Substrate Recognition by Two Members of Glycoside Hydrolase Family 32 Involved in Fructo-oligosaccharide Metabolism in Lactobacillus acidophilus NCFM
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Two fructo-oligosaccharide active enzymes, BfrA and ScrB, of glycoside hydrolase family 32 are found inLactobacillus acidophilus NCFM and belong to phylogenetic clusters of bacterial β-fructosidase and sucrose 6-phosphatehydrolase, respectively, involved in the intracellular metabolism of kesto-oligosaccharides [β-D-fructofuranosyl-(2,1)]n-β-D-fructofuranosyl-(2,1)-α-D-glucopyranose (n, 1-3) and sucrose. Recombinant ScrB, produced in Escherichia coli,showed 20-1500-fold higher catalytic efficiency (kcat/Km) toward sucrose than raffinose [α-D-galactopyranosyl-(1,6)-α-D-glucopyranosyl-(1,2)-β-D-fructofuranose] and kesto-oligosaccharides. At high concentration of sucrose (0.4-1.0 M) ScrBcatalysed formation of 1-kestose [β-D-fructofuranosyl-(2,1)-β-D-fructofuranosyl-(2,1)-α-D-glucopyranose] in 2.3-15% yield bytransglycosylation. Recombinant BfrA in contrast efficiently hydrolysed kesto-oligosaccharides, in particular 1-kestose with 70-300-fold higher kcat/Km than for sucrose and raffinose and the kcat/Km values decreased slightly with increasing degree of thepolymerization of the kesto-oligosaccharides. Neither BfrA nor ScrB degraded the β-(2,1)- and β-(2,6)-linked fructans, inulinand levan. The outcome of the present detailed specificity analysis of the β-fructosidase and sucrose 6-phosphate hydrolasetowards fructo-oligosaccharides combined with modelling of BfrA and ScrB, using the structure of Thermotoga maritima β-fructosidase as template and superimposition of β-fructose from a complex with Aspergillus awamori exo-inulinase, sucroseand 1-kestose from complexes with Cichorium intybus fructan β-(2,1)-fructosidase/1-exohydrolase, suggested that loop 1 inthe fifth blade of the characteristic GH32 β-propeller catalytic domain controls the specificity toward 1-kestose. Furthermore,a ScrB His71 is strictly conserved in the first blade of sucrose 6-phosphate hydrolase and is suggested to recognise thephosphate group of the α-glucose 6-phosphate moiety in the substrate sucrose 6-phosphate at subsite +1.乳酸菌Lactobacillus acidophilus NCFM でのフルクトオリゴ糖代謝に関与する２つの糖質加水分解酵素（BfrA, β- フルクトシダーゼ; ScrB, スクロース6- リン酸ハイドロラーゼ）の基質認識機構を明らかにすることを目的とし、各種組み換え酵素を用いてフルクトオリゴ糖に対する加水分解反応の速度論的解析を行った。ScrB はフルクトオリゴ糖よりスクロースに対して高い加水分解活性を示し、BfrA は短鎖なフルクトオリゴ糖に対して特異性を示すことを明らかにした。加えて、両酵素の基質特異性に関与する領域（BfrA ループ１）およびアミノ酸残基（ScrB His71）を、ホモロジーモデリング法により推定した。
- 2012-03-00
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関連論文
- 乳酸菌由来糖質加水分解酵素ファミリー32タンパク質の基質認識機構の解明
- プレバイオティクスとして有用な新規機能性オリゴ糖の生産開発
- Substrate Recognition by Two Members of Glycoside Hydrolase Family 32 Involved in Fructo-oligosaccharide Metabolism in Lactobacillus acidophilus NCFM
- プレバイオティクスとして有用な新規機能性オリゴ糖の生産開発(糖質関連酸素化学シンポジウム)