## 4 Discussion  
### Sequence and Structure of Clit62J4  
The most abundant extracellular enzyme of _Clostridium parapurtiticum_ J4 is chitinase Chit62J4 with predominant exochitinib activity and belonging to the GH18 family with the retaining mechanism. Mature Chit62J4 comprises four domains: the catalytic domain, two copies of a Fn3 domain, and the carbohydrate-binding module ChtBD3 (chitin-binding domain 3). This domain structure is not uncommon in chitinases, the most similar enzyme being Chil8C from _Clostridium parapurtiticum_ M21, however, with different catalytic domain and specificity. A sequence search against known structures in the Protein Data Bank does not provide significant hits.  
The catalytic domain of Chit62J4 shares the highest sequence identity (79%) with single domain chitinase D from _Clostridium botulinum_ B str. Eklund 17B (NCBIYP_001885576.1). Of the chitinases with known 3D structures, the catalytic domain of Chit62J4 shows the highest similarity (75% identity) to the single domain chitinase ChiNCTU2 from _Bacillus cereus_ NCTU2 [36]. Thus, while the complete domain structure of Chit62J4 most resembles that of Chi18C, the catalytic properties should resemble those of ChiNCTU2. The explanation for this \"catalytic domain replacement\" remains elusive. The accessory domains likely contribute to better binding and directionality of the substrate with respect to the catalytic site and support catalysis on fibers.  
The 90-residue Fn3/Big3 domains (residues 363-442 and 449-548) share 63% identity and show 44-57% identity to the closest homologous sequences in chitinases, e.g., in Chi18C. Pfam [37] classifies the first Fn3 domain into the Cadherin family or family DUF4397 (\"domain of unknown function in bacteria, archaea, and eukaryotes\") and the second domain as part of the CARDB family (\"cell adhesion domain found in bacteria\"). In our general sequence search against all protein records, the first Fn3 domain is most similar to two domains from _Clostridium beijerinckii_ NCIMB 8052 carbohydrate-binding family V/XII protein (residues 229-317, 58% identity and 135-222, 49%). The second domain search leads to similar results. The complete two-domain sequence does not produce any significant hits, making the Fn3-Fn3 combination of domains in Chit62J4 unique.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Enzyme** & **Compound Added into the Reaction Trial** & \(k_{\text{cat}}\) (s\({}^{-1}\)) & \(K_{\text{m}}\) (mM) \\ \hline rChit62J4 & none & 31.5 \(\pm\) 0.9 & 0.24 \(\pm\) 0.02 \\ \hline \multirow{6}{*}{rChit62J4} & 5 mM glucosamine & 30.2 \(\pm\) 0.9 & 0.36 \(\pm\) 0.04 \\  & 5 mM glucose & 31.2 \(\pm\) 0.9 & 0.36 \(\pm\) 0.04 \\  & 5 mM MgCl\({}_{2}\) & 29.6 \(\pm\) 0.6 & 0.18 \(\pm\) 0.02 \\  & 5 mM EDTA & 31.9 \(\pm\) 0.8 & 0.19 \(\pm\) 0.02 \\  & 5 mM ZnSO\({}_{4}\) & 31.1 \(\pm\) 0.6 & 0.16 \(\pm\) 0.01 \\  & 5 mM CaCl\({}_{2}\) & 30.3 \(\pm\) 0.6 & 0.14 \(\pm\) 0.01 \\ \hline rChit62J4 cat & none & 31.4 \(\pm\) 0.8 & 0.19 \(\pm\) 0.02 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Kinetic parameters of rChit62J4 influenced by selected compounds. Standard deviations are given.  
The C-terminal domain ChtBD3 (557-601) is 60% identical with a carbohydrate-binding module of chitinase A1 from _Bacillus circulans_[38], a domain found by [PERSON] et al. [39] to enhance the enzymatic activity by interaction with insoluble chitin, while its interaction with soluble chitin or chitin derivatives was not observed.