Koji – the only one domesticated fungi: characterization, enzymology, and use

Authors

  • Dušan Straka Slovak University of Agriculture in Nitra, AgroBioTech Research Centre, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, Tel.: +421917393664 Author https://orcid.org/0009-0008-4229-519X
  • Lukáš Hleba Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Science, Institute of Biotechnology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, Tel.: +421904189191 Author https://orcid.org/0000-0001-8244-6548
  • Štefan Ailer Slovak University of Agriculture in Nitra, Faculty of Horticulture and Landscape Engineering, Institute of Horticulture, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, Tel.: +421376415804 Author https://orcid.org/0000-0001-8447-5377

DOI:

https://doi.org/10.5219/scifood.63

Keywords:

koji, fermentation, microbial enzymes, Aspergillus oryzae

Abstract

Koji mold (Aspergillus oryzae) is the only known domesticated fungal species, playing a central role in traditional East Asian fermented foods such as soy sauce, miso, and sake. This filamentous fungus is highly valued for its exceptional enzymatic capabilities, as it secretes diverse hydrolytic enzymes, including proteases and amylases. A. oryzae possesses 65 endopeptidase and 69 exopeptidase genes, supporting efficient protein degradation. In contrast, A. sojae contains 83 endopeptidase and 67 exopeptidase genes. Key enzymes include alkaline protease (optimal at pH 9.0 and 40 °C), neutral protease I (broad specificity), and acid protease (optimal at pH 3.7, 39 kDa). α-Amylase and glucoamylase production are also prominent; the former exhibits thermal stability up to 75 °C, while the latter displays optimal activity at 60 °C and has a molecular weight of between 60–70 kDa. Genomic studies reveal that A. oryzae contains three amylase genes, in contrast to only one in A. sojae, which correlates with its superior saccharification performance. Importantly, A. oryzae is genetically incapable of producing aflatoxin due to critical mutations in the aflR regulatory gene and deletions within the aflatoxin biosynthesis cluster, particularly in Group 1 and 2 strains that dominate industrial use. Koji mold is traditionally cultivated through solid-state fermentation, primarily on steamed, polished rice, under controlled conditions (30–35°C, 90% humidity). This review provides a comprehensive overview of the enzymology, genomic adaptations, and fermentation technologies associated with A. oryzae, emphasizing its unique domestication, safety profile, and industrial relevance in enzyme production and sustainable food biotechnology.

References

1. Avwioroko, O. J., Anigboro, A. A., Unachukwu, N. N., & Tonukari, N. J. (2018). Isolation, identification Christensen, T., Woeldike, H., Boel, E., Mortensen, S. B., Hjortshoej, K., Thim, L., & Hansen, M. T. (1988). High Level Expression of Recombinant Genes in Aspergillus Oryzae. Nature Biotechnology, 6(12), 1419–1422. https://doi.org/10.1038/nbt1288-1419

2. Yamashita, H. (2021). Koji Starter and Koji World in Japan. Journal of Fungi, 7(7), 569. https://doi.org/10.3390/jof7070569

3. Machida, M., Yamada, O., & Gomi, K. (2008). Genomics of Aspergillus oryzae: Learning from the History of Koji Mold and Exploration of Its Future. DNA Research, 15(4), 173–183. https://doi.org/10.1093/dnares/dsn020

4. Matsushima, K., Chang, P.-K., Yu, J., Abe, K., Bhatnagar, D., & Cleveland, T. E. (2001). Pre-termination in aflR of Aspergillus sojae inhibits aflatoxin biosynthesis. Applied Microbiology and Biotechnology, 55(5), 585–589. https://doi.org/10.1007/s002530100607

5. Ehrlich, K. C., Chang, P.-K., Yu, J., & Cotty, P. J. (2004). Aflatoxin Biosynthesis Cluster GenecypAIs Required for G Aflatoxin Formation. Applied and Environmental Microbiology, 70(11), 6518–6524. https://doi.org/10.1128/aem.70.11.6518-6524.2004

6. Cary, J. W., Wright, M., Bhatnagar, D., Lee, R., & Chu, F. S. (1996). Molecular characterization of an Aspergillus parasiticus dehydrogenase gene, norA, located on the aflatoxin biosynthesis gene cluster. Applied and Environmental Microbiology, 62(2), 360–366. https://doi.org/10.1128/aem.62.2.360-366.1996

7. Zhou, R., & Linz, J. E. (1999). Enzymatic Function of the Nor-1 Protein in Aflatoxin Biosynthesis in Aspergillus parasiticus. Applied and Environmental Microbiology, 65(12), 5639–5641. https://doi.org/10.1128/aem.65.12.5639-5641.1999

8. Yu, J., Bhatnagar, D., & Cleveland, T. E. (2004). Completed sequence of aflatoxin pathway gene cluster in Aspergillus parasiticus1. FEBS Letters, 564(1–2), 126–130. https://doi.org/10.1016/s0014-5793(04)00327-8

9. Yabe, K., & Nakajima, H. (2004). Enzyme reactions and genes in aflatoxin biosynthesis. Applied Microbiology and Biotechnology, 64(6), 745–755. https://doi.org/10.1007/s00253-004-1566-x

10. Trail, F., Chang, P. K., Cary, J., & Linz, J. E. (1994). Structural and functional analysis of the nor-1 gene involved in the biosynthesis of aflatoxins by Aspergillus parasiticus. Applied and Environmental Microbiology, 60(11), 4078–4085. https://doi.org/10.1128/aem.60.11.4078-4085.1994

11. Tominaga, M., Lee, Y.-H., Hayashi, R., Suzuki, Y., Yamada, O., Sakamoto, K., Gotoh, K., & Akita, O. (2006). Molecular Analysis of an Inactive Aflatoxin Biosynthesis Gene Cluster in Aspergillus oryzae RIB Strains. Applied and Environmental Microbiology, 72(1), 484–490. https://doi.org/10.1128/aem.72.1.484-490.2006

12. Yu, J., Chang, P. K., Cary, J. W., Bhatnagar, D., & Cleveland, T. E. (1997). avnA, a gene encoding a cytochrome P-450 monooxygenase, is involved in the conversion of averantin to averufin in aflatoxin biosynthesis in Aspergillus parasiticus. Applied and Environmental Microbiology, 63(4), 1349–1356. https://doi.org/10.1128/aem.63.4.1349-1356.1997

13. Ito, K., & Matsuyama, A. (2021). Koji Molds for Japanese Soy Sauce Brewing: Characteristics and Key Enzymes. Journal of Fungi, 7(8), 658. https://doi.org/10.3390/jof7080658

14. Sato, A., Oshima, K., Noguchi, H., Ogawa, M., Takahashi, T., Oguma, T., Koyama, Y., Itoh, T., Hattori, M., & Hanya, Y. (2011). Draft Genome Sequencing and Comparative Analysis of Aspergillus sojae NBRC4239. DNA Research, 18(3), 165–176. https://doi.org/10.1093/dnares/dsr009

15. Ishihara K., Honma N., Matsumoto I., Imai S., Nakazawa S., & Iwafuchi H. (1996). Comparison of Volatile Components in Soy Sauce (Koikuchi Shoyu) Produced Using Aspergillus sojae and Aspergillus oryzae. Nippon Shokuhin Kagaku Kogaku Kaishi, 43(9), 1063–1074. https://doi.org/10.3136/nskkk.43.1063

16. Kim, K. U., Kim, K. M., Choi, Y.-H., Hurh, B.-S., & Lee, I. (2019). Whole genome analysis of Aspergillus sojae SMF 134 supports its merits as a starter for soybean fermentation. Journal of Microbiology, 57(10), 874–883. https://doi.org/10.1007/s12275-019-9152-1

17. Nakadai, T., Nasuno, S., & Iguchi, N. (1972). The Action of Peptidases fromAspergillus oryzaein Digestion of Soybean Proteins. Agricultural and Biological Chemistry, 36(2), 261–268. https://doi.org/10.1080/00021369.1972.10860243

18. Gao, X., Yin, Y., Yan, J., Zhang, J., Ma, H., & Zhou, C. (2019). Separation, biochemical characterization and salt‐tolerant mechanisms of alkaline protease from Aspergillus oryzae. Journal of the Science of Food and Agriculture, 99(7), 3359–3366. https://doi.org/10.1002/jsfa.9553

19. Li, S., Hu, Y., Hong, Y., Xu, L., Zhou, M., Fu, C., Wang, C., Xu, N., & Li, D. (2015). Analysis of the Hydrolytic Capacities of Aspergillus oryzae Proteases on Soybean Protein Using Artificial Neural Networks. Journal of Food Processing and Preservation, 40(5), 918–924. https://doi.org/10.1111/jfpp.12670

20. M, R. R., & Yepuru, S. K. (2018). Production of alkaline protease from Aspergillus oryzae isolated from seashore of Bay of Bengal. Journal of Applied and Natural Science, 10(4), 1210–1215. https://doi.org/10.31018/jans.v10i4.1905

21. Xu, D., Li, C., Wang, Y., Sun, L., Zhao, H., & Zhao, M. (2013). Characterisation of acid proteases from a fusant and its progenitors spergillus oryzae HN3042 and Aspergillus niger CICC2377. International Journal of Food Science & Technology, 48(4), 678–684. https://doi.org/10.1111/j.1365-2621.2012.03142.x

22. Sekine, H. (1976). Neutral proteinases I and II of Aspergillus sojae. Action on various substrates. Agricultural and Biological Chemistry, 40(4), 703–709. https://doi.org/10.1271/bbb1961.40.703

23. Nakadai, T., Nasuno, S., & Iguchi, N. (1973). Purification and Properties of Neutral Proteinase II from Aspergillus oryzae. Agricultural and Biological Chemistry, 37(12), 2703–2708. https://doi.org/10.1271/bbb1961.37.2703

24. Nakadai, T., & Nasuno, S. (1977). The action of acid proteinase from Aspergillus oryzae on soybean proteins. Agricultural and Biological Chemistry, 41(2), 409–410. https://doi.org/10.1271/bbb1961.41.409

25. Vishwanatha, K., Appurao, A., & Singh, S. (2009). Characterisation of acid protease expressed from Aspergillus oryzae MTCC 5341. Food Chemistry, 114(2), 402–407. https://doi.org/10.1016/j.foodchem.2008.09.070

26. Liu, J., Xia, W., Abdullahi, A. Y., Wu, F., Ai, Q., Feng, D., & Zuo, J. (2014). Purification and Partial Characterization of an Acidic α-Amylase from a Newly IsolatedBacillus subtilisZJ-1 that may be Applied to Feed Enzyme. Preparative Biochemistry and Biotechnology, 45(3), 259–267. https://doi.org/10.1080/10826068.2014.907184

27. Nayab, D.-, Akhtar, S., Bangash, N., Nisa, W.-, Hayat, M. T., Zulfiqar, A., Niaz, M., Qayyum, A., Syed, A., Bahkali, A. H., & Elgorban, A. M. (2022). Production of Glucoamylase from Novel Strain of Alternaria Alternata under Solid State Fermentation. BioMed Research International, 2022(1). https://doi.org/10.1155/2022/2943790

28. Devanthi, P. V. P., & Gkatzionis, K. (2019). Soy sauce fermentation: Microorganisms, aroma formation, and process modification. Food Research International, 120, 364–374. https://doi.org/10.1016/j.foodres.2019.03.010

29. Jünger, M., Mittermeier-Kleßinger, V. K., Farrenkopf, A., Dunkel, A., Stark, T., Fröhlich, S., Somoza, V., Dawid, C., & Hofmann, T. (2022). Sensoproteomic Discovery of Taste-Modulating Peptides and Taste Re-engineering of Soy Sauce. Journal of Agricultural and Food Chemistry, 70(21), 6503–6518. https://doi.org/10.1021/acs.jafc.2c01688

30. Yu, H., Jiang, L., Gao, L., Zhang, R., Zhang, Y., Yuan, S., Xie, Y., & Yao, W. (2024). High-intensity ultrasound promoted the maturation of high-salt liquid-state soy sauce: A mean of enhancing quality attributes and sensory properties. Food Chemistry, 438, 138045. https://doi.org/10.1016/j.foodchem.2023.138045

31. Costa, S., Summa, D., Zappaterra, F., Blo, R., & Tamburini, E. (2021). Aspergillus oryzae Grown on Rice Hulls Used as an Additive for Pretreatment of Starch-Containing Wastewater from the Pulp and Paper Industry. Fermentation, 7(4), 317. https://doi.org/10.3390/fermentation7040317

32. Dutt, S., Goel, V., Garg, N., Choudhury, D., Mallick, D., & Tyagi, V. (2019). Biocatalytic Aza‐Michael Addition of Aromatic Amines to Enone Using α‐Amylase in Water. Advanced Synthesis & Catalysis, 362(4), 858–866. https://doi.org/10.1002/adsc.201901254

33. Avwioroko, O. J., Anigboro, A. A., Unachukwu, N. N., & Tonukari, N. J. (2018). Isolation, identification and in silico analysis of alpha-amylase gene of Aspergillus niger strain CSA35 obtained from cassava undergoing spoilage. Biochemistry and Biophysics Reports, 14, 35–42. https://doi.org/10.1016/j.bbrep.2018.03.006

34. Budhadev, H. (2023). Bioinformatics Analysis of α-Amylase Three-Dimensional Structure in Aspergillus oryzae. International Journal of Research Publication and Reviews, 4(9), 868–874. https://doi.org/10.55248/gengpi.4.923.52600

35. Sanghamitra Mallik, S. M. (2015). Optimization of Solid State Fermentation Conditions and Characterization of Thermostable Alpha Amylase from Bacillus subtilis (ATCC 6633). Journal of Bioprocessing & Biotechniques, 05(04). https://doi.org/10.4172/2155-9821.1000218

36. Hata, Y., Ishida, H., Ichikawa, E., Kawato, A., Suginami, K., & Imayasu, S. (1998). Nucleotide sequence of an alternative glucoamylase-encoding gene (glaB) expressed in solid-state culture of Aspergillus oryzae. Gene, 207(2), 127–134. https://doi.org/10.1016/s0378-1119(97)00612-4

37. Zambare, V. (2010). Solid State Fermentation of Aspergillus oryzae for Glucoamylase Production on Agro residues. International Journal of Life Sciences, 4, 16–25. https://doi.org/10.3126/ijls.v4i0.2892

38. Dubey, A. K., Suresh, C., Kavitha, R., Karanth, N. G., & Umesh-Kumar, S. (2000). Evidence that the glucoamylases and α‐amylase secreted by Aspergillus niger are proteolytically processed products of a precursor enzyme. FEBS Letters, 471(2–3), 251–255. https://doi.org/10.1016/s0014-5793(00)01410-1

39. Pandey, A. (1995). Glucoamylase Research: An Overview. Starch - Stärke, 47(11), 439–445. https://doi.org/10.1002/star.19950471108

40. Michelin, M., Ruller, R., Ward, R. J., Moraes, L. A. B., Jorge, J. A., Terenzi, H. F., & Polizeli, M. de L. T. M. (2007). Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii. Journal of Industrial Microbiology & Biotechnology, 35(1), 17–25. https://doi.org/10.1007/s10295-007-0261-1

41. Zong, X., Wen, L., Wang, Y., & Li, L. (2022). Research progress of glucoamylase with industrial potential. Journal of Food Biochemistry, 46(7). https://doi.org/10.1111/jfbc.14099

42. Rajagopalan, S., & Modak, J. M. (1995). Evaluation of relative growth limitation due to depletion of glucose and oxygen during fungal growth on a spherical solid particle. Chemical Engineering Science, 50(5), 803–811. https://doi.org/10.1016/0009-2509(94)00452-w

43. Witteveen, C. F. B., & Visser, J. (1995). Polyol pools inAspergillus niger. FEMS Microbiology Letters, 134(1), 57–62. https://doi.org/10.1111/j.1574-6968.1995.tb07914.x

44. Okuda, M. (2019). Rice used for Japanese sake making. Bioscience, Biotechnology, and Biochemistry, 83(8), 1428–1441. https://doi.org/10.1080/09168451.2019.1574552

45. Kanauchi, M. (2013). SAKE alcoholic beverage production in Japanese food industry. Food industry, 39–63. http://dx.doi.org/10.5772/53153

46. Yoshida, T. (2018). Technology development of saké fermentation in Japan. In The First International Symposium on Insight into the World of Indigenous Fermented Foods for Technology Development.

47. Suto, M., & Kawashima, H. (2024). Water-soluble ions and nitrogen and oxygen stable isotope ratios in nitrate in sake in Akita, Japan. LWT, 198, 115963. https://doi.org/10.1016/j.lwt.2024.115963

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2025-07-23

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Koji – the only one domesticated fungi: characterization, enzymology, and use. (2025). Scifood, 19(1), 426-436. https://doi.org/10.5219/scifood.63

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