## 4 Discussion  
Cellulose is one of the most important industrial enzymes that allows the utilization of the vast amount of biomass, especially lignocellulosic-based biomass, on earth. To date, a lot of cellulases have been reported; however, there are many limitations in developing technologies that can effectively apply cellulase in biomass decomposition [8; 9]. In the case of lignocellulosic substrates, since the lignin-hemicellulose-cellulose forms a tight complex, it is difficult for the enzyme to access the substrate for its action. In addition, cellulase enzyme activity can be influenced by various factors in the process such as inhibition by the final product, inactivation by high temperature, solvent or pH change, nonspecific binding with lignin, and irreversible adsorption on heterogeneous substrates. Therefore, it is necessary to screen as many cellulases as possible, with various biochemical characteristics, to broaden their application in degrading lignocellulosic materials. In this study, we identified a marine bacterium, _Celluolphaga_ sp. J9-3, with an ability to degrade cellulose and we characterized a cellulase (endo-\(\beta\)-1,4-glucanase) from its culture broth. According to the SignalP 5.0 program ([[http://www.cbs.dtu.dk/services/SignalP/](http://www.cbs.dtu.dk/services/SignalP/)]([http://www.cbs.dtu.dk/services/SignalP/](http://www.cbs.dtu.dk/services/SignalP/))) [25] the protein, CeAl\({}_{93}\), was expected, at a probability of 0.9994, to have a lipoprotein signal peptide (Sec/SPII) [26] with a cleavage site between Ser-19 and Cys-20. However, NH\({}_{2}\)-terminal amino acid sequencing revealed that the mature protein was cleaved between Ser-52 and Asn-53, indicating a unique pattern of cleavage. There are currently two reports of cellulases from the genus _Celluolphaga_ in PubMed ([[https://www.ncbi.nlm.nih.gov/pubmed/](https://www.ncbi.nlm.nih.gov/pubmed/)]([https://www.ncbi.nlm.nih.gov/pubmed/](https://www.ncbi.nlm.nih.gov/pubmed/))). [PERSON] et al. [27] reported a novel endoplasmic, Celal_2753 (Mw = 44 kDa), from the psychrophilic bacterium, _Celluolphaga alicola_ IC166(T). It showed maximum activity at 40 \({}^{\circ}\)C and pH 6. It was resistant to various detergent treatments, including SDS, and could be stimulated by NaCl and KCl. Moreover, it maintained 55% of its initial activity after boiling for 5 min. Thus, it will be useful for the degradation of hemicelluloses in very harsh conditions. Another study reported only CMCase production by _Celluolphaga lytic_ LBH-14 using rice bran, without any information regarding enzymatic properties [28]. Although many genes have been annotated as GH5 cellulases in the genus _Celluolphaga_, only one cellulase (Celal_2753), which shared 51% identity in  
Figure 5: Analysis of CeAl\({}_{93}\) enzyme hydrolyses of various substrates by thin layer chromatography. **(A)** Chromatogram of the carboxymethylcellulose hydrolysate produced by CeAl\({}_{93}\) according to reaction time at pH 7.5 and 37 \({}^{\circ}\)C. The presence of smeared bands under the cellobiose band indicates that CeAl\({}_{93}\) is acting as an endo-type glucanase, leading to cellobiose; **(B)** chromatogram of various cellooligosaccharide hydrolyses produced by CeAl\({}_{93}\) after reacting for 24 h at pH 7.5 and 37 \({}^{\circ}\)C. Cellooligosaccharides larger than cellobiose gave the common product of cellobiose, indicating that CeAl\({}_{93}\) can hydrolyze substrates to cellobiose. C1, D-glucose; C2, cellobiose; C3, cellotriose; C4, cellotetraose; C5, cellopenttaose; C6, cellohexaose. CMC; carboxymethylcellulose. its amino acid sequence with CeAl\({}_{93}\), was identified. CeAl\({}_{93}\) was quite distinct from Cela\({}_{-}\)2753, especially in that it had a higher optimum temperature (60 \({}^{\circ}\)C), it was highly sensitive to EDTA and SDS, and it required Co\({}^{2+}\) as a cofactor. Although most cellulases are not affected by the presence of the Co\({}^{2+}\) ion, some have been reported to be stimulated by Co\({}^{2+}\).