Improvement of the efficiency of transglycosylation catalyzed by α-galactosidase from Thermotoga maritima by protein engineering

Abstract

At high concentrations of p-nitrophenyl-α-D-galactopyranoside (pNPGal) as a substrate, its hydrolysis catalyzed by α-galactosidase from Thermotoga maritima (TmGalA) is accompanied by transglycosylation resulting in production of a mixture of (α1,2)-, (α1,3)-, and (α1,6)-p-nitrophenyl (pNP)-digalactosides. Molecular modeling of the reaction stage preceding the formation of the pNP-digalactosides within the active site of the enzyme revealed amino acid residues which modification was expected to increase the efficiency of transglycosylation. Upon the site-directed mutagenesis to the predicted substitutions of the amino acid residues, genes encoding the wild type TmGalA and its mutants were expressed in E. coli, and the corresponding enzymes were isolated and tested for the presence of the transglycosylating activity in synthesis of different pNP-digalactosides. Three mutants, F328A, P402D, and G385L, were shown to markedly increase the total transglycosylation as compared to the wild type enzyme. Moreover, the F328A mutant displayed an ability to produce a regio-isomer with the (α1,2)-bond at yield 16-times higher than the wild type TmGalA.

Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

“…Such mutations led to 4-16-fold increases in the yield of transglycosylation products, although in absolute terms the amounts produced were modest [182]. Indeed, the introduction of other mutations (P402D and F328A) into the + 1 region of the active site of a family GH36 α-D-galactosidase (subsite + 1) actually led to an enlargement of the entrance to the active site and consequently influenced the orientation of the bound acceptor.…”

Characterization of Properties and Transglycosylation Abilities of Recombinant α-Galactosidase from Cold-Adapted Marine Bacterium Pseudoalteromonas KMM 701 and Its C494N and D451A Mutants

“…To avoid loss of information about minor transglycosylation products, the proton ( 1 H) and carbon ( 13 C) NMR spectra were analyzed without separating the reaction mixtures into individual compounds. The 1 H and 13 C signals of the anomeric atoms of the substrates and the products were assigned according to the respective reference data [ 20 , 21 , 22 ], as well as by H,H-Correlation Spectroscopy (COSY) and Heteronuclear Single-Quantum Correlation (HSQC) experiments. The identified signals are shown in Table 5 .…”

“…The transglycosylation properties are well-studied for the highly thermoresistant GH 36 α- d -galactosidase from the hyperthermophilic bacterium Thermotoga maritima (TmGal36A). This enzyme catalyzes an autocondensation reaction with pNP-α-Gal as a substrate, forming substituted (1→2)-α-, (1→3)-α- and (1→6)-α-linked Gal 2 -pNP [ 22 ]. In total, the wild TmGal36A can produce up to 5.5% transglycosylation products.…”

“…In total, the wild TmGal36A can produce up to 5.5% transglycosylation products. The mechanism of the hydrolysis and synthesis in TmGal36A is not favorable for the formation and breaking of the (1→4)-α-O-glycosidic linkage [ 22 ], unlike α- d -galactosidases from human intestine [ 34 , 35 , 36 , 37 ] and α-PsGal from marine bacterium.…”

Synthesis of galactosyl glycerol from guar gum by transglycosylation of α-galactosidase from Aspergillus sp. MK14

“…Some a-galactosidases have transglycosylation action, and new products containing a-galactosyl residues have been reported. In the transglycosylation, either melibiose or pNP-a-D-galactopyranoside is used as the substrate (donor), leading to the synthesis of various oligosaccharides and alkyl galactosides (Bobrov et al, 2013;Hashimoto et al, 1995;Hinz et al, 2005;Mi et al, 2007;Simerska et al, 2006;Spangenberg et al, 2000;Van Laere et al, 1999). The enzyme from Candida guilliermondii has broad acceptor specificity to monosaccharides, disaccharides and alcohols (Hashimoto et al, 1995).…”