New alkalophilic ß-galactosidase with high activity in alkaline pH region from Teratosphaeria acidotherma AIU BGA-1.

A ß-d-galactosidase exhibiting high activity in the alkaline pH region was purified from Teratosphaeria acidotherma AIU BGA-1, which we previously isolated as a unique fungal producer of three acidophilic and one alkalophilic ß-d-galactosidases (Isobe et al., J. Biosci. Bioeng., 116, 171-174, 2013). The enzyme was stable in the pH range 7.5-10.0 and exhibited optimal activity at pH 8.0 and 60°C. The enzyme hydrolyzed 2-nitrophenyl ß-d-galactopyranoside, 4-nitrophenyl ß-d-galactopyranoside, and lactose, and the Km values were estimated to be 0.349 mM, 0.488 mM, and 701 mM, respectively. Chelating reagents (EDTA and o-phenanthroline) and metals (Cu2+and Ni2+) inhibited the enzyme activity, and Mn2+ was a good activator. The enzyme also exhibited transgalactosylation activity for lactose. The enzyme's molecular mass was estimated to be 180 kDa, and its structure was monomeric. Thus, the enzymatic and physicochemical characteristics of the alkalophilic ß-galactosidase in this study clearly differed from those of the previously known alkalophilic ß-d-galactosidases.

Novel acidophilic ß-galactosidase with high activity at extremely acidic pH region from Teratosphaeria acidotherma AIU BGA-1.

A ß-galactosidase exhibiting maximal activity at pH 1.0 was purified from Teratosphaeria acidotherma AIU BGA-1. The enzyme had a molecular mass of 180 kDa and consisted of two heterosubunits of 120 kDa and 66 kDa. The N-terminal amino acid sequence of the large subunit was found to be SPNLQDIVTVDGESY. These physicochemical properties differed from those of other microbial ß-galactosidases. At pH values of 1.5 and pH 4.5, the enzyme exhibited its highest activity at temperatures of 70°C and 80°C, respectively. Thus, the enzyme exhibited the lowest optimal pH and highest optimal temperature among the microbial ß-galactosidases thus reported. The enzyme retained more than 80% of its original activity in the pH range from 2.0 to 8.0 by incubation at 50°C for 30 min. The enzyme hydrolyzed 4-nitrophenyl-ß-D-fucopyranoside, 2-nitrophenyl-ß-D-galactopyranoside, and 4-nitrophenyl-ß-D-galacto-pyranoside at relative reaction rates of 100, 59, and 24, respectively, at pH 1.5, and its affinity for ß-D-galactopyranosides was higher than that for ß-D-fucopyranosides. The enzyme also efficiently hydrolyzed lactose in milk and whey from yoghurt at pH 1.5.

Acidophilic fungus, Teratosphaeria acidotherma AIU BGA-1, produces multiple forms of intracellular ß-galactosidase.

Teratosphaeria acidotherma AIU BGA-1 isolated from an acidic and high temperature hot spring produced four intracellular ß-D-galactosidases with different pH activity profiles, in which three forms were acidophilic and stable from extremely acidic to neutral pH region. The other one was alkalophilic and unstable in the acidic pH region.

Characterization of new ß-galactosidase from acidophilic fungus, Teratosphaeria acidotherma AIU BGA-1.

The ß-galactosidase exhibiting high activity from an extremely acidic pH region to neutral pH region was efficiently purified from an acidophilic fungus, Teratosphaeria acidotherma AIU BGA-1, using affinity chromatography with Toyopearl resins immobilized 4-aminophenyl-ß-d-galactopyranoside. The enzyme was stable in the pH range from 1.5 to 7.0, and exhibited optimal activity at pH 2.5-4.0 and 70°C. 2-Nitrophenyl-ß-d-galactopyranoside, 4-nitrophenyl-ß-d-galactopyranoside and lactose were rapidly hydrolyzed, and the apparent Km values were estimated to be 0.19 mM, 1.2 mM and 170 mM, respectively. Thus, the enzyme can be used in the wide pH range for hydrolysis of lactose. The molecular mass of the enzyme was estimated to be 140 kDa with two hetero subunits of 86 kDa and 50 kDa. The N-terminal amino acid sequence of the small subunit was found to be NTRMIIFNDK. These enzymatic and physicochemical characteristics are remarkably different from those of the previously known ß-galactosidases.