Identification and characterization of a novel β-galactosidase from Victivallis vadensis ATCC BAA-548, an anaerobic fecal bacterium

Abstract

Victivallis vadensis ATCC BAA-548 is a Gram-negative, anaerobic bacterium that was isolated from a human fecal sample. From the genomic sequence of V. vadensis, one gene was found to encode agarase; however, its enzymatic properties have never been characterized. The gene encoding the putative agarase (NCBI reference number ZP_01923925) was cloned by PCR and expressed in E. coli Rosetta-gami by using the inducible T(7) promoter of pET28a(+). The expressed protein with a 6×His tag at the N-terminus was named His6-VadG925 and purified as a soluble protein by Ni(2+)-NTA agarose affinity column chromatography. The purification of the enzyme was 26.8-fold, with a yield of 73.2% and a specific activity of 1.02 U/mg of protein. The purified His6-VadG925 produced a single band with an approximate MW of 155 kDa, which is consistent with the calculated value (154,660 Da) including the 6×His tag. Although VadG925 and many of its homologs were annotated as agarases, it did not hydrolyze agarose. Instead, purified His(6)-VadG925 hydrolyzed an artificial chromogenic substrate, p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-D-galactopyranoside. The optimum pH and temperature for this β-galactosidase activity were pH 7.0 and 40°C, respectively. The K(m) and V(max) of His6-VadG925 towards p-nitrophenyl-β-D-galactopyranoside were 1.69 mg/ml (0.0056 M) and 30.3 U/mg, respectively. His6-VadG925 efficiently hydrolyzed lactose into glucose and galactose, which was demonstrated by TLC and mass spectroscopy. These results clearly demonstrated that VadG925 is a novel β-galactosidase that can hydrolyze lactose, which is unusual because of its low homology to validated β-galactosidases.

Gut microbiota in obstructive sleep apnea–hypopnea syndrome: disease-related dysbiosis and metabolic comorbidities

“…However, the non-OSAHS patients in this enterotype may maintain constant health, leading to the speculation that Prevotellaceae, Bacteroidetes , and Victivallis spp. are putative SCFA-producing bacteria [41,42] and occur to a lesser degree in patients with OSAHS than in normal controls.…”

Biochemical Characteristics and Substrate Degradation Pattern of a Novel Exo-Type β-Agarase from the Polysaccharide-Degrading Marine Bacterium Flammeovirga sp. Strain MY04

“…To assay the smallest substrate, various oligosaccharides were reacted with the enzyme rAgaO, and the digests were analyzed by TLC as described above. D-Glucose and D-galactose (1.0 mg/ml) were used as standard markers to assay the lactose-degrading activity as described previously (39).…”

Amy63, a novel type of marine bacterial multifunctional enzyme possessing amylase, agarase and carrageenase activities

“…The PCR products were gel-purified and cloned into pET-30a(+) vector. The positive recombinant was confirmed by sequencing and transformed into E. coli BL21 (DE3) cells, and induced with isopropyl-β-thiogalactopyranoside (IPTG) at a final concentration of 0.2 mM at 16 °C for 16 h. The recombinant Amy63 was purified as described before 39 . Briefly, the protein was purified with three-column step procedure: HisTrap TM HP column (GE Healthcare), HiTrap TM Q HP column (GE Healthcare) and Hiload TM 16/600 superdex TM 200 column (GE Healthcare).…”

“…The reaction was cooled using flowing water. And the absorbance at 540 nm (OD 540 ) was then recorded 39 . Enzyme activity (U) was defined as the amylase enzyme that liberated 1 μmol of D-glucose per minute.…”