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Amylolytic Enzymes Acquired from L-Lactic Acid Producing Enterococcus faecium K-1 and Improvement of Direct Lactic Acid Production from Cassava Starch

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Abstract

An amylolytic lactic acid bacterium isolate K-1 was isolated from the wastewater of a cassava starch manufacturing factory and identified as Entercoccus faecium based on 16S rRNA gene sequence analysis. An extracellular α-amylase was purified to homogeneity and the molecular weight of the purified enzyme was approximately 112 kDa with optimal pH value and temperature measured of 7.0 and 40 °C, respectively. It was stable at a pH range of 6.0–7.0, but was markedly sensitive to high temperatures and low pH conditions, even at a pH value of 5. Ba2+, Al3+, and Co2+ activated enzyme activity. This bacterium was capable of producing 99.2% high optically pure L-lactic acid of 4.3 and 8.2 g/L under uncontrolled and controlled pH at 6.5 conditions, respectively, in the MRS broth containing 10 g/L cassava starch as the sole carbon source when cultivated at 37 °C for 48 h. A control pH condition of 6.5 improved and stabilized the yield of L-lactic acid production directly from starch even at a high concentration of starch at up to 150 g/L. This paper is the first report describing the properties of purified α-amylase from E. faecium. Additionally, pullulanase and cyclodextrinase activities were also firstly recorded from E. faecium K-1.

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References

  1. Bano, S., Qader, S. A. U., Aman, A., Syed, M. N., & Azhar, A. (2011). Purification and characterization of novel alpha-amylase from Bacillus subtilis KIBGE HAS. AAPS PharmSciTech, 12, 255–261.

    Article  CAS  Google Scholar 

  2. Hmidet, N., Maalej, H., Haddar, A., & Nasri, M. (2010). A novel alpha-amylase from Bacillus mojavensis A21: purification and biochemical characterization. Applied Biochemistry and Biotechnology, 162, 1018–1030.

    Article  CAS  Google Scholar 

  3. Morlon-Guyot, J., Mucciolo-Roux, F., Sanoja, R. R., & Guyot, J. P. (2001). Characterization of the L. manihotivorans alpha-amylase gene. DNA Sequence, 12, 27–37.

    Article  CAS  Google Scholar 

  4. Vishnu, C., Seenayya, G., & Reddy, G. (2000). Direct conversion of starch to L(+) lactic acid by amylase producing Lactobacillus amylophilus GV6. Bioprocess Engineering, 23, 155–158.

    Article  CAS  Google Scholar 

  5. Giraud, E., Champailler, A., & Raimbault, M. (1994). Degradation of raw starch by a wild amylolytic strain of Lactobacillus plantarum. Applied and Environmental Microbiology, 60, 4319–4323.

    CAS  Google Scholar 

  6. Freer, S. N. (1993). Purification and characterization of the extracellular alpha-amylase from Streptococcus bovis JB1. Applied and Environmental Microbiology, 59, 1398–1402.

    CAS  Google Scholar 

  7. Petrov, K., Urshev, Z., & Petrova, P. (2008). L(+)-lactic acid production from starch by a novel amylolytic Lactococcus lactis subsp lactis B84. Food Microbiology, 25, 550–557.

    Article  CAS  Google Scholar 

  8. Kanpiengjai, A., Rieantrakoonchai, W., Pratanaphon, R., Pathom-aree, W., Lumyong, S., & Khanongnuch, C. (2014). High efficacy bioconversion of starch to lactic acid using an amylolytic lactic acid bacterium isolated from Thai indigenous fermented rice noodles. Food Science and Biotechnology, 23, 1541–1550.

    Article  CAS  Google Scholar 

  9. Shibata, K., Flores, D. M., Kobayashi, G., & Sonomoto, K. (2007). Direct L-lactic acid fermentation with sago starch by a novel amylolytic lactic acid bacterium, Enterococcus faecium. Enzyme and Microbial Technology, 41, 149–155.

    Article  CAS  Google Scholar 

  10. Aguilar, G., Morlon-Guyot, J., Trejo-Aguilar, B., & Guyot, J. P. (2000). Purification and characterization of an extracellular alpha amylase produced by Lactobacillus manihotivorans LMG 18010, an amylolytic lactic acid bacterium. Enzyme and Microbial Technology, 27, 406–413.

    Article  CAS  Google Scholar 

  11. Kanpiengjai, A., Lumyong, S., Nguyen, T.-H., Haltrich, D., & Khanongnuch, C. (2015). Characterization of a maltose-forming α-amylase from an amylolytic lactic acid bacterium Lactobacillus plantarum S21. Journal of Molecular Catalysis B: Enzymatic, 120, 1–8.

    Article  CAS  Google Scholar 

  12. Sambrook, J., & Russell, D. W. (2001). Molecular clonging: a laboratory mannual (3rd ed.). New York: Cold Spring Harbor Press.

    Google Scholar 

  13. Lane, D. J. (1991). in Nucleic acid techniques in bacterial systematic, (Stackebrandt & Goodfellow, M., eds.), Chichester, United Kingdom, pp. 115–175.

  14. Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W., & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389–3402.

    Article  CAS  Google Scholar 

  15. Tamura, K., Dudley, J., Nei, M., & Kumar, S. (2007). MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 24, 1596–1599.

    Article  CAS  Google Scholar 

  16. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28, 350–356.

    Article  CAS  Google Scholar 

  17. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.

    Article  CAS  Google Scholar 

  18. Mussatto, S. I., Fernandes, M., Mancilha, I. M., & Roberto, I. C. (2008). Effects of medium supplementation and pH control on lactic acid production from brewer’s spent grain. Biochemical Engineering Journal, 40, 437–444.

    Article  CAS  Google Scholar 

  19. Guyot, J.-P., Calderon, M., & Morlon-Guyot, J. (2000). Effect of pH control on lactic acid fermentation of starch by Lactobacillus manihotivorans LMG 18010T. Journal of Applied Microbiology, 88, 176–182.

    Article  CAS  Google Scholar 

  20. Petrova, P., Petrov, K., & Stoyancheva, G. (2013). Starch-modifying enzymes of lactic acid bacteria—structures, properties, and applications. Starch-Stärke, 65, 34–47.

    Article  CAS  Google Scholar 

  21. Vishnu, C., Naveena, B. J., Altaf, M., Venkateshwar, M., & Reddy, G. (2006). Amylopullulanase—a novel enzyme of L. amylophilus GV6 in direct fermentation of starch to L(+) lactic acid. Enzyme and Microbial Technology, 38, 545–550.

    Article  CAS  Google Scholar 

  22. Wasko, A., Polak-Berecka, M., & Targonski, Z. (2011). Purification and characterization of pullulnase from Lactococcus lactis. Preparative Biochemistry & Biotechnology, 41, 252–261.

    Article  CAS  Google Scholar 

  23. Eom, H. J., Moon, J. S., Seo, E. Y., & Han, N. S. (2009). Heterologous expression and secretion of Lactobacillus amylovorus alpha-amylase in Leuconostoc citreum. Biotechnology Letters, 31, 1783–1788.

    Article  CAS  Google Scholar 

  24. Wasko, A. (2010). A new protein of α-amylase activity from Lactococcus lactis. Journal of Microbiology and Biotechnology, 20, 1307–1313.

    Article  CAS  Google Scholar 

  25. Dheeran, P., Kumar, S., Jaiswal, Y. K., & Adhikari, D. K. (2010). Characterization of hyperthermostable alpha-amylase from Geobacillus sp. IIPTN. Applied Microbiology and Biotechnology, 86, 1857–1866.

    Article  CAS  Google Scholar 

  26. Sajedi, R. H., Naderi-Manesh, H., Khajeh, K., Ahmadvand, R., Ranjbar, B., Asoodeh, A., & Moradian, F. (2005). A Ca-independent α-amylase that is active and stable at low pH from the Bacillus sp. KR-8104. Enzyme and Microbial Technology, 36, 666–671.

    Article  CAS  Google Scholar 

  27. Mollania, N., Khajeh, K., Hosseinkhani, S., & Dabirmanesh, B. (2010). Purification and characterization of a thermostable phytate resistant α-amylase from Geobacillus sp. LH8. International Journal of Biological Macromolecules, 46, 27–36.

    Article  CAS  Google Scholar 

  28. Uma Maheswar Rao, J. L., & Satyanarayana, T. (2007). Purification and characterization of a hyperthermostable and high maltogenic α-amylase of an extreme thermophile Geobacillus thermoleovorans. Applied Biochemistry and Biotechnology, 142, 179–193.

    Article  CAS  Google Scholar 

  29. Doukyu, N., Yamagishi, W., Kuwahara, H., Ogino, H., & Furuki, N. (2007). Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25. Extremophiles, 11, 781–788.

    Article  CAS  Google Scholar 

  30. Vishnu, C., Seenayya, G., & Reddy, G. (2002). Direct fermentation of various pure and crude starchy substrates to L (+) lactic acid using Lactobacillus amylophilus GV6. World Journal of Microbiology and Biotechnology, 18, 429–433.

    Article  CAS  Google Scholar 

  31. Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K., & Chauhan, B. (2003). Microbial α-amylases: a biotechnological perspective. Process Biochemistry, 38, 1599–1616.

    Article  CAS  Google Scholar 

  32. Lam, M. M., Seemann, T., Bulach, D. M., Gladman, S. L., Chen, H., Haring, V., Moore, R. J., Ballard, S., Grayson, M. L., & Johnson, P. D. (2012). Comparative analysis of the first complete Enterococcus faecium genome. Journal of Bacteriology, 194, 2334–2341.

    Article  CAS  Google Scholar 

  33. Lam, M. M., Seemann, T., Tobias, N. J., Chen, H., Haring, V., Moore, R. J., Ballard, S., Grayson, L. M., Johnson, P. D., & Howden, B. P. (2013). Comparative analysis of the complete genome of an epidemic hospital sequence type 203 clone of vancomycin-resistant Enterococcus faecium. BMC Genomics, 14, 595.

    Article  CAS  Google Scholar 

  34. Qin, X., Galloway-Peña, J. R., Sillanpaa, J., Roh, J. H., Nallapareddy, S. R., Chowdhury, S., Bourgogne, A., Choudhury, T., Muzny, D. M., Buhay, C. J., Ding, Y., Dugan-Rocha, S., Liu, W., Kovar, C., Sodergren, E., Highlander, S., Petrosino, J. F., Worley, K. C., Gibbs, R. A., Weinstock, G. M., & Murray, B. E. (2012). Complete genome sequence of Enterococcus faecium strain TX16 and comparative genomic analysis of Enterococcus faecium genomes. BMC Microbiology, 12, 135.

    Article  CAS  Google Scholar 

  35. Kopit, L. M., Kim, E. B., Siezen, R. J., Harris, L. J., & Marco, M. L. (2014). Safety of the surrogate microorganism Enterococcus faecium NRRL B-2354 for use in thermal process validation. Applied and Environmental Microbiology, 80, 1899–1909.

    Article  Google Scholar 

Download references

Acknowledgment

This work was financially supported by Chiang Mai University’s 50th anniversary scholarship program. Authors also acknowledge JSPS-NRCT (Core-to-Core Program “Establishment of an International Research Core for New Bio-research Fields with Microbes from Tropical Areas”). Mr. Russell K. Hollis at English Department, Faculty of Humanity, Chiang Mai University, was also acknowledged for his kindness in English editing.

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Authors and Affiliations

  1. Division of Biotechnology, School of Agro-Industry, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, 50100, Thailand

    Kridsada Unban, Apinun Kanpiengjai & Chartchai Khanongnuch

  2. Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa, 761-0795, Japan

    Goro Takata & Keiko Uechi

  3. Department of Chemical Engineering, Systems Biology and Tissue Engineering Research Center, National Chung Cheng University, Minhsiung, Chiayi, 621, Taiwan

    Wen-Chien Lee

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  1. Kridsada Unban
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Correspondence to Chartchai Khanongnuch.

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Unban, K., Kanpiengjai, A., Takata, G. et al. Amylolytic Enzymes Acquired from L-Lactic Acid Producing Enterococcus faecium K-1 and Improvement of Direct Lactic Acid Production from Cassava Starch. Appl Biochem Biotechnol 183, 155–170 (2017). https://doi.org/10.1007/s12010-017-2436-1

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  • DOI: https://doi.org/10.1007/s12010-017-2436-1

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