Enzymatic Analysis Of The Ferulic Acid Esterase Of Clostridium Thermocellum And Construction Of Its Plant Efficient Expression Vector

In gramineous cell wall,ferulic acid can covalently cross link with hemicellulose and lignin to form the lignin-ferulic acid-arabinoxylan complexs,which is an important molecular basis for the formation of a solid anti-degradation barrier to lignocellulose degrading enzymes.Heterologous expression of ferulic acid esterase(FAE)in plant cells can hydrolyze the ester bond between ferulic acid and ligonocellulose macromolecules,improve the degeradability of biomass and therefore reduce its cost of enzymatic conversion.However,the mesophilic FAE activity expressed in plant cells usually disturbs the normal growth and development of host plants or reduces the stress resistance.Genic transformation using the thermophilic FAE coding gene is an alternative strategy to overcome these problems.In this study,the a thermophilic FAE enzyme from Clostridium thermocellum was characterized,and then its plant effiecient expression vectors were constructed.The main results are as follows:1)The coding gene sequence for the thermophilic ferulic acid esterase catalytic domain(FAE)within XynZ protein from Clostridium thermocellum as well as for this catalytic domain adding an extra carbohydrate binding module 6(FAE-CBM6)was cloned.The gene constructed with pET22b(+)and expressed in E.coli BL21(DE3)cells successfully,which produced a target recombinant protein with a molecular weight of 29.0 KDa and 45.0 KDa,respectively.2)The characteristics of recombinant FAE and FAE-CBM6 enzymes were analyzed.The optimum reaction temperature for FAE and FAE-CBM6 was 60 ? and 70 ?,respectively,and the optimum pH being pH 6.0 and pH 7.0.The FAE activity kept stable in the range of pH 5.0-pH 9.0,while FAE-CBM6 was in pH 4.0-pH 9.0.The relative enzyme activity of FAE was still over 60% after incubating for 2 h at 70 ? or 75 ?,and that of FAE-CBM6 was more than 80% after incubating for 2 h at 70 ?.Mn2 + and Zn2 + had positive effects on FAE activity,but Mg2+,Cu2+ and Ni2+ gave birth to negative effects(p<0.05).On the other hand,Cu2+ and Zn2+ had significantly negative effects on FAE-CBM6 activity.Generally,under the same reaction condition,FAE-CBM6 presented higer catalytic activity than FAE,suggesting that the CBM6 domain plays an important role in improving the ferulic acid activity of FAE.3)The codon sequence for FAE-CBM6 was optimized according to the codon usage bias of maize(Zea mays)and then the optimized sequence was synthesized.Based on this,several kinds of plant efficient expression vectors of thermophilic ferulic acid esterase gene were constructed through combining the gene sequence with a strong Ubiquitin promoter in Gramineae,an ? translation enhancer and different signal peptide coding sequences for protein subcellular localization.