Structure And Function Of The Module Recombinants Of Multifunctional Amylase OPMA-G

Multifunctional amylases (MFA), including new pullulanase, maltogenic amylase and cycle-maltodextrinase, as a new member of a-amylase family, can utilize cheap starch as raw materials to generate isomaltooligosaccharide which had important physiological function. Isomaltooligosaccharide has been recognized as the growth promoting factor of bifidobacterium, referred to as "bifidus factor". Our laboratory screened and isolated a producing amylase strain Bacillus sp.ZW2531-1from the soil, it can transformate starch to oligosaccharides, including maltose, maltotriose, the isomaltotriose and isomaltotetrose, but no glucose.In this paper the module recombinant OPMA-GFG gene and C-terminal domain OPMA-GC gene were amplified based on the wt-multifunctional amylase gene OPMA-G and the C-terminal truncated mutant OPMA-GT gene, and then the expression vectors pET28a-OPMA-GFG and pET28a-OPMA-GC were constructed. Finally these vectors were transformed into the expression host E.coli BL21(DE3) to express the target proteins. All expression products existed as the inclusion bodies and the yield ratio of OPMA-G, OPMA-GT, OPMA-GC were40%, the yield ratio of OPMA-GFG was23%by SDS-PAGE. The insoluble expressed proteins were dissolved with8M urea-including solution and then purified by Ni2+affinity chromatography. The purities of the purified proteins were up to95%by SDS-PAGE analysis. The target proteins were precipitated by Ammonium sulfate precipitation method, and then dissolved in10mM, pH10PBS buffer solution.The enzyme activities of OPMA-G,OPM A-G+OPMA-GT, OPMA-GT, OPMA-GFG, OPMA-GT+OPMA-GC and OPMA-GC were determined under the same number of molecules for each kind of enzyme. Compared with wild-type OPMA-G the activity of the module recombinant OPMA-GFG was increased and the activity of the system OPMA-GT+OPMA-GC was decreased. OPMA-G+OPMA-GT had the highest relative activity, whereas OPMA-GC did not show any activity. By measuring optimum temperature and thermal stability of these recombinants, we found that the optimum reaction temperature of OPMA-GFG and OPMA-GT+OPMA-GC was50℃which was consistent with that of wild type OPMA-G, but the optimum reaction temperature of OPMA-GT and the system OPMA-G+OPMA-GT decreased to40℃and30℃, respectively. Incubated at30-80℃with Ih to measure the residual vitality, the result indicate that OPMA-G, OPMA-GT,OPMA-GFG, OPMA-GT+OPMA-GC were maintained in more than70%at30℃-50℃, and module of recombinant OPMA-G+OPMA-GT was more than40%at30℃-50℃. The optimum pH for OPMA-G, OPMA-GT, OPMA-GT+OPMA-GC was pH7.5, for OPMA-GFG was pH7.0and for OPMA-G+OPMA-GT was pH8.0. In addition, OPMA-G, OPMA-GT and OPMA-GT+OPMA-GC could maintain more than50%activity between pH6.0-8.0, while OPMA-GFG and OPMA-G+OPMA-GT could do it between pH3.0-8.0. The five species of enzymes were Ca2+-independent enzymes and they showed the same substrate and product specificity. The most suitable substrate for them was still soluble starch and the catalyzed reaction products included maltose, maltotriose, isomaltotriose and isomaltotetrose, but no glucose. Finally, the enzyme reaction kinetics analysis showed that compared with wt-OPMA-G, OPMA-GT, OPMA-GFG and OPMA-GT+OPMA-GC had a higher Km value, while both of OPMA-GT and OPMA-GT+OPMA-GC had a lower kcat value, and OPMA-GFG had a higher kcat value.With the enzymatic parameters of multifunctional amylase OPMA-G and the different module recombinant enzymes, the following conclusions could be obtained: ①The C-terminal domain had a significant effect on the enzyme stability, substrate binding capacity and the catalytic efficiency, but such contribution of the C-terminal domain mainly relied on the covalently reconstruction with other OPMA-G catalytic domain which contains the typical TIM barrel domain of amylase.②One reason for the decrease of long chain substrate (starch)-binding capacity of OPMA-GFG was that OPMA-GFG had two catalytic centers (or two TIM barrels) which might be adjacent each other in three dimensional space and cause additional steric hindrance. Another possible reason for the substrate-binding decrease was that the existence of the additional catalytic center affected oligomerization of the enzyme.③Though OPMA-G+OPMA-GT and OPMA-GFG had the same contents at the sub-molecular level, OPMA-G and OPMA-GT were only non-covalent united together, so the enzyme activity and kinetic parameters of the united system were similar to that of wt OPMA-G but different from OPMA-GFG, which also proved that the change of catalytic efficiency of the recombinant OPMA-GFG resulted from the additional steric hindrance.④Though these five species of recombinants have different catalytic efficiency and catalytic characteristics, they showed the similar substrate and product specificities, which indicated that the change of the structure or module content in these enzymes could significantly affect their catalytic efficiency and the enzymatic properties, but could not obviously affect their catalytic properties.With the development of Molecular Enzymology and Structural Biology, we will profoundly and comprehensively understand the molecular structure and function of the known enzymes to date, which would play an important role for developing new enzyme sources or improving the known enzymes.