## 3 Results  
Anaerobic cultivation yielded approximately 0.5 mg of crChit62J4 per liter of culture, after purification providing \(\sim\) 0.1 mg of protein per liter of initial culture volume. The identity and purity of crChit62J4 were confirmed by MS analysis and reliable sequence match. The Chit62J4 gene codes for 601 amino acids with a total theoretical MW = 65414.9 and pI = 5.81 (Figure 1, Supplementary Figure S5). This includes a signal sequence identified by the prediction services as residues 1-29, i.e., the \(N\)-terminus of the mature enzyme sequence being AQSL [33; 34].  
The theoretical mass of mature Chit62J4 (residues 30-601) of 62284.9 and pI of 5.2 correspond to the experimental values for crChit62J4: 62.4 kDa (MALDI-TOF, Supplementary Figure S3) and pI of 4.9 (chromatofocusing). The DLS results for crChit62J4 indicate a monodisperse solution with hydrodynamic radius 3.65 nm (\(\sim\) 130 kDa for globular protein) corresponding to a dimer of Chit62J4. If Chit62J4 had an extremely elongated shape, it may also roughly correspond to a monomer of the enzyme.  
Crude Chit62J4 is active on pNGG and pNGGG (against pNGGG \(\sim\)10\(\times\) lower, data not shown), with no activity toward pNG. On these chromogenic substrates, the enzyme performs mostly as an exochitinase, on pNGG exhibiting \(k_{\text{cat}}\) 13.5 s\({}^{-1}\), \(K_{\text{m}}\) = 0.57 mM and catalytic efficiency \(k_{\text{cat}}/K_{\text{m}}\) = 23,684 M\({}^{-1}\)s\({}^{-1}\), optimal pH 5.5 and temperature 60 \({}^{\circ}\)C (Supplementary Figure S6). The enzyme is also active on colloidal chitin. In the presence of standard antimyoctics Griseofulvin, Amfotericine, and Clotrimazole (5 mM) it retains its full activity.  
The predicted membrane translocation signal sequence at the \(N\)-terminus of the native Chit62J4 is followed by an amino acid sequence corresponding to a four-domain protein. Based on the sequence alignment with other known chitinases (Supplementary Figure S7), we propose the following domain structure of mature Chit62J4: the N-terminal catalytic domain, two re-iterated Fn3/Big3 domains, and the C-terminal chitin binding module ChtBD3 (chitin-binding domain type 3). The overall structure resembles that of chitinase Chi18C from _Clostidium paraprutritcium_ M21 [35].  
The kinetic parameters for both rChit62J4 and rChit62J4 cat toward pNGG were determined as \(K_{m}\) = 0.24 \(\pm\) 0.02 mM, \(k_{\text{cat}}\) = 31.5 \(\pm\) 0.9 s\({}^{-1}\) and \(K_{m}\) = 0.19 \(\pm\) 0.02 mM, \(k_{\text{cat}}\) = 31.4 \(\pm\) 0.8 s\({}^{-1}\), respectively. The influence of several groups of compounds expected to affect activity was tested. In the case of the catalytic domain, the substrate affinity increased slightly compared to the complete mature enzyme. The affinity also increased in the presence of 5 mM MgCl\({}_{2}\), 5 mM EDTA, 5 mM ZnSO\({}_{4}\), and 5 mM CaCl\({}_{2}\) (Table 1; Supplementary Figures S8 and S9). Conversely, the substrate affinity decreased in the presence of 5 mM glucosamine or 5 mM glucose. The TLC results showed that both the intact enzyme and its catalytic domain cleaved chitohexaose as well as colloidal chitin producing chitobiose as the end product (Supplementary Figure S4).