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Identification and Functional Characterization of an α-Amylase with Broad Temperature and pH Stability from Paenibacillus sp.

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Abstract

Amylases are important industrial enzymes that have been applied widely in the food, detergent, and pulp industries and fermentation processes. In the present study, a gene encoding an alpha-amylase from the genomic DNA library of Paenibacillus sp. was identified and characterized. The amylase gene designated amy1 was shown to consist of 1,980 bp and shared sequence identity towards α-amylase genes from other Bacillus sp. The deduced amino acid sequence for Amy1 indicated 80 % sequence identity with other Bacillus strains. Heterologous expression of recombinant Amy1 in Escherichia coli BL21(DE3) facilitated the recovery of this protein in soluble form. Enzyme kinetic data revealed Amy1 to have a K m of 23.83 mg/mL and K cat of 48.74 min−1 and K cat /K m of 2 min−1 mg−1 mL−1 for starch. The activity of this protein was found to be enhanced by Mn2+, and furthermore, Amy1 remained active at a broad pH range (4–10) and temperature (30–90 °C). The ability of Amy1 to act on food waste under broad temperature and pH conditions, together with its ability to produce simple sugars, shows many advantages for further application in the food industry.

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References

  1. Amann, E., Ochs, B., & Abel, K. J. (1988). Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene, 69, 301–314.

    Article  CAS  Google Scholar 

  2. Anto, H., Trivedi, U., & Patel, K. (2006). α-Amylase production by Bacillus cereus MTCC 1305 using solid-state fermentation. Food Technology and Biotechnology, 44, 241–245.

    CAS  Google Scholar 

  3. Arikan, B. (2008). Highly thermostable, thermophilic, alkaline, SDS and chelator resistant amylase from a thermophilic Bacillus sp. isolate A3-15. Bioresource Technology, 99, 3071–3076.

    Article  CAS  Google Scholar 

  4. Asgher, M., Asad, M. J., Rahman, S. U., & Legge, R. L. (2007). A thermostable α-amylase from a moderately thermophilic Bacillus subtilis strain for starch processing. Journal of Food Process Engineering, 79, 950–955.

    Article  CAS  Google Scholar 

  5. Burhan, A., Nisa, U., Gokhan, C., Omer, C., Ashabil, A., & Osman, G. (2003). Enzymatic properties of a novel thermophilic, alkaline and chelator resistant amylase from an alkalophilic Bacillus sp. isolate ANT-6. Process Biochemistry, 38, 1397–1403.

    Article  CAS  Google Scholar 

  6. Gangadharan, D., Sivaramakrishnan, S., Nampoothiri, K. M., Sukumaran, R. K., & Pandey, A. (2008). Response surface methodology for the optimization of alpha amylase production by Bacillus amyloliquefaciens. Bioresource Technology, 99, 4597–4602.

    Article  CAS  Google Scholar 

  7. Goyal, N., Gupta, J. K., & Soni, S. K. (2005). A novel raw starch digesting thermostable α-amylase from Bacillus sp.I-3 and its use in the direct hydrolysis of raw potato starch. Enzyme and Microbial Technology, 37, 723–734.

    Article  CAS  Google Scholar 

  8. 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 

  9. Konsoula, Z., & Liakopoulou-Kyriakides, M. (2007). Co-production of alpha-amylase and beta-galactosidase by Bacillus subtilis in complex organic substrates. Bioresource Technology, 98, 150–157.

    Article  CAS  Google Scholar 

  10. Morgan, F. J., & Priest, F. G. (1981). Characterization of thermostable α-amylase from Bacillus licheniformis NCTB 6346. Journal of Applied Bacteriology, 50, 104–114.

    Article  Google Scholar 

  11. Nielsen, J. E., & Borchert, T. V. (2000). Protein engineering of bacterial α-amylases. Biochimica et Biophysica Acta, 1543, 253–274.

    Article  CAS  Google Scholar 

  12. Pancha, I., Jain, D., Shrivastav, A., Mishra, S., Shethia, B., Mishra, S., et al. (2010). A thermoactive α-amylase from a Bacillus sp. isolated from CSMCRI salt farm. International Journal of Biological Macromolecules, 47, 288–91.

    Article  CAS  Google Scholar 

  13. Prakash, O., & Jaiswal, N. (2009). Alpha-amylase: an ideal representative of thermostable enzymes. Applied Biochemistry and Biotechnology, 160, 2401–14.

    Article  Google Scholar 

  14. Rajagopalan, G., & Krishnan, C. (2010). Hyper-production of alpha-amylase from agro-residual medium with high-glucose in SSF using catabolite derepressed Bacillus subtilis KCC103. Journal of Basic Microbiology, 50, 336–343.

    Article  CAS  Google Scholar 

  15. Sajedi, R. H., Naderi-Manesh, H., Khajeh, K., Ahmadvand, R., Ranjbar, B., Asoodeh, A., et al. (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 

  16. Sambrook, J., Fritsch, E., & Maniatis, T. (1998). Molecular cloning: a laboratory manual (2nd ed.). Cold Spring Harbor: Cold Spring Harbor Laboratory.

    Google Scholar 

  17. Saxena, R. K., Dutt, K., Agarwal, L., & Nayyar, P. (2007). A highly thermostable and alkaline amylase from a Bacillus sp. PN5. Bioresource Technology, 98, 260–265.

    Article  CAS  Google Scholar 

  18. Shanmughapriya, S., Kiran, G. S., Selvin, J., Gandhimathi, R., Baskar, T. B., Manilal, A., et al. (2009). Optimization, production, and partial characterization of an alkalophilic amylase produced by sponge associated marine bacterium Halobacterium salinarum MMD047. Biotechnology and Bioprocess Engineering, 14, 67–75.

    Article  CAS  Google Scholar 

  19. Sodhi, H. K., Sharma, K., Gupta, J. K., & Soni, S. K. (2005). Production of a thermostable α-amylase from Bacillus sp. PS-7 by solid state fermentation and its synergistic use in the hydrolysis of malt starch for alcohol production. Process Biochemistry, 40, 525–534.

    Article  CAS  Google Scholar 

  20. Souza, P. M., & Magalhães, P. O. (2010). Application of microbial α-amylase in industry—a review. Brazilian Journal of Microbiology, 41, 850–861.

    Google Scholar 

  21. Svensson, B. (1994). Protein engineering in the α-amylase family: catalytic mechanism, substrate specificity, and stability. Plant Molecular Biology, 25, 141–157.

    Article  CAS  Google Scholar 

  22. Tanyildizi, M. S., Ozer, D., & Elibol, M. (2005). Optimization of α-amylase production by Bacillus sp. using response surface methodology. Process Biochemistry, 40, 2291–2296.

    Article  CAS  Google Scholar 

  23. Tsusaki, K., Watanabe, H., Yamamoto, T., Nishimoto, T., Chaen, H., & Fukuda, S. (2012). Purification and characterization of highly branched α-glucan-producing enzymes from Paenibacillus sp. PP710. Bioscience, Biotechnology, and Biochemistry, 76, 721–731.

    Article  CAS  Google Scholar 

  24. Ul-Haq, I., Hameed, U., Mahmood, Z., & Javed, M. M. (2012). Solid state fermentation for the production of α-amylase by Paenibacillus amylolyticus. Pakistan Journal of Botany, 44, 341–346.

    Google Scholar 

  25. van der Maarel, M. J., van der Veen, B., Uitdehaag, J. C., Leemhuis, H., & Dijkhuizen, L. (2002). Properties and applications of starch-converting enzymes of the alpha-amylase family. Journal of Biotechnology, 94, 137–155.

    Article  Google Scholar 

  26. Yang, Y. H., Joo, H. S., Lee, K., Liou, K. K., Lee, H. C., Sohng, J. K., et al. (2005). Novel method for detection of butanolides in Streptomyces coelicolor culture broth, using a His-tagged receptor (ScbR) and mass spectrometry. Applied and Environmental Microbiology, 71, 5050–5055.

    Article  CAS  Google Scholar 

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Acknowledgments

This work at Konkuk University was partially supported by a National Research Foundation of Korea grant funded by the Korean Government (NRF-2011-619-E0002). This subject is also partially supported by the Korea Ministry of Environment as a “Converging Technology Project (201-101-007)” and as an “Eco-Innovation Project (405-112-0382).” This research was also supported by the 2012 KU Brain Pool of Konkuk University.

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Correspondence to Yung-Hun Yang.

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Thangamani Rajesh and Yong Hyun Kim contributed equally to this work.

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Rajesh, T., Kim, Y.H., Choi, YK. et al. Identification and Functional Characterization of an α-Amylase with Broad Temperature and pH Stability from Paenibacillus sp.. Appl Biochem Biotechnol 170, 359–369 (2013). https://doi.org/10.1007/s12010-013-0197-z

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