Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus

Abstract

When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces a-galactosidase I which is different from a-galactosidase II which it produces when grown on galactose, melibiose, raffinose, or stachyose. We have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9 to 6.4 for a-galactosidase I, 6.3 to 6.5 for a-galactosidase II). However, a-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000, whereas a-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. a-Galactosidase I has a lower affinity for melibiose, raffinose, and stachyose (K. values of 20.8, 98.1, and 8.5 mM, respectively) than does a-galactosidase II (Km values of 2.3, 5.9, and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase. The increase in specific activity of a-galactosidase which was associated with growth on guar gum was due to an increase in the specific activity of enzyme I. Low, constitutive levels of enzyme II also were produced. By contrast, enzyme II was the only a-galactosidase thgt was detectable in bacteria which had been grown on galactose, melibiose, raffinose, or stachyose. Bacteroides ovatus, a gram-negative obligate anaerobe, is a member of the resident microflora of the human colon (15). This organism can ferment a variety of a-galactosides such as melibiose, raffinose, stachyose, and guar gum (7, 18), all of which contain a(1-6)-linked galactose residues (Fig. 1). All of these a-galactosides are found in human foods. Raffinose and stachyose occur naturally in beans and other vegetables, whereas guar gum is added to foods as an emulsifier. Guar gum also is being tested for possible use in reducing the rate of glucose absorption by diabetics (8, 14). Except for melibiose, these a-galactosides are not degraded and absorbed during transit through the small intestine. Thus, they may serve as natural sources of carbohydrate for B. ovatus in the colon. Although many species of colon bacteria can ferment melibiose and raffinose, only a few species can ferment galactomannans such as guar gum (18, 19). During a prelim

Principal characteristics of α‐galactosidase from Leuconostoc mesenteroides subsp. mesenteroides

“…2). These results are similar to those determined by GHERARDINI et al (1985) in Lactobacillus. The fact that the pH optimum of Leuconostoc strains is close to neutral, may be desirable for the action of this enzyme to degrade a-D-galactosides present in food.…”

“…Most a-gal are stable over a broad range of acidity. In general, bacterial a-gal has a pH optimum in the range of 6.0 to 7.5 (KAWANURA et al 1976, GHERARDINI et al 1985, while the pH optimum of the fungal and yeast a-gal is more acidic, from 3.5 to 5.0 (ULEZLO andZAPROMETOVA 1982, OHTAKARA et al 1984). The pH optimum determined in Leuc.…”

“…(1985). The enzyme was also purified and characterized in Bacteriodes ovatus (GHERARDINI et al 1985). These works reported two different triniers of a-gal (I and 11) which have a monomeric molecular weight of 85,000 and 80,500 respectively.…”

A β‐mannan utilization locus in Bacteroides ovatus involves a GH36 α‐galactosidase active on galactomannans

“…The particle size increases to~1 lm after 16 h when the extent of galactose release reached 90%. [32,37]. An overlay of the modelled tetrameric BoGal36A active site with that of the active site of the RFOS-hydrolysing AgaA a-galactosidase reveals likely architectural differences between BoGal36A and other subgroup I GH36 enzymes.…”

α‐Galactosidases of Penicillium simplicissimum: production, purification and characterization of the gene encoding AGLI

“…P. simplicissimum AGLII differed completely from the other two α-galactosidases produced, as it was larger. Thus AGLII resembled more the bacterial, yeast or Aspergillus αgalactosidases than the other Penicillium enzymes [21,38,39,[43][44][45][46]. Furthermore, AGLII possessed significantly higher specific activity on the basis of the pNPG assay than AGLI and AGLIII, and was more resistant towards endproduct inhibition.…”