## 4 Discussion
### Adaptation of Clostridium Chitinase to Environment  
Chitinases of this family have optimal temperatures in a wide range of 30-60 \({}^{\circ}\)C and optimal pH mostly in the range 5.0-8.0 with several examples of extremes at pH 4.0 and 9.0 [35,46]. Within related enzymes, there is no example of a chitinase with such a high-temperature stability and at the same time such a low pH optimum. The catalytic domains of ChiNCTU2 and Chit62J4 are highly similar and according to our structure model (see below) the core of the catalytic site should be almost identical. Their temperature optima are equal, however, the Chit62J4 pH optimum of 5.5 contrasts with that of ChiNCTU2 at pH 7.0. This significant difference is, nevertheless, in agreement with the surface electrostatics (Supplementary Figure S10) and the theoretical isoelectric points of their catalytic domains. More acidic Chit62J4 (negative surface potential, pI 4.9) has a lower pH optimum than ChiNCTU2 with pI 6.0. The theoretical and experimental (native conditions) pI of complete mature Chit62J4 are 5.2 and 4.9, respectively. In both enzymes, the pH optimum is several tenths to one degree above the respective pI. Given the high identity of the active sites, the difference in pH optima between ChiNCTU2 and Chit62J4 results rather from the overall protein behavior at a certain pH than from any particular variations in the active site. Our calculations of electrostatics distributions at the measured pH optima show for both ChiNCTU2 and Chit62J4 typical chitinase patterns (Supplementary Figure S10), with no significant positive potential and relatively high negative potential at the active center. Chitin chains, often partially deacetylated, can carry positive charge and positive potential near the active site would block substrate binding, while strong negative potential will lead to nonproductive binding. Calculations performed at the exchanged pH points for the two proteins clearly show extreme electrostatics (Supplementary Figure S10). Therefore, the optimization with respect to the acidity of the environment in these enzymes was achieved by adjusting the overall molecular electrostatics, coded by the amino acid composition (different) and three-dimensional organization (similar) without any interference directly in the active site. ChiNCTU2 originates from a soil bacterium, while Chit62J4 is active in the human colon. pH within a healthy human colon slowly increases from 5.5 at the  
Figure 2: Sequence alignment of Chit62J4 from _C. parapupritritum_J4, chitinase D from _C. botulinum_ and chitinase ChiNCTU2 from _B. cereus_. Identities are marked by black background. Expected catalytic residues are marked by a red line; residues forming the \(-2\) to +2 subsites (according to [PERSON] et al. [36]) of the substrate binding site are marked by colored lines and site numbering. beginning to 7.0 at its end [47]. The _Roseburia_/_E. rectale_ group of bacteria in the study by [PERSON] et al. (2009) [6] competed well at lower pH for polysaccharide substrates. It follows from this that if _Clostritium_ needs to compete for insoluble fiber substrates, it must compete at decreased pH in the proximal part of the colon. Thus, the optimization for chitinolytic activity at lower pH is in full agreement with the microbiome environmental conditions. The highest bacterial growth occurs in the first colon section, and it can be expected that _C. paraputrificum_ J4 is exposed to lower pH than _B. cereus_, under competition with other microbiome bacteria. Adjustment of extracellular Chit62J4 to the acidic environment of the human colon therefore explains the differences observed on the molecular level. The Chit62J4 adaptation happened away from the actual catalytic site. Similar shifts of pH optima, realized by the overall protein composition rather than active site changes, were seen also in other enzymatic systems, e.g., in non-specific nucleases [48].