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In Vivo Regulation of Alcohol Dehydrogenase and Lactate Dehydrogenase in Rhizopus Oryzae to Improve l-Lactic Acid Fermentation

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Abstract

Rhizopus oryzae is becoming more important due to its ability to produce an optically pure l-lactic acid. However, fermentation by Rhizopus usually suffers from low yield because of production of ethanol as a byproduct. Limiting ethanol production in living immobilized R. oryzae by inhibition of alcohol dehydrogenase (ADH) was observed in shake flask fermentation. The effects of ADH inhibitors added into the medium on the regulation of ADH and lactate dehydrogenase (LDH) as well as the production of cell biomass, lactic acid, and ethanol were elucidated. 1,2-diazole and 2,2,2-trifluroethanol were found to be the effective inhibitors used in this study. The highest lactic acid yield of 0.47 g/g glucose was obtained when 0.01 mM 2,2,2-trifluoroethanol was present during the production phase of the pregrown R. oryzae. This represents about 38% increase in yield as compared with that from the simple glucose fermentation. Fungal metabolism was suppressed when iodoacetic acid, N-ethylmaleimide, 4,4′-dithiodipyridine, or 4-hydroxymercury benzoic acid were present. Dramatic increase in ADH and LDH activities but slight change in product yields might be explained by the inhibitors controlling enzyme activities at the pyruvate branch point. This showed that in living R. oryzae, the inhibitors regulated the flux through the related pathways.

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References

  1. Wang, L., Zhao, B., Liu, B., Yu, B., Ma, C., Su, F., et al. (2010). Efficient production of l-lactic acid from corncob molasses, a waste by-product in xylitol production, by a newly isolated xylose utilizing Bacillus sp. strain. Bioresource Technology, 101, 7908–7915.

    Article  CAS  Google Scholar 

  2. Thongchul, N., & Yang, S.-T. (2003). Controlling filamentous fungal morphology by immobilization on a rotating fibrous matrix to enhance oxygen transfer and L(+)-lactic acid production by Rhizopus oryzae. In B. C. Saha (Ed.), Fermentation biotechnology (pp. 36–51). Oxford: Oxford University Press.

    Chapter  Google Scholar 

  3. Wang, L., Zhao, B., Li, F., Xu, K., Ma, C., Tao, F., et al. (2010). Highly efficient production of d-lactate by Sporolactobacillus sp. CASD with simultaneous enzymatic hydrolysis of peanut meal. Applied Microbiology and Biotechnology. doi:10.1007/s00253-010-2904-9.

    Google Scholar 

  4. Hofvendahl, K., & Hahn-Hagerdal, B. (2000). Factor affecting the fermentative lactic acid production from renewable resources. Enzyme and Microbial Technology, 26, 87–107.

    Article  CAS  Google Scholar 

  5. Wang, L., Zhao, B., Liu, B., Yang, C., Yu, B., Li, Q., et al. (2010). Efficient production of l-lactic acid from cassava powder by Lactobacillus rhamnosus. Bioresource Technology, 101, 7895–7901.

    Article  CAS  Google Scholar 

  6. Hsieh, C. M., Yang, F.-C., & Iannotti, E. L. (1999). The effect of soy protein hydrolyzates on fermentation by Lactobacillus amylovorus. Process Biochemistry, 34, 173–179.

    Article  CAS  Google Scholar 

  7. Silva, E. M., & Yang, S.-T. (1995). Continuous production of lactic acid from acid whey by Lactobacillus helveticus in a fibrous-bed bioreactor. Journal of Biotechnology, 41, 59–70.

    Article  CAS  Google Scholar 

  8. Tsai, S. P., & Moon, S.-H. (1998). An integrated bioconversion process for production of l-lactic acid from starchy potato feedstocks. Applied Biochemistry and Biotechnology, 70–72, 417–428.

    Article  Google Scholar 

  9. Woiciechowski, A. L., Soccol, C. R., Ramos, L. P., & Pandey, A. (1999). Experimental design to enhance the production of l-(+)-lactic acid from steam-exploded wood hydrolysate using Rhizopus oryzae in a mixed-acid fermentation. Process Biochemistry, 34, 949–955.

    Article  CAS  Google Scholar 

  10. Zhou, Y., Dominguez, J. M., Cao, N., Du, J., & Tsao, G. T. (1999). Optimization of l-lactic acid production from glucose by Rhizopus oryzae ATCC52311. Applied Biochemistry and Biotechnology, 77–79, 401–407.

    Article  Google Scholar 

  11. Cui, Y. Q., van der Lans, R. G. J. M., & Luyben, K. C. A. M. (1997). Effect of agitation intensities on fungal morphology of submerged fermentation. Biotechnology and Bioengineering, 55, 715–726.

    Article  CAS  Google Scholar 

  12. Cui, Y. Q., van der Lans, R. G. J. M., & Luyben, K. C. A. M. (1998). Effects of dissolved oxygen tension and mechanical forces on fungal morphology in submerged fermentation. Biotechnology and Bioengineering, 57, 409–419.

    Article  CAS  Google Scholar 

  13. Skory, C. D., Freer, S. N., & Bothast, R. J. (1998). Production of l-lactic acid by Rhizopus oryzae under oxygen limiting conditions. Biotechnology Letters, 20, 191–194.

    Article  CAS  Google Scholar 

  14. Bai, F., Wang, L., Huang, H., Xu, J., Caesar, J., Ridgway, D., et al. (2001). Oxygen mass-transfer performance of low viscosity gas-liquid-solid system in a split-cylinder airlift bioreactor. Biotechnology Letters, 23, 1109–1113.

    Article  CAS  Google Scholar 

  15. Bai, D.-M., Jia, M.-Z., Zhao, X.-M., Ban, R., Shen, F., Li, X.-G., et al. (2003). L(+)-lactic acid production by pellet-form Rhizopus oryzae R1021 in a stirred tank fermentor. Chemical Engineering Science, 58, 785–791.

    Article  CAS  Google Scholar 

  16. Dong, X. Y., Bai, S., & Sun, Y. (1996). Production of l(+)-lactic acid with Rhizopus oryzae immobilized in polyurethane foam cubes. Biotechnology Letters, 18, 225–228.

    Article  CAS  Google Scholar 

  17. Hang, Y. D., Hamamci, H., & Woodams, E. E. (1989). Production of l(+)-lactic acid by Rhizopus oryzae immobilized in calcium alginate gels. Biotechnology Letters, 11, 119–120.

    Article  CAS  Google Scholar 

  18. Park, E. Y., Kosakai, Y., & Okabe, M. (1998). Efficient production of l-(+)-lactic acid using mycelial cotton-like flocs of Rhizopus oryzae in an air-lift bioreactor. Biotechnology Progress, 14, 699–704.

    Article  CAS  Google Scholar 

  19. Sun, Y., Li, Y.-L., & Bai, S. (1999). Modeling of continuous l(+)-lactic acid production with immobilized R. oryzae in an airlift bioreactor. Biochemical Engineering Journal, 3, 87–90.

    Article  CAS  Google Scholar 

  20. Tamada, M., Begum, A. A., & Sadi, S. (1992). Production of l(+)-lactic acid by immobilized cells of Rhizopus oryzae with polymer supports prepared by gamma-ray-induced polymerization. Journal of Fermentation and Bioengineering, 74, 379–383.

    Article  CAS  Google Scholar 

  21. Yang, C. W., Lu, Z., & Tsao, G. T. (1995). Lactic acid production by pellet-form Rhizopus oryzae in a submerged system. Applied Biochemistry and Biotechnology, 51–52, 57–71.

    Article  Google Scholar 

  22. Yin, P., Yahiro, K., Ishigaki, T., Park, Y., & Okabe, M. (1998). l(+)-Lactic acid production by repeated batch culture of Rhizopus oryzae in air-lift bioreactor. Journal of Fermentation and Bioengineering, 85, 96–100.

    Article  CAS  Google Scholar 

  23. Taber, R. L. (1998). The competitive inhibition of yeast alcohol dehydrogenase by 2,2,2-trifluoroethanol. Biochemical Education, 26, 239–242.

    Article  CAS  Google Scholar 

  24. Zheng, S.-Y., Xu, D., Wang, H.-R., Li, J., & Zhou, H.-M. (1997). Kinetics of irreversible inhibition of yeast alcohol dehydrogenase during modification by 4,4′-dithiodipyridine. International Journal of Biological Macromolecules, 20, 307–313.

    Article  CAS  Google Scholar 

  25. Yoneya, T., & Sato, Y. (1979). Alcohol dehydrogenase from Rhizopus javanicus. Applied and Environmental Microbiology, 37, 1073–1078.

    CAS  Google Scholar 

  26. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. The Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  27. Pritchard, G. G. (1971). An NAD+-independent l-lactate dehydrogenase from Rhizopus oryzae. Biochimica et Biophysica Acta, 250, 25–34.

    CAS  Google Scholar 

  28. Skory, C. D. (2000). Isolation and expression of lactate dehydrogenase genes from Rhizopus oryzae. Applied and Environmental Microbiology, 66, 2343–2348.

    Article  CAS  Google Scholar 

  29. Singlevich, T. E., & Barboriak, J. J. (1971). Pyrazole-induced inhibition of yeast alcohol dehydrogenase. Biochemical Pharmacology, 20, 2087–2089.

    Article  CAS  Google Scholar 

  30. Thongchul, N., Navankasattusas, S., & Yang, S. T. (2010). Production of lactic acid and ethanol by Rhizopus oryzae integrated with cassava pulp hydrolysis. Bioprocess and Biosystems Engineering, 33, 407–416.

    Article  CAS  Google Scholar 

  31. Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., & Van Wart, H. E. (1990). Multiple modes of activation of latent human fibroblast collagenase: evidence for the role of a Cys73 active-site zinc complex in latency and a “cysteine switch” mechanism for activation. Biochemistry. Proceedings of the National Academy of Sciences. USA, 87, 364–368.

    Article  CAS  Google Scholar 

  32. Winslow, J. W. (1981). The reaction of sulfhydryl groups of sodium and potassium ion-activated adenosine triphosphatase with N-ethylmaleimide. The relationship between ligand-dependent alterations of nucleophilicity and enzymatic conformation states. The Journal of Biological Chemistry, 256, 9522–9531.

    CAS  Google Scholar 

  33. Chotisubha-anandha, N., Thitiprasert, S., Tolieng, V., & Thongchul, N. (2011). Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor. Bioprocess and Biosystems Engineering, 34, 163–172.

    Article  CAS  Google Scholar 

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Acknowledgments

This work has been supported by Thailand Research Fund and the Graduate School of Chulalongkorn University via the TRF Master Research Grant (MRG-WII51E053). Partial funding from the National Research University Project of Commission on Higher Education (CHE) and the Ratchadapiseksomphot Endowment Fund (AM1026A) is also acknowledged. The authors appreciated US Department of Agriculture for providing the fungal strain used in this study. We thank Professor Shang-Tian Yang, Ph.D. (Department of Chemical and Biomolecular Engineering, The Ohio State University, USA) for his valuable guidance and comments throughout lactic acid fermentation study. English proof reading and fruitful suggestion provided by Professor Anthony Whalley, Ph.D. (School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, UK) are highly appreciated.

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Authors and Affiliations

  1. Biotechnology Program, Faculty of Science, Chulalongkorn University, Bangkok, Thailand

    Sitanan Thitiprasert

  2. Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, Bangkok, Thailand

    Sarintip Sooksai & Nuttha Thongchul

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  1. Sitanan Thitiprasert
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  2. Sarintip Sooksai
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  3. Nuttha Thongchul
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Correspondence to Nuttha Thongchul.

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Thitiprasert, S., Sooksai, S. & Thongchul, N. In Vivo Regulation of Alcohol Dehydrogenase and Lactate Dehydrogenase in Rhizopus Oryzae to Improve l-Lactic Acid Fermentation. Appl Biochem Biotechnol 164, 1305–1322 (2011). https://doi.org/10.1007/s12010-011-9214-2

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  • DOI: https://doi.org/10.1007/s12010-011-9214-2

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