47[Noda Institute for Scientific Research GRANT]2006 Young Investigator Research Grant
Outline of Research Result

Application of Salt-tolerant Glutaminase from Aspergillus
oryzae to Soy Sauce Fermentation
Kazuaki YOSHIMUNE
Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology

Research aims
Glutaminase is one of the most important enzymes in soy
sauce fermentation, since it hydrolyzes L-glutamine to produce L-glutamic acid, which is a highly savory amino acid.1)
However, glutaminases of A. oryzae that have been reported, are markedly inhibited by the high salt concentrations present in the soy sauce fermentation process.2) Asalt-tolerant glutaminase was shown to exhibit high ability
in the production of L-glutamic acid in a model reaction ofsoy sauce fermentation.3) Thus, salt-tolerant glutaminase issuitable for application in soy sauce fermentation. We havefound a novel gene from A. oryzae RIB40 encoding a glutaminase (AoGls) that shows a higher salt-tolerance than theother known glutaminases from A. oryzae.4) In this research, effects of growth conditions on expression ofA. oryzae glutaminases, including AoGls, were investigatedfor the final goal of applying the glutaminases to soy sauce
fermentation.
Methods
A. oryzae RIB40 was obtained from National ResearchInstitute of Brewing. The strain was cultured at 30°C in awheat bran medium (wheat bran moistened with the same
weight of potassium phosphate buffer (pH 7.2)), a malt extract medium (1% malt extract and 0.1% yeast extract), orLB medium (1% peptone, 0.5% yeast extract, and 1%
sodium chloride). Hydrochloric acid or sodium hydroxide
was used for pH adjustment for the media.
The wheat bran culture was extracted with twice its
weight of 10 mM tris(hydroxymethyl)aminomethane (Tris,pH 9.0) for over night at 4°C and then filtered throughgauze. The filtrate was centrifuged and used as the extracellular glutaminase. The mycelia were harvested from themalt extract or LB agar medium that was covered with cellophane to separate them from the debris. The mycelia werefrozen by liquid nitrogen and disrupted with the same
weight of sea sand and 10 mM Tris (pH 9.0) in a mill, followed by centrifugation. The supernatant was used as theintracellular glutaminase.
Glutaminase activity was assayed by determining the formation of L-glutamic acid with L-glutamate dehydrogenaseas previously described.5) The reaction mixture contained
100 mM Tris (pH7.5) and 30 mM L-glutamine. The reaction
was carried out at 30°C for 10 min and stopped by boilingfor 3 min followed by centrifugation. The supernatant wasadded to the reaction mixture for L-glutamic acid determination. One unit of glutaminase was defined as the amountof enzyme which catalyze the formation of 1 mol of L-glutamic acid per minute. The protein concentration was determined by BCA protein assay kit (PIERCE).
An expression of AoGls was determined by immunoblot
analysis. Anti AoGls antibodies were prepared by immunization of a rabbit with purified histidine tagged AoGls
that was overproduced in E. coli. Protein samples were subjected to SDS-PAGE and the proteins in the gel were transferred onto PVDF membrane. The anti-AoGls antibodies
were used as a primary antibody and a horseradish peroxidase-conjugated anti-rabbit IgG (Immunstar GAR, Biorad)as a secondary antibody, and an Immunstar GAR-HRP detection kit (Biorad) and a Hyperfilm ECL (GE HealthcareBioscience) were used for the antibody detection.
Results1) Extracellular glutaminase
The extracellular enzyme fraction prepared from thewheat bran culture showed a little activity (5 mU/mg). This
activity decreased to 76% after an incubation at 4°C for
24 h even at pH 9.0, at which extracellular enzymes produced by A. oryzae were found to be more stable than at pH
5.0 or pH 7.0. However, partially purified glutaminase byQ-Sepharose and Butyl-Toyopearl was stable for 2 weeks at4°C. These results suggest that the extracellular glutaminases tend to be digested by protease from A. oryzae
RIB40. No glutaminase activity was detected in the extracellular enzyme fractions prepared from A. oryzae RIB40
cultured in the malt extract nor LB liquid medium at pH 5,7, or 9. An addition of 50 mM L-glutamine to those liquid
media did not increase the glutaminase activity of the extracellular enzyme fractions. Instability of the extracelluar
glutaminases may be one of the reasons why no glutaminase activity was detected in the extracellular enzyme fractions from the liquid cultures.
2) Intracellular glutaminase
The intracellular enzyme fraction showed slightly higher
glutaminase activity than the extracellular enzyme fraction
did. The intracellular enzyme fraction from LB medium
culture showed higher glutaminase activity than that fromthe malt extract agar culture (less than 1 mU/mg). The high-48

est glutaminase activity (16 mU/mg) was obtained from LB
agar culture (pH 9.0). AoGls was not detected from 30 g of
the intracellular enzymes from LB agar culture (pH 9.0) byimmunoblot analyses with the anti-AoGls antibody. Since
the immunoblot analysis could detected 10 ng of AoGls andits specific activity is 730 U/mg,6) the major glutaminase responsible for the intracellular activity may not be AoGls.
For the application of the salt-tolerant glutaminase AoGls
to soy sauce fermentation, further investigation is necessary
to increase AoGls production, though there is a possibility
that the anti-AoGls antibody used in our experiments is unable to react with AoGls from A. oryzae RIB40.

ity was not obtained from the malt extract or LB liquid culture. The highest activity (16 mU/mg) was obtained fromthe intracellular enzyme fraction prepared from LB agar
culture (pH 9.0). AoGls was not detected from A. oryzae
cells by immunoblot analysis under the conditions used.
Further investigation is necessary to increase the productionof AoGls.

Conclusion3)

The culture conditions for A. oryzae RIB40 to show anextracellular or an intracellular glutaminase activity wereinvestigated. The culture conditions for production of thesalt-tolerant glutaminase AoGls were also examined by immunoblot analysis. The extracellular glutaminase activity
was obtained from the wheat bran culture, though the activ-References
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