Supplementary MaterialsSupporting Information 41598_2019_52136_MOESM1_ESM. Chia, CatD and CatD21 created (GlcNAc)2 and (GlcNAc)3C6, while CatD46 didn’t degrade the substrate (Fig.?5c and Supplementary Fig.?S10). Full-length Chia, CatD and CatD21 degraded chitosan and, besides short chitooligosaccharides, also produced (GlcNAc)>6 (Fig.?5d and Supplementary Fig.?S10). CatD46 did not affect any of the substrates. These results indicate that lack of CBD in the Chia does not inhibit its chitin- and chitosan-degrading activities, whereas further deletion Pozanicline of 46 amino acids from your C-terminus of the CatD caused loss of chitinolytic activity. Porcine Chia and its truncated forms as well as a pepsin preparation produced similar chitooligosaccharides Finally, the degradation products from chitosan by natural Chia or recombinant proteins and the most active pepsin preparation (S1, P7012) were compared. Several chitosan substrates differing in DD (DD 69%, 73%, 84% and 95%) were incubated with porcine Chia, its truncated forms, PA-Chia, PA-CatD or pepsin preparation as explained in Methods and the products were analyzed by the FACE. Chitosan substrates with DD of 69%, 73% and 84% were degraded to up to (GlcNAc)~20. The degradation pattern was similar for those enzymes (Fig.?6aCd and Supplementary Fig.?S11). The digestion efficiency decreased with increasing DD of chitosan and only a very limited Pozanicline degradation was observed in DD 95% (Fig.?6aCd). These results indicate that chitosan-degrading activity in the pepsin preparations is due to the chitinolytic activity of truncated Chia which can digest chitosan. Open in a separate window Number 6 Full-length and its truncated forms of Chia as well as pepsin preparation produced comparable chitooligosaccharides. Degradation products generated by incubation of wide range of DD chitosan; (a) 69%, (b) 73%, (c) 84% and (d) 95% of DD with full-length Chia (Chia), Rabbit polyclonal to IFIH1 truncated Chia (CatD), PA-Chia, PA-CatD or the pepsin preparation (Pep) at pH 4.0 were analyzed by FACE. The images of (aCd) were cropped from dotted lines on original full-length gel images shown in Supplementary Fig.?S11. Furthermore, the ability of porcine Chia and the pepsin preparation to degrade (GlcNAc)5 as well as (GlcN)5 and (GlcN)6 was investigated. None of the GlcN substrates was digested, while (GlcNAc)5 was completely degraded into (GlcNAc)2 and GlcNAc monomer (Fig.?S12). These results indicate that the chitosan-degrading activity of porcine Chia and the pepsin preparation appears to be mediated by chitinolytic rather than by Pozanicline chitosanolytic activity with no effect on GlcN-GlcN bonds. Discussion In this study, we investigated the chitosan-degrading Pozanicline (chitosanase) activity in the commercially available porcine pepsin preparations, which have been used for enzymatic production of chitooligosaccharides. We detected residual active fragments (CatD) derived from Chia displaying chitosan-degrading activity similar to those of the full-length and C-terminally truncated porcine Chia. Functional properties of recombinant mouse CatD are comparable with those of full-length Chia30. Natural porcine CatD, obtained by incubation of Chia with purified pepsin, showed a comparable degradation activity against chitin and chitosan by the full-length enzyme. This activity is present even in a further truncated form of CatD by up to 24 amino acids, while the deletion of 46 amino acids leads to activity loss. These results suggested that at least five of the six cysteines conserved in the catalytic Pozanicline domain31 are sufficient to keep the proper tertiary structure required for chitinolytic activity and/or chitin/chitosan substrates recognition. Porcine Chia and pepsin preparation degraded chitosan substrates with DD of 69C84%. However, the digestion efficiency decreased with increasing DD and only a very limited degradation was observed in DD of 95% chitosan suggesting the absence of a chitosanase activity of Chia. To investigate the mechanism of chitosan degradation by porcine Chia, (GlcNAc)5 as well as (GlcN)5 and.