Characterization of galactooligosaccharides produced by β-galactosidase immobilized onto magnetized Dacron

Abstract

a b s t r a c tBioconversions using enzymes immobilized in magnetic supports present significant advantages due to the easy separation of the enzyme from the reaction mixture and the simplicity and low cost of the support preparation. The characterization of the oligosaccharide mixture obtained by the action of b-galactosidase covalently attached, via glutaraldehyde, to a hydrazideeDacronemagnetite composite is presented. The fractionation of the oligosaccharide mixture by high performance liquid chromatography, followed by the analysis of the purified compounds by mass spectrometry and nuclear magnetic resonance spectroscopy permitted the identification of glucose, galactose, lactose and a hexose disaccharide containing a 1/6 linkage. Also, the following GOS were identified: b-D-Galp-(1/6)-b-D-Galp-(1/4)-Glcp, b-D-Galp-(1/4)-[b-D-Galp-(1/6)]-Glcp and b-D-Galp-(1/6)-b-D-Galp-(1/6)-b-D-Galp-(1/4)-Glcp. When GOS yield (26.2%) and kinetics of biotransformation of lactose by the Dacron immobilized b-galactosidase were compared with values obtained for the enzyme immobilized in other magnetic supports, similar behaviour was observed.

Structural and functional stabilization of protein entities: state-of-the-art

“…Notwithstanding the fact that multimeric proteins are intrinsically more stable than monomeric ones due to their rigidity when compared to their monomeric counterparts [3], under certain experimental conditions such subunit-subunit multi-interactions may be weakened leading to dissociation of subunits and concomitant rapid inactivation of the multimeric protein [8]. Hence, prevention of subunit dissociation is the first goal when stabilization of such highly interesting protein entities is sought [8,45,[95][96][97][102][103][104][105][106][107].…”

Hydrophilic interaction liquid chromatography coupled to mass spectrometry for the characterization of prebiotic galactooligosaccharides

“…Nevertheless, the use of mass spectrometric (MS) detectors coupled to HPLC systems has considerably enriched the field of oligosaccharide analysis, allowing the determination of their molecular weight [18]. Multi-stage mass spectrometry (MS n ) can also provide structural information; however, scarce studies have been still carried out about its utility for the characterization of neutral oligosaccharides [19,20]. Moreover, the addition of appropriate metals to HPLC mobile phases to form complexes with carbohydrates or their previous derivatization (peracetylation or permethylation) is usually required to facilitate the sequential identification of residues by MS [21].…”

“…Characterization of different GOS has been generally carried out by the combination of a great variety of analytical methodologies (methylation analysis followed by GC-MS, NMR, HPAEC-PAD-MS, ESI-MS) with previous fractionation of the oligosaccharides (yeast treatment, SEC, HILIC) [7,8,20]. HILIC-MS has been used for the analysis of GOS previously fractionated by cation exchange chromatography to determine their molecular weights [22].…”

Analysis, structural characterization, and bioactivity of oligosaccharides derived from lactose

“…GOS synthesized from lactose and previously purified by LC were analyzed by Neri et al. using ESI(+) and MS n ( n = 2,3) on a LTQ mass spectrometer. The initial ESI‐MS analysis determined the presence of di‐, tri‐, and tetrasaccharides; while the ESI‐MS/MS experiments allowed the detection of major fragment ions at m / z 305, 275, and 245 in the disaccharide fraction which were attributed to the cross‐ring fragmentation of two glucose units linked by a α(1→6) type linkage .…”