| Pectin is a natural polymer riched in polygalacturonic acid, and it is an important component of plant cell walls. Pectin methylesterase catalyses the demethoxylation of the homogalacturonan chain of pectin, and therefore it has the activity to hydrosis the pectin. The pectin methylesterase from Aspergillus niger can efficiently control the transparency and turbidity of the juice in the juice industrial production. However, A. niger can produce a variety of pectinase, thus leading to that it very difficult to purification of pectin methylesterase from these enzyme mixtures due to the similar biochemical properties of these enzymes. Hence, the recombinant expression technology is a feasible and effective method for the production of pectin methylesterase. On the other hand, pectin methylesterase from A. flavus is one of the pathogenic factors when A. flavus infects the plant. Consequently, the infestation of A. flavus to crops can be controlled through the inhibition of enzymatic activity. Moreover, the catalytic mechanism of fungal pectin methylesterase is different from bacterial or plant and the crystal structure of fungal pectin methylesterase have not been reported. Therefore, that makes it to be necessary to study the structure and function of the fungal pectin methylesterase. Here, we select pectin methyl esterases from A. niger and A. flavus as sample, and then reseach the recombinant expression and characterization of these two enzymes.1. Cloning,expression,purification and characterization of pectin methylesterase from Aspergillus nigerAfter cloning of the gene of pectin methylesterase from A. niger (ANPME), the recombinant pectin methylesterase was expressed in Pichia pastoris GS115as a secretory form by pGAPZa vector, and moreover, this method can avoid the inconvenience of industrial methanol induction. The recombinant ANPME was purified by anion exchange, followed by cation exchange chromatography, giving an overall yield of28.0%and the specific activity of342U/mg. The Km, Vmax and kcat values of the enzyme with respect to pectin were8.6mmol/L,1.376mmol/(minxmg)and8.26×103S-1respectively. It was stable at30-50℃and pH4.0-6.0,and had optimal activity at50℃and pH4.8. Cations such as K+, Mg2+, Ni2+Mn2+and Co2+slightly inhibited its activity, whereas Na+had no effect. 2. Recombinant expression,purification and inhibitor screening of pectin methylesterase from Aspegillus flavusThe genen of pectin methylesterase from A. flavus (AFPME) was expressed in P. pastoris GS115by using the vector of pPIC9. After two-step purification by affinity chromatography and ion exchange chromatography, the purified AFPME was appeared as a single band in SDS-PAGE. The recombinant AFPME had optimal activity at55℃and pH4.8. Moreover, it was found that AFPME show higher catalytic activity on pectin substrate with middle DM degree. It was also found that epigallocatechin gallate (EGCG) and polyphenols60(PP60) extracted had the ability to inhibit the activity of AFPME.3. Study of prokaryotic expression by E.coli and crystallization conditions of pectin methylesterases from Aspergillus niger and Aspergillus flavusThe recombinant ANPME and AFPME in P. pastoris showed the inhomoeneous glycosylation. The inhomoeneous glycosylation interferenced the crystallization of protein, and therefore, E. coli expression system was used to produce these two enzymes. It was found that AFPME was expressed as inclusion bodies in E. coli, and ANPME was expressed as soluble protein in E. coli. ANPME was purified by one-step affinity chromatography with the purity of more than95%. After screening of crystal growth conditions using Hampton crystallization kit, three conditions was found to be suitable for the crystal growth of ANPME, and ANPME was found to be growth as micro-crystals under these three conditions. |
PDF Full Text Request