Ethanol fermentation and protein characterization of Zymomonas mobilis cloned with extracellular sucrase gene

• Main Authors: I-Hsiang Chan, 詹議翔
• Other Authors: Wen-Chien Lee
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/70765139104679065845

碩士 === 國立中正大學 === 化學工程研究所 === 90 === Zymomonas mobilis is a Gram-negative bacterium that is known as an efficient ethanol producer. It uses an exclusively fermentative metabolism through the Entner-Doudoroff pathway under anaerobic conditions to metabolize glucose, fructose, or sucrose. On sucrose-based substrate, it may be postulated that sucrose is first hydrolyzed to form glucose and fructose, and then, these monosaccharides are uptaken by the cell. The ethanol yield from sucrose is lower due to the formation of by-products, mainly levan and sorbitol. In this study, we used the strain of Zymomonas mobilis CCRC 10809 cloned with extracellular sucrase gene (SacC gene). The recombinant plasmid was pZM-SacC, which is a 7.7 kb molecule and codes for the chloramphenicol resistance gene (Cmr) as a selectable marker gene. Compared with the batch ethanol fermentation of wild-type Zymomonas mobilis CCRC 10809 on different concentrations of sucrose, the recombinant Z. mobile led to higher ethanol yields. The ethanol yield of recombinant strain could keep at 0.334 to 0.357 g/g (73.1 to 78.1﹪of the theoretical value). But the ethanol yield of wild-type strain was reduced from 0.346 to 0.285 g/g (75.7 to 62.3 ﹪of the theoretical value) by the increase of sucrose concentration from 150 to 200 g/L. At the same sucrose concentration, the ethanol yield of recombinant cell was always higher than that of wild-type cell; especially at the sucrose concentration of 200 g/L, the difference in ethanol yield was greater than 0.049 g/g. A comparison of results from the two-dimensional gel electrophoresis of extracellular proteins secreted by wild-type and recombinant strains indicates that the recombinant strain did not secret more amount of extracellular sucrase than the wild-type. This may be why the ethanol yield by the recombinant strain wasn’t significantly higher than that of the wild-type. In this study, the stoichiometric and kinetic models for Zymomonas mobilis fermentation using sucrose as the sole carbon source system were also developed. According to the calculation based on mass balance, the specific sucrose hydrolyze rate (αqS) was higher than the specific levan formation rate (βqS). That meant the total activity of extracellular and intracellular sucrase was higher than the activity of extracellular levansucrase. When the αqS/βqS values were calculated based on the time of the largest sucrose-hydrolyzing rate, the recombinant strain was significantly higher than the wild-type strain. It meant the recombinant strain had a highly extracellular sucrase activity, which was helpful for sucrose hydrolysis to form glucose and fructose, and then, for the cell to assimilate these sugars easily. In the model simulation, the experimental data of sucrose, biomass and ethanol concentrations were fitted to the kinetic model. Modeling work on the kinetics of ethanol fermentation by Zymomonas mobilis on sucrose will be helpful for scaling-up such ethanol-producing process.