As a multifunctional enzyme, 4-α-glycosyltransferase (4αGT) finds diverse applications in the starch industry, including processes such as starch modification and the production of trehalose, maltooligosaccharides, large-ring cyclodextrins, glycogen, and other starch-derived products.In the industrial preparation of trehalose from starch, the disproportionation of 4αGT plays a crucial role in reducing the amount of glucose, maltose, and other by-products, thereby increasing the yield of trehalose.This study employed a semi-rational design strategy to engineer 4αGT from Synechocystis sp.PCC 6803 for enhanced activity.The HotSpot Wizard online tool and the spatial residue location method were used to guide the selection of mutation hotspots.Totally 18 000 converters with high throughput using the ability of 4αGT were screened to hydrolyze starch to produce a transparent ring.A site that plays a key role in the activity of 4αGT was identified.Saturation mutagenesis at this site yielded the activity-enhanced mutant A470F with disproportionation activity that was 17% higher than that of the wild-type.Moreover, A470F showed an improvement over the wild-type in trehalose preparation under industrialized conditions.The results of substrate channel and force analyses near the mutation site provide theoretical support for the activity modification of 4αGT and novel insights for improving manufacturing efficiency in the starch sugar industry.
XIE Jingwen
,
LI Chaohui
,
WEI Meng
,
ZHAO Wei
,
ZHANG Jinxiang
. Semi-rational design to enhance the disproportionation activity of 4-α-glycosyltransferase and its application in industrial production[J]. Food and Fermentation Industries, 2024
, 50(23)
: 45
-52
.
DOI: 10.13995/j.cnki.11-1802/ts.038159
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