Vol 9 No1 - Journal of Cell and Molecular Biology - Haliç Üniversitesi

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Expression of L. edodes cel7A gene in E.coli 59 Figure 5. Effects of pH and temperature on the activities of recombinant CEL7A. A) pH profile was determined by incubating the enzyme at 50 0 C for 1 h at varying pHs (sodium acetate buffer pH 4-6, sodium phosphate buffer pH 6-7 and glycine buffer pH 9). B) Temperature profile was determined by incubating the enzyme in 0.05M sodium phosphate buffer pH 7 for 1 h at different temperatures. Crude extract of an E. coli expressing the cel7A gene was examined for its ability to hydrolyze various cellulosic substrates at pH 7 and 50 0 C (Table 2). It could hydrolyze Avicel, Filter paper, pNP-Lac and pNP-Cell, but activity towards carboxymethylcellulose (CMC) was much lower compared to Avicel and Filter paper. These results strongly indicate that CEL7A has cellobiohydrolase activity. Table 2. Activities of the recombinant enzyme toward different cellulosic substrates at pH 7.0 and 50 0 C Substrate Activity (U/mg total protein) Avicel 21.4 ± 1.4 Filter paper 23.2 ± 0.3 CMC 2.9 ± 0.5 pNP-Lac 4.1 ± 0.08 pNP-Cell 3.8 ± 0.1
60 Sabira TAIPAKOVA et al. Discussion CBH enzymes are key components in fungal cellulase systems, and their functional activity is critical for consolidated bioprocessing for bioethanol production. For example, CBHs make up to 80% of the total mass for the T. reesei system, and CBH plays a particularly important role, making up 60% of the total mass (van Zyl et al., 2007). A number of cellulase genes from bacteria and fungus have been cloned and expressed in yeast or E. coli (Qiao et al., 2004; Hong et al., 2001, Hong et al., 2003). However fungal and bacterial endoglucanase (EG) production in recombinant strains was more successful than CBH production (van Zyl et al., 2007). This is not surprising considering that EG enzymes usually have specific activities of 2 to 3 orders higher in magnitude on synthetic and amorphous cellulose substrates, such as carboxymethylcellulose (CMC), in comparison to CBHs. Thus it is easier to measure the presence of even small amounts of heterologous EG compared to CBHs. Expression of fungal cellulases in E. coli as non-glycosylated forms has been attempted with limited success. Only a few cellulose genes, such as cbhI and egl3 of the well-studied fungus T. reesei, were expressed in E. coli, but the productivity of cellulases was very low (Ekino et al., 1999). In this study, we have successfully expressed the cel7A gene of L. edodes in E. coli under the control of the T7 promoter. SDS–PAGE and Western blotting analysis showed that CEL7A constitutes a major protein produced in E. coli with a molecular weight of 53,5 kDa, which agrees with the predicted size from the deduced amino acid sequence. Amino acid sequencing of the putative recombinant protein by MALDI-TOF and its analysis using NCBI BLAST indicated that the enzyme contained putative conserved domains of glycosyl hydrolase family 7. As shown in Figure 4A plasmid vector pET11d/cel7A expresses two additional bands which in SDS-PAGE migrate as 53 kDa and 49 kDa proteins. Since the difference in molecular weight between these two proteins is approximately 5 kDa we may suggest that the CEL7A protein was partially degraded possibly during cell lysis. The cel7A enzyme contains 430 aa catalytic domain and 57 aa C-terminal cellulose-binding domain (CBD) and serine/threonine-rich linker sequence (Lee et al., 2001). We may speculate that the proteolytic degradation of 57 aa C-terminal CBD and linker sequence results in truncated 49 kDa protein. Indeed, western blot analysis confirms the presence of low molecular weight CEL7A proteins which migrate below the full-length recombinant protein in SDS-PAGE suggesting proteolytic degradation of CEL7A during expression and/or cell lysis. Furthermore we hypothesize that the linker region of E. coli-expressed CEL7A might be more susceptible to proteolysis as compared to glycosylated form of this protein expressed in yeast cells (unpublished observation). It seems likely that glycosylated linker region in native CEL7A of L. edodes protects the enzyme from proteolytic degradation nevertheless further investigations are required. The substrate specificities indicated that CEL7A contains cellobiohydrolase activity since it could hydrolyze crystalline cellulose (Avicel, Filter paper). Activity of cloned enzyme toward CMC was much lower than to Avicel and filter paper. These data are consistent with the established results that fungal CBH proteins have very limited action on substituted cellulose such as CMC or hydroxyethylcellulose (Basiria and Mishra, 1989; Kanokratana et al., 2002). Since most of the fungal cellulases are glycoproteins, the modification of the native protein by glycosylation has been reported to play an important role in synthesis, secretion, and stability of extracellular cellulases (Basiria and Mishra, 1989). Here we have demonstrated that CEL7A synthesized in E. coli without glycosylation has an enzymatical activity. This indicates that glycosylation is not necessary for enzymatic activity of CEL7A. In summary, a gene encoding CEL7A was successfully isolated from L. edodes strain N127 using RT-PCR technique. The determined nucleotide sequence is completely corresponded to the nucleotide sequence of the cel7A gene of L. edodes strain Stamets CS-2 which was described previously (Lee et al., 2001). The enzyme was successfully expressed in E. coli in an active form. Enzymatic properties of CEL7A were also determined. The optimal temperature for enzyme activity was 50 0 C and the optimal pH was 7.0. Acknowledgement This work was supported by grant from National Centre for Biotechnology of the Republic of Kazakhstan.
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