title: Fusion of self-assembling amphipathic oligopeptides with cyclodextrin glycosyltransferase improves 2-O-D-glucopyranosyl-L-ascorbic acid synthesis with soluble starch as the glycosyl donor.
abstract: In this study, we fused six self-assembling amphipathic peptides (SAPs) with cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans to catalyze 2-O-D-glucopyranosyl-L-ascorbic acid (AA-2G) production with cheap substrates, including maltose, maltodextrin, and soluble starch as glycosyl donors. The results showed that two fusion enzymes, SAP5-CGTase and SAP6-CGTase, increased AA-2G yields to 2.33- and 3.36-fold that of wild-type CGTase when soluble starch was used as a substrate. The cyclization activities of these enzymes decreased, while disproportionation activities increased. Enzymatic characterization of the two fusion enzymes was performed, and kinetics analysis of AA-2G synthesis confirmed the enhanced soluble starch specificity of SAP5-CGTase and SAP6-CGTase compared to that in the wild-type CGTase. As revealed by structure modeling of the fusion and wild-type CGTases, enhanced substrate-binding capacity may result from the increased number of hydrogen bonds present after fusion. This study demonstrates an effective protein fusion approach to improving the substrate specificity of CGTase for AA-2G synthesis. Fusion enzymes, especially SAP6-CGTase, are promising starting points for further development through protein engineering.
authors.forename: Ruizhi
authors.initials: R
authors.lastname: Han
authors.forename: Jianghua
authors.initials: J
authors.lastname: Li
authors.forename: Hyun-dong
authors.initials: HD
authors.lastname: Shin
authors.forename: Rachel R
authors.initials: RR
authors.lastname: Chen
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D001205:Ascorbic Acid / Q000031:analogs & derivatives* / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C065378:ascorbic acid 2-O-glucoside; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase; D001205:Ascorbic Acid