MOESM1 of Unique N-glycosylation of a recombinant exo-inulinase from Kluyveromyces cicerisporus and its effect on enzymatic activity and thermostability

Additional file 1: Materials and Methods about Site-Directed Mutagenesis, Protein Expression and Purification, Glycosidase Treatment, Zymogram Analysis, Enzymes Activity Assays and Mass Spectrometry Analysis. Figure S1. LC-MS/MS spectrum analysis of the glycosylation sites within the wild-type rKcINU1. The sample was treated by trypsin followed with PNGase F digestion and then subjected to LC-MS/MS. The peak with m/z of 976.95, 694.66, 942.49, 1188.59 and 817.90 confirmed the presence of deamidation modification on (A) Asn-362, (B) Asn-370, (C) Asn-399, (D) Asn-467 and (E) Asn-526, respectively (substitution of Asn with Glu residue). Each peptide sequence was shown in the panel and the arrows indicated the N-glycosylation sites. Figure S2. LC-MS/MS spectrum analysis of the glycosylation sites within the variant Mut. The sample was treated by trypsin followed with PNGase F digestion and then subjected to LC-MS/MS. The peak with m/z of 621.32, 1298.60, 1298.60, 990.45, 990.45 and 874.37 confirmed the presence of deamidation modification on (A) Asn-9, (B) Asn-147, Asn-153 (C) Asn-197, Asn-203 and (D) Asn-233, respectively (substitution of Asn with Glu residue). Each peptide sequence was shown in the panel and the arrows indicated the N-glycosylation sites. Figure S3. Positive ion ESI-MS analysis of N-linked glycan chains for the single-sited mutants of (A) N362Q, (B) N370Q, (C) N399Q, (D) N467Q and (E) N526Q, respectively. The peak of m/z 1905.17 confirmed the presence of the high mannose oligosaccharide (Man)7(GlcNAc)2. Series of peaks by 162 Da (the mass of anhydrohexose) demonstrated the glycoform heterogeneity. Solid squares and circles represented N-acetylglucosamine (GlcNAc) and Mannose (Man), respectively. Table S1. Primers of the site-directed mutagenesis. Table S2. The enzyme activity of the wildtype rKcINU1 treated with Endo F1