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.

journal_name

Appl Environ Microbiol

authors

Han R,Li J,Shin HD,Chen RR,Liu L,Du G,Chen J

doi

10.1128/AEM.01249-14

subject

Has Abstract

pub_date

2014-08-01 00:00:00

pages

4717-24

issue

15

eissn

0099-2240

issn

1098-5336

pii

AEM.01249-14

journal_volume

80

pub_type

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