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.