BmSuc1, a novel animal-type β-fructofuranosidase (β-FFase, EC 3.2.1.26) encoding gene, was cloned and identified for the first time in the silkworm, Bombyx mori. BmSuc1 was specifically and highly expressed in the midgut and silk gland of Bombyx mori. Until now, the function of BmSuc1 in the silk gland was unclear. In this study, it was found that the expression changes of BmSuc1 in the fifth instar silk gland were consistent with the growth rate of the silk gland. Next, with the aid of the CRISPR/Cas9 system, the BmSuc1 locus was genetically mutated, and homozygous mutant silkworm strains with truncated β-FFase (BmSUC1) proteins were established. BmSuc1 mutant larvae exhibited stunted growth and decreased body weight. Interestingly, the molecular weight of part of Sericin1 (Ser1) in the silk gland of the mutant silkworms was reduced. The knockout of BmSuc1 reduced the sericin content in the silkworm cocoon shell, and the mechanical properties of the mutant line silk fibers were also negatively affected. These results reveal that BmSUC1 is involved in the synthesis of Ser1 protein in silk glands and helps to maintain the homeostasis of silk protein content in silk fibers and the mechanical properties of silk fibers, laying a foundation for the study of BmSUC1 regulation of silk protein synthesis in silk glands.

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.