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Deciphering characteristics of the designer cellulosome from Bacillus subtilis WB800N via enzymatic analysis

Lin, Chia-Chi, Yap, Cally Joe San, Kan, Shu-Chen, Hsueh, Nai-Chi, Yang, Liang-Yu, Shieh, Chwen-Jen, Huang, Chieh-Chen, Liu, Yung-Chuan
Biochemical engineering journal 2017 v.117 pp. 147-155
Bacillus subtilis, cellobiose, cellulose, cellulosome, clones, endo-1,4-beta-glucanase, enzymatic reactions, genes, high performance liquid chromatography, pellets, xylanases
Recombinant Bacillus subtilis WB800N harboring pGETS 118 designer cellulosome genes was applied to the artificial cellulosome production. To investigate the characteristics of cellulosome assembly in the supernatant, three free enzymes clones, i.e., endoglucanase, exoglucanase and xylanase, were constructed. The samples of the respective free enzymes and the cellulosome were collected and examined via various enzymatic assays. In comparison of specific activity of anchored cellulosome and the free enzymes on cell pellets, it was found that only anchored cellulosome exhibited the activities. No activity was detected on the cell pellets of the three free enzyme clones. This indicated that the designer cellulosome subunits must be properly self-assembled into a substantial complex capable of the cellulose degradation. To study the cellulose hydrolytic behavior, the best specific activities of endoglucanase and xylanase obtained in the supernatant cellulosome were 3.62±0.02U/mg and 1.97±0.06U/mg, respectively. The end products of the enzyme reaction detected via HPLC analysis showed that oligosaccharide and cellobiose were the major products in the hydrolytic reactions. In the kinetic studies, the Vmax value (8.98mM/h) for endoglucanase in the supernatant cellulosome was higher than pellets (0.603mM/h). However, the endoglucanase of pellets gave a lower Km value of 0.359g/L, indicating a higher substrate affinity than that in the supernatant (11.87g/L).