40NISR Research GRANT
2007 Research Grant

Effective synthesis of bio-nylon materials using Bacillus megaterium
Makoto ASHIUCHI
Department of Agriculture, Kochi University

Research aims
Poly-g -glutamate (PGA) is a biopolymer with a nylonlike backbone, and some experiments regarding the esterification of its a -carboxyl side chains indicate that PGA substantially exhibits nylon-like properties.1) Unlike chemicalnylons, PGA with multiple chirotopic carbons possesses
fair biodegradability and other useful functionalities.2) PGAis thus considered as a bionylon. Edible PGA is obtained
from the mucilage of Japanese natto food.3) Unfortunately,
it is not quality-controlled in D- and L-glutamate sequences(D ratios, 60%15%; L ratios, 40%15%) and molecularsize distribution (Mw, 10,000 to 1,000,000):2) the demeritsof natto PGA from the industrial point of view. Stereo-regular PGA (L-rich form) was recently isolated from archaeal
exopolymers,4) but the low productivity of archaeal PGA
may be fatal in ensuring its feasibility. Therefore, further
research for useful PGA producers has been requested.
A most important determinant for PGA stereochemistryis glutamate racemase, which virtually serves for the formation of intercellular D-glutamate pools.2) The natto
starters of Bacillus subtilis indeed produced the enzyme inabundance;2) however, Bacillus megaterium showed only alittle D-glutamate-supplying activity,5) viz., a point of resemblance to L-rich PGA-producing archaea. This study wasaimed at the effective synthesis of stereo-regular PGA as anapplicable bionylon material, and here the PGA productivity of B. megaterium was investigated.
MethodsB. megaterium WH320 (MoBiTec Co., Göttingen) wasused in the experiments, and the synthesis of PGA was carried out as follows. First, growing cells were inoculated
at an appropriate concentration (8 g L1) in a standard
medium (pH 7.0) containing 5% sucrose (as a sugar), 2% Lglutamate (as a substrate), 5% NaCl (as a salt), 1%(NH4)2SO4, 0.27% KH2PO4, 0.42% Na2HPO4, 0.5%MgSO4 · 7H2O, and MS vitamin solution (PhotoTechnology
Laboratories, KS) and incubated at 30°C for 5 days.
PGA was isolated by the methods of Park et al,6) and afraction of the isolated PGA was hydrolyzed with distilled
HCl (6 M) at 105°C for 8 h in vacuo.2) The total amount ofglutamate monomers thus generated, which is applicable tothe calculation of the polymer yield, was determined bymeans of high-performance liquid chromatography with achiral separation column;2) typically, D-glutamate is more

rapidly eluted than L-glutamate. The standard curve for Dand L-glutamate (showing the relationships of their amounts
and the peak area on the high-performance liquid chromatography profiles): yD-Glu2.97x (fmol) and yL-Glu2.91x(fmol), where x represents each peak area.5) On the otherhand, a fraction of the isolated PGA was subjected to SDSPAGE and specifically visualized by the gel staining withmethylene blue (a basic dye).3)
Results
Optimization of substrate concentration. An essential
substrate for the synthesis of B. megaterium PGA was anamino acid, glutamate. Based on the tests under various
concentrations (0% to 10%), its optimum concentration wasdefined as 2%. In the standard medium containing 2% Lglutamate, the maximum volumetric yield of B. megaterium
PGA was 8.6 g L1; this value is almost as high as that ofnatto PGA.2) On the other hand, B. megaterium could not
utilize D-glutamate effectively in contrast to some B. subtilis strains.7,8)
Furthermore, the stereochemistry of B. megaterium PGA
was analyzed. If L-glutamate was applied as a substrate,stereo-regular PGA, in which the L ratio reached a maximum of 95%, can be synthesized consistently.5) In contrast,
when D-glutamate (e.g., 2%) was used, a random copolymerof PGA with a DL ratio of 45 : 55 was generated.
Effects of salt and osmotic stress. As shown in Fig. 1,B. megaterium dramatically accumulated PGA in salt-richliquid media (2% NaCl). It is also noteworthy that B.
megaterium PGA with a greater molecular size (Mw,2,000,000) was obtained from a medium containing ahigher salt concentration (Fig. 1 upper, an SDS-PAGE gel).
In general, salt stress causes osmotic (due to high soluteconcentrations) as well as ionic stress (due to over-accumulation of salt in cells). The effect of osmotic stress on B.
megaterium PGA production was examined by the use ofsorbitol (0.1 M to 1.0 M)7) instead of NaCl. As a result, PGA
was not accumulated in media under any sorbitol concentrations tested, suggesting that the PGA productivity of B.
megaterium is affected by ionic-than-osmotic stress.
Effect of sugar-solute concentration. To further examine the effect of sugar-solute, the structural features of B.
megaterium PGA produced at various concentrations of sucrose (0% to 10%) were examined. There was no differencein the molecular size distribution of PGA, whereas the effect on the polymer stereochemistry was not negligible. The

41Fig. 1. Salt-responsive PGA production by B. megaterium.5) Upper, SDSPAGE and visualization of PGA. Lower, PGA accumulation in the standard medium containing the indicated concentration of NaCl (0% to 10%).

addition of a sugar-solute (preferably 5% or at least
2%) to media allowed for the production of PGA with ahigh content of L-glutamate (viz., 90% ). In fact, if asugar-less liquid medium was used, the L ratio of B. megaterium PGA was decreased to 75%. Indeed, the DL ratio ofBacillus PGA often was shown to be affected by coexistingmetal ions;2) however, this is the first example showing thatthe apparently similar phenomena are induced by a sugarsolute as well.
Ability for sugar utilization. Probably, sugar plays animportant role in B. megaterium PGA production. Further
research for such sugar effects thus helps to develop an effective synthesis system for stereo-regular PGA. In thiswork, B. megaterium was found to utilize glucose, fructose,xylose, arabinose, galactose, sucrose, maltose, cellobiose,lactose, and starch (5% each). Cellobiose is a main digestive product of cellulose, whereas xylose and arabinose aregenerated by hemicellulose decomposition.
Identification of preferable sugars. Besides sucrose, theeffects of other sugars on B. megaterium PGA production
were examined. In this work, the PGA productivity in thepresence of sucrose was defined as 100% (Fig. 2, bar ②),
and the L ratio of the polymer was about 92%. As shown in
an SDS-PAGE gel of Fig. 2 (upper), there were no differences in the molecular size distribution among all PGAs
synthesized here. When a sugar-less liquid medium wasused (Fig. 2, bar ① ), the PGA yield reduced by half.
Among sugars tested, arabinose (bar ⑥) and fructose (bar⑦) significantly increased the PGA yield; the productivity
was about 1.8-fold higher than that under the standard conditions (bar ②). Considering the effectiveness of the stereoregular PGA synthesis, it seems likely that the use of arabinose is superior to those of other sugars; the L ratios (%) of
the polymers synthesized are indicated in the parentheses ofFig. 2.

Fig. 2. Sugar effects on B. megaterium PGA production. Upper, SDSPAGE and visualization of PGA. Lower, Relative PGA productivity
against the use of various sugars: ①, sugar-less; ②, sucrose; ③, glucose;④, xylose; ⑤, galactose; ⑥, arabinose; ⑦, fructose; and ⑧, lactose. The
values in the parentheses represent the L ratios (%) of the synthesized
PGA.

Conclusion
The study showed that B. megaterium PGA production is
induced by ionic-than-osmotic stress and verified the feasibility of the addition of a high concentration of salt to theculture media. In addition, the optimization of the substrateand sugar concentrations in media and the identification of
the preferable sugars allowed for the effective synthesis ofstereo-regular PGA, a promising bionylon material. As thepreferable sugars identified are essentially the same as
small products derived from woody biomass and garbage,B. megaterium will serve as one of the most useful microorganisms to realize a strategy for biomass polymer refinery.
The development of a genetically engineered B. megaterium that exhibits a high biomass-degradation activity isnow in progress.
References
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