4-α-糖基转移酶(4-alpha-glucanotransferase, 4αGT)是一种多功能的糖基转移酶,在淀粉工业上具有广泛的应用,如淀粉改性及海藻糖、麦芽寡糖、大环糊精、糖原等淀粉深加工产品的制备。其中,以淀粉为原料制备海藻糖的工业生产过程中,借助4αGT的歧化活性,能够减少葡萄糖、麦芽糖等副产物的含量,从而提升海藻糖产量。在该研究中,采用半理性设计策略对Synechocystis sp.PCC 6803来源4αGT进行了改造。研究基于HotSpot Wizard在线工具及残基所处空间位置选择突变热点,利用4-α-糖基转移酶水解淀粉产生透明圈的能力对18 000个转化子进行高通量筛选,确定了一个对4αGT酶活性起着关键性作用的位点,对其进行饱和突变,最终获得活性提升的突变体A470F,歧化活性较野生型提升了17%。在工业化生产的条件下,突变体A470F在海藻糖制备上,较野生型也有所提升。通过对突变位点附近底物通道及作用力进行分析,为4αGT的活性改造提供了理论支持,并且为淀粉糖工业的制造效率提升提供了新思路。
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.
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