## 3 Results
### Identification of Cellulase Enzymes and their Genes  
The NH\({}_{2}\)-terminal amino acid sequence of the purified protein was determined to be N-N-T-E-Q-T-V-V-D-A-Y-G. Genomic sequencing analysis of J9-3 revealed that the bacterium had a circular chromosome composed of 3,806,639 bp, with 32.08% G+C content. From the 3300 coding sequences, we identified one gene encoding a polypeptide that was perfectly matched with the NH\({}_{2}\)-terminal amino acid sequence of the purified protein. This gene was registered in GenBank under accession no. MN33592. According to its nucleotide sequence, the corresponding protein was composed of 359 amino acids (Mw = 40,738 Da). Moreover, the NH\({}_{2}\)-terminal amino acid sequence revealed that the premature form of the protein was cleaved between Ser-52 and Asn-53, thus yielding a mature protein with 307 amino acids (Mw = 34,951 Da), which was consistent with our SDS-PAGE results (Figure 4A).  
Figure 4: Purification and biochemical characterization of an extracellular cellulase, CeAl\({}_{93}\), from the culture broth of _Cellulophag_ sp.!9-3. (**A**) Sodium dodecyl sulfate-polyacrylamideamide gel electrophoresis of protein samples during purification. Lane M, protein molecular weight markers; 1, 75% ammonium sulfate precipitate; 2, sample after DEAE Sepharose Fast Flow column chromatography; 3, sample after Superdex 75 column chromatography; (**B**) substrate specificity of CeAl\({}_{93}\) toward various polysaccharides; (**C**) effect of temperature on CeAl\({}_{93}\) enzyme activity toward carboxymethylcellulose. \(\blacktriangle\), optimum temperature; \(\blacksquare\), thermostability; (**D**) effect of pH on CeAl\({}_{93}\) enzyme activity toward carboxymethylcellulose. \(\blacksquare\), 50 mM MOPS (pH 6.0-7.0); \(\odot\), 50 mM Tris-Cl (pH 7.0-9.0); \(\blacktriangle\), 50 mM glycine-NaOH (pH 9.0-10.0); (**E**) effect of metal ions, ethylenediaminetetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) on CeAl\({}_{93}\) enzyme activity toward carboxymethylcellulose. The final concentration of each chemical used is indicated under the figure. The maximum enzyme activity in (**B**)–(**D**) and the enzyme activity without chemicals in (E) was set to 100% when calculating relative activities. In (**B**)–(**E**), all data shown are mean values from at least three replicate experiments.  
Based on amino acid sequences, several homologous proteins with more than 90% identity were retrieved using the BlastP program. These proteins were all listed as putative cellulases (endoglucanase) belonging to glycosyl hydrolase family 5 (GH5) of Carbohydrate-Active enZYmes Database [22]. They included _Celluophaga_ sp. W5C (99% identity with WP_077398709.1), _Celluophaga lytica_ (95% identity with WP_013619852.1), _Celluophaga_ sp. RHA_52 (95% identity with WP_144709822.1), _Celluophaga gejensis_ (95% identity with WP_034644593.1), _Celluophaga_ sp. QY3 (92% identity with ADB80152.1), and _Celluophaga fricicola_ (90% identity with WP_072302686.1). However, the enzymatic properties of these proteins have not been characterized. Therefore, studies on the enzymatic properties of the purified cellulase were performed.