Purification and Some Properties of .ALPHA.-Galactosidase from Pseudomonas fluorescens H-601.

Abstract

Pseudomonas fluorescens H-601, isolated from soil, produces a-galactosidase.The enzyme was purified to homogeneity by disc electrophoresis after column chromatographies on Butyl-Toyopearl 650M, DEAE-Toyopearl 650M, and Toyopearl HW-55F. The enzyme had a molecular weight of 390,000 by gel filtration with Toyopearl HW-55F and 86,000 by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 6.3. The enzyme was most active at pH 6.0-7.0 and at 45°C and stable up to 40°C at pH 6.5 for 15min of incubation. The enzyme hydrolyzed PNP a-galactoside, ONPa-galactoside, melibiose, raffinose, and stachyose at the relative velocities of 100, 59, 28, 13, and 12. The enzyme had strong transfer activity and wide acceptor specificity.The a-galactosidase (a-D-galactoside galactohydrolase, EC 3.2.1.22) are a group of exo-type carbohydrases, which release agalactose from the non-reducing end side of the substrates such as synthetic a-galactosides and oligosaccharides having a-galactosidic bonds. The enzyme also catalyzes transgalactosylation as well as having hydrolysis action.1>2) a-Galactosidase has been used to remove raffinose and stachyose in soymilk processing and raffinose in sugar beet molasses by its hydrolytic action.3'4) Recently, oligosaccharides having a-galactosidic bonds such as raffinose and stachyose have attracted attention as strong bifldus growth factors. However, no study has been done on the synthesis of those oligosaccharides with the transgalactosylation of a-galactosidase. We have been working on the synthesis of various oligosaccharides and glycosides using transglycosylation of microbial enzymes such as cyclomaltodextrin glucanotransferase,5'6) amylomaltase,7) debranching enzyme,8) /?-

Cloning of the Gene Encoding a Novel Thermostable α-Galactosidase from Thermus brockianus ITI360

“…␣-Galactosidases catalyze the hydrolysis of ␣-1,6-linked ␣-galactose residues from oligosaccharides such as melibiose (galactose-␣-1,6-glucose), raffinose (galactose-␣-1,6-sucrose), and stachyose (galactose ␣-1,6-raffinose) and from polymeric galactomannans (37,38,48,49,58). Some ␣-galactosidases are also known to catalyze transgalactosylation, especially at a high concentration of substrate (21,22). ␣-Galactosidases have been isolated from a variety of eucaryotes and bacteria.…”

Three α‐Galactosidase Genes of Trichoderma reesei Cloned by Expression in Yeast

“…Most biochemical analyses of rx-galactosidases have been carried out using the small substrates p-nitrophenyl-a-D-galactopyranoside, melibiose, raffinose and stachyose. The substrate specificities of a-galactosidases can vary, for example the activity of the enzymes produced by Pseudomonasfluorescens (Hashimoto et al, 1991) and Candida guilliermondii (Hashimoto et al, 1995) decrease with the number of sugar units in these substrates. The capability of some a-galactosidases to release a-galactosyl side groups from galactomannans has also been studied.…”