Effect of loop-grafting on the activity, stability and regioselectivity of Priestia megaterium levansucrase using two inulosucrases as loop donors.

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Tác giả: Marcel Groß, Maria Elena Ortiz-Soto, Kilian Schmid, Jürgen Seibel, Caterina Weber

Ngôn ngữ: eng

Ký hiệu phân loại: 331.128 Placement

Thông tin xuất bản: Netherlands : International journal of biological macromolecules , 2025

Mô tả vật lý:

Bộ sưu tập: NCBI

ID: 739596

Active-site loops of enzymes modulate activity, stability, regioselectivity and substrate specificity. This study examines the impact of loop transplantation on Priestia megaterium levansucrase Pm-SacB, which produces levan-type, β-(2,6)-linked fructooligosaccharides. Inulosucrases from Lactobacillus johnsonii (Lj-InuJ) and Halalkalicoccus jeotgali (Hje-Inu), which synthesize β-(2,1)-linked inulin fructans, served as donors of eight active-site loops. Most Lj-InuJ-based loop-grafting enzymes produce smaller oligosaccharides than Pm-SacB, and variants carrying a Lj-InuJ loop 3, whether alone or in combination with grafted-loops 7 and 8, exhibit inulin-type oligosaccharides 1-kestose and 1-nystose among their products. Pm-SacB does not synthesize inulin-type oligosaccharides other than 1-kestose but can elongate them via β-(2,6)-linkages. A construct containing Lj-InuJ loops 3, 7 and 8, along with the mutations S372N, K373R and H423Y (variant Lj-Loop3-7-8**), has impaired 6-kestose and levan synthesis, accumulates 14-fold more 1-kestose than Pm-SacB and outperforms other variants in the synthesis of 1-nystose. The enzyme exhibits similar thermal stability to Pm-SacB, but its catalytic efficiency is sevenfold lower. The product profile of Lj-Loop3-7-8** could not be replicated by mutating reported levan- and inulin-binding residues in Pm-SacB, suggesting that modifying neighboring residues, in addition to those involved in FOS-binding may be necessary to gain access to levansucrases with unique properties.
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