Quan Gan,Xinwei Zhang,Daobo Zhang,Liang Shi,Yue Zhou,Tongtong Sun,Song Jiang,Junshan Gao,Yan Meng
Sucrose is the most commonly transported sugar in plants and is easily assimilated by insects to fulfill the requirement of physiological metabolism. BmSuc1 is a novel animal β-fructofuranosidase (β-FFase, EC 3.2.1.26) - encoding gene that was firstly cloned and identified in silkworm , Bombyx mori . BmSUC1 was presumed to play an important role in the silkworm-mulberry enzymatic adaptation system by effectively hydrolyzing sucrose absorbed from mulberry leaves. However, this has not been proved with direct evidence thus far. In this study, we investigated sucrose hydrolysis activity in the larval midgut of B. mori by inhibition tests and found that sucrase activity mainly stemmed from β-FFase, not α-glucosidase. Next, we performed shRNA-mediated transgenic RNAi to analyze the growth characteristics of silkworm larvae and variations in glycometabolism in vivo in transgenic silkworms. The results showed that in the RNAi- BmSuc1 transgenic line, larval development was delayed, and their body size was markedly reduced. Finally, the activity of several disaccharidases alone in the midgut and the sugar distribution, total sugar and glycogen in the midgut, hemolymph and fat body were then determined and compared. Our results demonstrated that silencing BmSuc1 significantly reduced glucose and apparently activated maltase and trehalase in the midgut. Together with a clear decrease in both glycogen and trehalose in the fat body, we conclude that BmSUC1 acts as an essential sucrase by directly modulating the degree of sucrose hydrolysis in the silkworm larval midgut, and insufficient sugar storage in the fat body may be responsible for larval malnutrition and abnormal petite phenotypes.
Quan Gan,Xin Li,Xinwei Zhang,Lanlan Wu,Chongjun Ye,Ying Wang,Junshan Gao,Yan Meng
β-fructofuranosidase (β-FFase) belongs to the glycosyl-hydrolase family 32 (GH32), which can catalyze both the release of β-fructose from β-d-fructofuranoside substrates to hydrolyze sucrose and the synthesis of short-chain fructooligosaccharide (FOS).BmSuc1has been cloned and identified from the silkwormBombyx morias a first animal type of β-FFase encoding gene. It was hypothesized that BmSUC1 plays an important role in the silkworm-mulberry adaptation system. However, there is little information about the enzymatic core sites of BmSUC1. In this study, we mutated three amino acid residues (D63, D181, and E234) that represent important conserved motifs for β-FFase activity in GH32 to alanine respectively by using site-directed mutagenesis. Recombinant proteins of three mutants and wild type BmSUC1 were obtained by using a Bac-to-Bac/BmNPV expression system and BmN cells. Enzymatic activity, kinetic properties, and substrate specificity of the four proteins were analyzed. High Performance Liquid Chromatography (HPLC) was used to compare the hydrolyzing and transfructosylating activities between D181A and wtBmSUC1. Our results revealed that the D63A and E234A mutations lost activity, suggesting that D63 and E234 are key amino acid residues for BmSUC1 to function as an enzyme. The D181A mutation significantly enhanced both hydrolyzing and transfructosylating activities of BmSUC1, indicating that D181 may not be directly involved in catalyzation. The results provide insight into the chemical catalyzation mechanism of BmSUC1 inB. mori. Up-regulated transfructosylating activity of BmSUC1 could provide new ideas for usingB. moriβ-FFase to produce functional FOS.Keywords:Bombyx mori;β-fructofuranosidase;site-directed mutagenesis;hydrolysis;transfructosylation
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