Increasing the transglycosylation activity of α‐galactosidase from Bifidobacterium adolescentis DSM 20083 by site‐directed mutagenesis

Abstract

The alpha-galactosidase (AGA) from Bifidobacterium adolescentis DSM 20083 has a high transglycosylation activity. The optimal conditions for this activity are pH 8, and 37 degrees C. At high melibiose concentration (600 mM), approximately 64% of the enzyme-substrate encounters resulted in transglycosylation. Examination of the acceptor specificity showed that AGA required a hydroxyl group at C-6 for transglycosylation. Pentoses, hexuronic acids, deoxyhexoses, and alditols did not serve as acceptor molecules. Disaccharides were found to be good acceptors. A putative 3D-structure of the catalytic site of AGA was obtained by homology modeling. Based on this structure and amino acid sequence alignments, site-directed mutagenesis was performed to increase the transglycosylation efficiency of the enzyme, which resulted in four positive mutants. The positive single mutations were combined, resulting in six double mutants. The mutant H497M had an increase in transglycosylation of 16%, whereas most of the single mutations showed an increase of 2%-5% compared to the wild-type AGA. The double mutants G382C-Y500L, and H497M-Y500L had an increase in transglycosylation activity of 10%-16%, compared to the wild-type enzyme, whereas the increase for the other double mutants was low (4%-7%). The results show that with a single mutation (H497M) the transglycosylation efficiency can be increased from 64% to 75% of all enzyme-substrate encounters. Combining successful single mutants in double mutations did not necessarily result in an extra increase in transglycosylation efficiency. The donor and acceptor specificity did not change in the mutants, whereas the thermostability of the mutants with G382C decreased drastically.

Bifidobacterium carbohydrases‐their role in breakdown and synthesis of (potential) prebiotics

“…Similar results were found for the heterologously produced enzyme [123,125,126]. In contrast to the preferred formation of 1 6 linkages during the transfer reaction, Leder et al [122] showed that 1 3 linkages are hydrolyzed at a higher rate than 1 6 linkages.…”

“…For bifidobacteria only one attempt is reported. In this case the transglycosylation properties of a-galactosidase from B. adolescentis were changed by sitedirected mutagenesis [125]. The highest increase in transglycosylation activity obtained was 16% for one single mutant, whereas most of the other single mutants showed an increase of only 2 -5%.…”

Expression and Characterization of a Bifidobacterium adolescentis Beta-Mannanase Carrying Mannan-Binding and Cell Association Motifs

“…In line with this view, additional exo-acting enzymes (␤-mannosidases and ␣-galactosidases) or sugar phosphorylases (35) are likely to be needed to complete the degradation to monomers. An ␣-galactosidase from B. adolescentis has been expressed recombinantly (61). Its potential activity toward galactosyl substitutions of mannosides has, however, not been reported.…”

Enhancement of the Alcoholytic Activity of α-Amylase AmyA fromThermotoga maritimaMSB8 (DSM 3109) by Site-Directed Mutagenesis

“…From the structural point of view, the direct comparison through multiple-sequence alignments of liquefying and saccharifying enzymes, as well as maltogenic amylases, neopullulanases, and CGTases, that have important transglycosylation activities has allowed the identification of residues potentially involved in transglycosylation activity (20,30,54). In spite of the fact that the ␣-amylase AmyA from T. maritima is already a saccharifying enzyme, after a sequence and structure comparison, we were able to identify various residues that are important in saccharifying amylases and are not conserved in the T. maritima ␣-amylase, suggesting that their mutation may generate an even more saccharifying enzyme; actually, as alcoholysis can be considered a specific type of transfer reaction, this would probably result in a more alcoholytic enzyme.…”

“…Several reports in the literature indicate that transglycosylation activity can be introduced or modified by promoting the effective concentration of less polar substrates in the active site, either by decreasing the affinity for water (7,20,34) or by modifying the geometry of the active site to disable the activation of the catalytic water molecule (39) and thereby favoring the activation of other, bulkier acceptor groups. The importance of aromatic residues in substrate binding at the active site of glycosidases through stacking interactions is well documented (7), and it has been proven that the introduction or substitution of these kinds of residues can modify the affinity of the protein at the different substrate subsites, affecting both the product profile and the activity of the enzyme (40,45,50,54).…”