clarified by centrifugation at 4 • C for 30 min at 9000 × g [37]. Method 2: The cell pellet was ground into a fine powder in a mortar and pestle under liquid nitrogen, with addition of silicone beads.…”