Improved Transferase/Hydrolase Ratio through Rational Design of a Family 1 β-Glucosidase from Thermotoga neapolitana

Abstract

Alkyl glycosides are attractive surfactants because of their high surface activity and good biodegradability and can be produced from renewable resources. Through enzymatic catalysis, one can obtain well-defined alkyl glycosides, something that is very difficult to do using conventional chemistry. However, there is a need for better enzymes to get a commercially feasible process. A thermostable ␤-glucosidase from the well-studied glycoside hydrolase family 1 from Thermotoga neapolitana, TnBgl1A, was mutated in an attempt to improve its value for synthesis of alkyl glycosides. This was done by rational design using prior knowledge from structural homologues together with a recently generated model of the enzyme in question. Three out of four studied mutations increased the hydrolytic reaction rate in an aqueous environment, while none displayed this property in the presence of an alcohol acceptor. This shows that even if the enzyme resides in a separate aqueous phase, the presence of an organic solvent has a great influence. We could also show that a single amino acid replacement in a less studied part of the aglycone subsite, N220F, improves the specificity for transglycosylation 7-fold and thereby increases the potential yield of alkyl glycoside from 17% to 58%.

Efficient and Regioselective Synthesis of β-GalNAc/GlcNAc-Lactose by a Bifunctional Transglycosylating β- N -Acetylhexosaminidase from Bifidobacterium bifidum

“…The R T/H of BbhI was 1.53 for GalNAc-Lac or 1.46 for GlcNAc-Lac under optimal conditions, consistent with the reported range of glycosidase catalysis at 0.2 to 2.15 (50)(51)(52). Site-directed mutation of a thermostable ␤-glucosidase obtained from Thermotoga neapolitana increased the transferase/hydrolase ratio by 7-fold (50). Thus, the R T/H of BbhI can be improved in the future through molecular evolution.…”

“…The effects of substrate concentration, pH, temperature, and reaction time on the yield of the transglycosylation product were studied, and the optimal conditions for the maximum yields of GalNAc-Lac and GlcNAc-Lac were obtained. The R T/H of BbhI was 1.53 for GalNAc-Lac or 1.46 for GlcNAc-Lac under optimal conditions, consistent with the reported range of glycosidase catalysis at 0.2 to 2.15 (50)(51)(52). Site-directed mutation of a thermostable ␤-glucosidase obtained from Thermotoga neapolitana increased the transferase/hydrolase ratio by 7-fold (50).…”

Microbial β-Glucosidases: Screening, Characterization, Cloning and Applications

“…Understanding mechanism of transglycosylation and identification of amino acid residues playing central role in transglycosylation reactions hold a great importance in protein engineering for the development of β-glucosidase with high transglycosylation reaction. Replacement of asparagine residue to phenylalanine at position 220 of GH 1 β-glucosidase of Thermotoga neapolitana resulted in 7-fold increase in the transglycosylation reactions [142].…”

“…Transglycosylation fundamentally is an important reaction for production of many compounds such as aryl/alkyl-, poly glycosides, and synthetic oligosaccharides e.g., galacto-oligosaccharides and gentio-oligosaccharides [142]. Both glycosyltransferases and glycosidases can be utilized for catalyzing transglycosylation.…”

Marine enzymes and food industry: insight on existing and potential interactions

“…The mutations introduced resulted in minor changes in catalytic efficiency (as k cat /K M ), transition temperature and stability, as compared to the WT (Khan et al, 2011). Based on the knowledge gathered in this work, namely on structure-activity relation, further mutations of the TnBgl 1A enzymes were produced, aiming to further increase the yield of alkyl glucosidase in the transglycosylation reaction and bring the process closer to commercial use (Lundemo et al, 2013). Shifting the polar asparagine with non-polar phenylalanine led to a 7 fold increase in r s /r h ratio, allowing for a 57% yield in hexyl-β-glucoside from a 34 mM solution of pNPG in about 4 h. A site specific mutation was also recently used to increase the performance of β-glucosidase (GH1) from T. maritima on quercetin-3,4 -glucoside (Q3,4 ) and on quercetin-4 -glucoside (Q4 ).…”