Xylo- and cello-oligosaccharide oxidation by gluco-oligosaccharide oxidase from Sarocladium strictumand variants with reduced substrate inhibition

Abstract

BackgroundThe oxidation of carbohydrates from lignocellulose can facilitate the synthesis of new biopolymers and biochemicals, and also reduce sugar metabolism by lignocellulolytic microorganisms, reserving aldonates for fermentation to biofuels. Although oxidoreductases that oxidize cellulosic hydrolysates have been well characterized, none have been reported to oxidize substituted or branched xylo-oligosaccharides. Moreover, this is the first report that identifies amino acid substitutions leading to GOOX variants with reduced substrate inhibition.ResultsThe recombinant wild type gluco-oligosaccharide oxidase (GOOX) from the fungus Sarocladium strictum, along with variants that were generated by site-directed mutagenesis, retained the FAD cofactor, and showed high activity on cello-oligosaccharide and xylo-oligosaccharides, including substituted and branched xylo-oligosaccharides. Mass spectrometric analyses confirmed that GOOX introduces one oxygen atom to oxidized products, and 1H NMR and tandem mass spectrometry analysis confirmed that oxidation was restricted to the anomeric carbon. The A38V mutation, which is close to a predicted divalent ion-binding site in the FAD-binding domain of GOOX but 30 Å away from the active site, significantly increased the kcat and catalytic efficiency of the enzyme on all oligosaccharides. Eight amino acid substitutions were separately introduced to the substrate-binding domain of GOOX-VN (at positions Y72, E247, W351, Q353 and Q384). In all cases, the Km of the enzyme variant was higher than that of GOOX, supporting the role of corresponding residues in substrate binding. Most notably, W351A increased Km values by up to two orders of magnitude while also increasing kcat up to 3-fold on cello- and xylo-oligosaccharides and showing no substrate inhibition.ConclusionsThis study provides further evidence that S. strictum GOOX has broader substrate specificity than the enzyme name implies, and that substrate inhibition can be reduced by removing aromatic side chains in the -2 binding subsite. Of the enzyme variants, W351A might be particularly advantageous when oxidizing oligosaccharides present at high substrate concentrations often experienced in industrial processes.

Fusion of a Xylan-Binding Module to Gluco-Oligosaccharide Oxidase Increases Activity and Promotes Stable Immobilization

“…In particular, it is conceivable that the relatively rigid TP-rich linker caused local conformational change in the FAD-binding domain, as proposed for the V38A mutation at the N-terminal region of GOOX-VN, which also increases k cat on glucose, xylose and their oligosaccharides by up to 2-fold [7].…”

Direct comparison of gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability

“…The K m values of Ct CBM22A_Y300A on monosaccharides including glucose and xylose were also decreased by around 45%, resulting in higher catalytic efficiency (Table 1). For instance, k cat / K m values of Ct CBM22A_Y300A on glucose and xylose (173 and 27 mM −1 min −1 , respectively) are about 100% higher than those of Y300A (Table 1), and more than 600% higher than those reported for wild-type GOOX19. As Ct CBM22A does not bind xylose and glucose27, the observed improvement in K m and catalysis on monosaccharides, as well as slight thermal activation at 40 °C (Fig.…”

“…The GOOX variant Y300A displays a similar substrate profile to the wild-type enzyme; however, it demonstrates higher activity on glucose16 and lower substrate inhibition19. Therefore, Y300A was chosen for the current comparative analysis with GO.…”

“…Similar to MnCO, GOOX oxidizes oligosaccharides more effectively than monosaccharides16. To date, thirteen GOOX variants have been created through site-directed mutagenesis1619, where one variant (Y300A) shows higher activity on monosaccharides (including glucose and xylose) compared to the wild-type GOOX. The Y300A variant also retains activity on oligosaccharides and displays reduced substrate inhibition19.…”

Synthesis of Glycoconjugates Utilizing the Regioselectivity of a Lytic Polysaccharide Monooxygenase

“…These enzymes use different mechanisms to generate oxidations on mono-, oligo-and/or polysaccharides. These oxidation reactions produce either carbonyls or carboxylic acids and, for poly-and oligomeric substrates, these reactions involve enzymes such as LPMOs 5,8,9,10 , cellobiose dehydrogenase (CDH) 11,12 , galactose oxidase 13 , pyranose dehydrogenase 14,15 and glucooligosaccharide oxidases 16 . Together, these enzymes enable a range of oxidative functionalizations 17 and offer several possibilities for subsequent glycoconjugation 18 .…”