4 Discussion
A large number of AGLs were found in mammals, plants, and microorganisms. Regarding the production of rAGL, up to now, most bacterial enzymes from Sulfolobus tokodaii[49], Thermoplasma acidophilum[50], Pyrobaculum aerophilum[51], and Bifidobacterium longum[52], were overexpressed in E. coli , and several AGLs from plants and fungi like barley[48], Rice [53],Aspergillus niger [54], were successfully produced in P. pastoris . In the previous work, efforts have been made to produce the rAGL of glycoside hydrolase family GH31 from higher plants in E. coli , but resulted in no or rather low enzyme activity [55-56]. It was not until 2006 that the AGL from barley (Hordeum vulgare ) with fully activity was produced in P. pastoris for the first time[47-48]. Since then, P. pastoris system that is widely used to produce heterologous proteins from various organisms involving viruses, bacteria, fungi, plants, animals and humans[57], also greatly promotes the heterologous production of AGL (Table 2).
Table 2
Previously, when two isoforms of ONG2 were purified from dry rice seeds, native AGL from O. sativa has been characterized[53, 58]. In this study, rAGL corresponding to the ONG2 was found only one bandlarger than 97.2 KDa (Fig. 3), probably due to the lack of post-translational proteolysis occurring inPichia . The Km value of rAGL for maltose was 61 mM, which indicates that the affinity to maltose is lower than that of native ONG2-I and ONG2-II, which are 2.1 mM and 2.3 mM respectively. As well, rAGL appeared to possess lower specific activity (49.83 U/mg) than that of the ONG2-I (122U/mg) and ONG2-II (124 U/mg), although the assay conditions were not completely the same. Comparison of biochemical characteristics of native AGLs from O. sativa and recombinant enzyme from P. pastoris indicated that they had similar optimal pH and remained active in a wide pH range. Native AGLs in plants prefer to be close to the temperature of the growing environment. However, rAGL was stable at up to 55 °C and the highest optimal temperature (65 °C) of enzyme activity, which demonstrates better thermal stability than native informs, which was stable as high as 40 °C, and completely lost its activity after incubation at 60 °C for 10 min. It should be noted that thermal stability of rAGL is an important factor related to its potential applications in biotechnology.
Thanks to the transglycosylation activity, AGLs are potentially to be effectively useful in the preparation of AA-2G (Fig. 7). The production of AA-2G was facilitated under optimized conditions (40°C and pH 6.0) by a truncated AGL from A. niger and the highest yield of AA-2G reached 0.528±0.04 g/L [19]. As far as we know, the yield of AA-2G in the present study represents the highest by AGLs reported so far, reaching 8.7±0.4 g/L at 37 °C and pH 4.0. Among the results of reactions, a typical ‘n’-shaped kinetic profile of the AA-2G formation was observed. By understanding the glucosidase-catalyzed glycosylation process in water-rich environment[59], it can be concluded that glycosylation is dominant at the beginning of reaction, which leads to rapid synthesis of AA-2G. At the same time, the synthesized AA-2G started to degrade with time. In the batch system, the AA conversion is thermodynamically restricted, and the catalytic efficiency is mainly impacted by the selectivity of AGL’s glycosylation/hydrolysis. In the case of the different maltose/AA·Na ratios (Fig. 6c), it can be concluded that high glycosyl donor (maltose) content helped to slow down the rate of hydrolysis and therefore have an influence on the catalytic efficiency. However, the amount of rAGL only has the effect on the reaction rate but does not affect the equilibrium of glycosylation and hydrolysis (Fig. 6d). In order to maximize the product accumulation, the decomposition of AA at inappropriate pH and temperatures should never be neglected (Fig. 6a and 6b).
Figure 7
In summary, AGL from O. sativa was expressed in P. pastoris , which proved to be beneficial in fermentation process, allowing the production of enough enzyme and promoting enzymatic synthesis of AA-2G by AGLs. To date, the full potential of rAGL has not been fully realized in this study and further work is needed to improve enzyme activity for transglycosylation, such as controlling the conditions of glycosylation reactions [60] and enzyme technology to create a glycosynthase [61]. These studies will provide the basis for the application of rAGL in AA-2G industrial production.