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Int. J. Mol. Sci. 2018, 19(3), 683; https://doi.org/10.3390/ijms19030683

D181A Site-Mutagenesis Enhances Both the Hydrolyzing and Transfructosylating Activities of BmSUC1, a Novel β-Fructofuranosidase in the Silkworm Bombyx mori

1
School of Life Sciences, Anhui Agricultural University, Hefei 230036, China
2
Anhui International Joint Research and Development Center of Sericulture Resources Utilization, 130 West Changjiang Road, Hefei 230036, China
*
Author to whom correspondence should be addressed.
Received: 19 January 2018 / Revised: 14 February 2018 / Accepted: 24 February 2018 / Published: 28 February 2018
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

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). BmSuc1 has been cloned and identified from the silkworm Bombyx mori as 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 in B. mori. Up-regulated transfructosylating activity of BmSUC1 could provide new ideas for using B. mori β-FFase to produce functional FOS. View Full-Text
Keywords: Bombyx mori; β-fructofuranosidase; site-directed mutagenesis; hydrolysis; transfructosylation Bombyx mori; β-fructofuranosidase; site-directed mutagenesis; hydrolysis; transfructosylation
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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Gan, Q.; Li, X.; Zhang, X.; Wu, L.; Ye, C.; Wang, Y.; Gao, J.; Meng, Y. D181A Site-Mutagenesis Enhances Both the Hydrolyzing and Transfructosylating Activities of BmSUC1, a Novel β-Fructofuranosidase in the Silkworm Bombyx mori. Int. J. Mol. Sci. 2018, 19, 683.

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