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Heterologous Expression and Characterization of a Novel Mesophilic Maltogenic α-Amylase AmyFlA from Flavobacterium sp. NAU1659

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Abstract

A novel amylase AmyFlA from Flavobacterium sp. NAU1659, AmyFlA, was cloned and expressed in Esherichia coli. Based on phylogenetic and functional analysis, it was identified as a novel member of the subfamily GH13_46, sharing high sequence identity. The protein was predicted to consist of 620 amino acids, with a putative signal peptide of 25 amino acids. The enzyme was able to hydrolyze soluble starch with a specific activity of 352.97 U/mg at 50 °C in 50 mM phosphate buffer (pH 6.0). The Km and Vmax values of AmyFlA were respectively 3.15 mg/ml and 566.36 µmol·ml−1·min−1 under optimal conditions. Its activity towards starch was enhanced by 63% in the presence of 1 mM Ca2+, indicating that AmyFlA was a Ca2+-dependent amylase. Compared to the reported maltogenic amylases, AmyFlA produced a lower variety of intermediate oligosaccharides at the start of the reaction so that the product mixture contained a higher proportion of maltose. These results indicate that AmyFlA may be potential application value in the production of high-maltose syrup.

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References

  1. Wang, S., Li, C., Copeland, L., Niu, Q., & Wang, S. (2015). Starch retrogradation: A comprehensive review. Comprehensive Reviews in Food Science Food Safety, 14, 568–585.

    Article  CAS  Google Scholar 

  2. Sjöö, M., & Nilsson, L. (2017). Starch in food: Structure, function and applications. ed. Woodhead Publishing.

    Google Scholar 

  3. Pan, S., Ding, N., Ren, J., Gu, Z., Li, C., Hong, Y., Cheng, L., Holler, T. P., & Li, Z. (2017). Maltooligosaccharide-forming amylase: Characteristics, preparation, and application. Biotechnology Advances, 35, 619–632.

    Article  CAS  PubMed  Google Scholar 

  4. Smits, A., Kruiskamp, P., Van Soest, J., & Vliegenthart, J. (2003). The influence of various small plasticisers and malto-oligosaccharides on the retrogradation of (partly) gelatinised starch. Carbohydrate Polymers, 51, 417–424.

    Article  CAS  Google Scholar 

  5. Chegeni, M., & Hamaker, B. (2015). Induction of differentiation of small intestinal enterocyte cells by maltooligosaccharides. The FASEB Journal, 29, 596514.

    Article  Google Scholar 

  6. Ielux, J., Franco, O., Silva, M., Silvinski, T., Bloch Jr, C., Rigden, J., & de Sa, M. G. (2000). Purification, biochemical characterization and partial primary structure of a new a-amylase inhibitor from Secale cereale (rye). The International Journal of Biochemistry & Cell Biology, 32, 1195–1204.

    Article  Google Scholar 

  7. Fulton, D. C., Stettler, M., Mettler, T., Vaughan, C. K., Li, J., Francisco, P., Gil, M., Reinhold, H., Eicke, S., & Messerli, G. (2008). β-Amylase4, a noncatalytic protein required for starch breakdown, acts upstream of three active β-amylases in Arabidopsis chloroplasts. The Plant Cell, 20, 1040–1058.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kumar, P., & Satyanarayana, T. (2009). Microbial glucoamylases: Characteristics and applications. Critical Reviews in Biotechnology, 29, 225–255.

    Article  CAS  PubMed  Google Scholar 

  9. Tanaka, A., & Hoshino, E. (1999). Study on the substrate specificity of α-amylases that contribute to soil removal in detergents. Journal of Surfactants Detergents, 2, 193–199.

    Article  CAS  Google Scholar 

  10. Feller, G., & Gerday, C. (2003). Psychrophilic enzymes: Hot topics in cold adaptation. Nature Reviews Microbiology, 1, 200–208.

    Article  CAS  PubMed  Google Scholar 

  11. Gupta, N., Beliya, E., Paul, J. S., Tiwari, S., Kunjam, S., & Jadhav, S. K. (2021). Molecular strategies to enhance stability and catalysis of extremophile-derived α-amylase using computational biology. Extremophiles, 25, 221–233.

    Article  CAS  PubMed  Google Scholar 

  12. Cappelli, A., Oliva, N., & Cini, E. (2020). A systematic review of gluten-free dough and bread: Dough rheology, bread characteristics, and improvement strategies. Applied Sciences, 10, 6559.

    Article  CAS  Google Scholar 

  13. Zafar, A., Aftab, M. N., Iqbal, I., ud, Din, Z., & Saleem, M. A. (2019). Pilot-scale production of a highly thermostable α-amylase enzyme from Thermotoga petrophila cloned into E. coli and its application as a desizer in textile industry. RSC Advances, 9, 984–992.

  14. Escaramboni, B., Núñez, E. G. F., Carvalho, A. F. A., & de Oliva Neto, P. (2018). Ethanol biosynthesis by fast hydrolysis of cassava bagasse using fungal amylases produced in optimized conditions. Industrial Crops and Products, 112, 368–377.

    Article  CAS  Google Scholar 

  15. Shad, M., Hussain, N., Usman, M., Akhtar, M. W., & Sajjad, M. (2023). Exploration of computational approaches to predict the structural features and recent trends in α-amylase production for industrial applications. Biotechnology and Bioengineering, 120, 2092–2116.

    Article  CAS  PubMed  Google Scholar 

  16. Farooq, M. A., Ali, S., Hassan, A., Tahir, H. M., Mumtaz, S., & Mumtaz, S. (2021). Biosynthesis and industrial applications of α-amylase: A review. Archives of Microbiology, 203, 1281–1292.

    Article  CAS  PubMed  Google Scholar 

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

  18. Zinck, S. S., Christensen, S. J., Sørensen, O. B., Svensson, B., & Meyer, A. S. (2023). Importance of inactivation methodology in enzymatic processing of raw potato starch: NaOCl as efficient α-amylase inactivation agent. Molecules, 28, 2947.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jespersen, H. M., Ann MacGregor, E., Henrissat, B., Sierks, M. R., & Svensson, B. (1993). Starch-and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes. Journal of Protein Chemistry, 12, 791–805.

    Article  CAS  PubMed  Google Scholar 

  20. Drula, E., Garron, M. L., Dogan, S., Lombard, V., Henrissat, B., & Terrapon, N. (2022). The carbohydrate-active enzyme database: Functions and literature. Nucleic Acids Research, 50, 571–577.

    Article  Google Scholar 

  21. 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 α-amylase family. Journal of Biotechnology, 94, 137–155.

    Article  PubMed  Google Scholar 

  22. Janeček, Š, Svensson, B., & MacGregor, E. A. (2014). α-Amylase: An enzyme specificity found in various families of glycoside hydrolases. Cellular and Molecular Life Sciences, 71, 1149–1170.

    Article  PubMed  Google Scholar 

  23. Stam, M. R., Danchin, E. G., Rancurel, C., Coutinho, P. M., & Henrissat, B. (2006). Dividing the large glycoside hydrolase family 13 into subfamilies: Towards improved functional annotations of α-amylase-related proteins. Protein Engineering Design and Selection, 19, 555–562.

    Article  CAS  Google Scholar 

  24. Mareček, F., & Janeček, Š. (2022). A novel subfamily GH13_46 of the α-amylase family GH13 represented by the cyclomaltodextrinase from Flavobacterium sp. 92. Molecules, 27, 8735.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Brown, H. A., DeVeaux, A. L., Juliano, B. R., Photenhauer, A. L., Boulinguiez, M., Bornschein, R. E., Wawrzak, Z., Ruotolo, B. T., Terrapon, N., & Koropatkin, N. M. (2023). BoGH13ASus from Bacteroides ovatus represents a novel α-amylase used for Bacteroides starch breakdown in the human gut. Cellular and Molecular Life Sciences, 80, 232.

    Article  CAS  PubMed  Google Scholar 

  26. Zhou, J., Li, Z., Zhang, H., Wu, J., Ye, X., Dong, W., Jiang, M., Huang, Y., & Cui, Z. (2018). Novel maltogenic amylase CoMA from Corallococcus sp. strain EGB catalyzes the conversion of maltooligosaccharides and soluble starch to maltose. Applied Environmental Microbiology, 84, 00152–00118.

    Article  ADS  Google Scholar 

  27. Min, B. C., Yoon, S. H., Kim, J. W., Lee, Y. W., Kim, Y. B., & Park, K. H. (1998). Cloning of novel maltooligosaccharide-producing amylases as antistaling agents for bread. Journal of Agricultural Food Chemistry, 46, 779–782.

    Article  CAS  PubMed  Google Scholar 

  28. Kim, Y. B., Yang, S. J., Lee, J. W., Cha, J., Hong, S. Y., Chung, S. H., Lee, S. T., & Rhim, S. L. (1996). Cloning and expression of an amylase gene from Streptomyces albus KSM-35 in Escherichia coli. Food Science Biotechnology, 5, 249–253.

    Google Scholar 

  29. Murakami, S., Nagasaki, K., Nishimoto, H., Shigematu, R., Umesaki, J., Takenaka, S., Kaulpiboon, J., Prousoontorn, M., Limpaseni, T., & Pongsawasdi, P. (2008). Purification and characterization of five alkaline, thermotolerant, and maltotetraose-producing α-amylases from Bacillus halodurans MS-2-5, and production of recombinant enzymes in Escherichia coli. Enzyme Microbial Technology, 43, 321–328.

    Article  CAS  Google Scholar 

  30. Auh, J. H., Lee, S. Y., Yoo, S. S., Son, H. J., Lee, J. W., Lee, S. J., Kim, Y. B., & Park, K. H. (2005). A novel maltopentaose-producing amylase as a bread antistaling agent. Food Science and Biotechnology, 14, 681–684.

    CAS  Google Scholar 

  31. Li, Z., Wu, J., Zhang, B., Wang, F., Ye, X., Huang, Y., Huang, Q., & Cui, Z. (2015). AmyM, a novel maltohexaose-forming α-amylase from Corallococcus sp. strain EGB. Applied Environmental Microbiology, 81, 1977–1987.

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Sun, Z., Li, D., Liu, P., Wang, W., Ji, K., Huang, Y., & Cui, Z. (2016). A novel L-asparaginase from Aquabacterium sp. A7-Y with self-cleavage activation. Antonie van Leeuwenhoek, 109, 121–130.

    Article  CAS  PubMed  Google Scholar 

  33. Yang, J., & Zhang, Y. (2015). I-TASSER server: New development for protein structure and function predictions. Nucleic Acids Research, 43, W174–W181.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Qin, Y., Huang, Z., & Liu, Z. (2014). A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: Molecular cloning, heterologous expression and biochemical characterization. Extremophiles, 18, 271–281.

    Article  CAS  PubMed  Google Scholar 

  35. Sanchez, A. C., Ravanal, M. C., Andrews, B. A., & Asenjo, J. A. (2019). Heterologous expression and biochemical characterization of a novel cold-active α-amylase from the antarctic bacteria Pseudoalteromonas sp. 2–3. Protein Expression and Purification, 155, 78–85.

  36. D’Elia, J. N. (1996). Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch. Journal of Bacteriology, 178, 7173–7179.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Fritzsche, H. B., Schwede, T., & Schulz, G. E. (2003). Covalent and three-dimensional structure of the cyclodextrinase from Flavobacterium sp. 92. European Journal of Biochemistry, 270, 2332–2341.

    Article  CAS  PubMed  Google Scholar 

  38. Dos Santos, F. C., & Barbosa-Tessmann, I. P. (2019). Recombinant expression, purification, and characterization of a cyclodextrinase from Massilia timonae Protein Expression and Purification, 154, 74–84.

    Article  CAS  Google Scholar 

  39. Janeček, Š. (2002). How many conserved sequence regions are there in the α-amylase family. Biologia, 57, 29–41.

    Google Scholar 

  40. 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 

  41. Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K. M., Soccol, C. R., & Pandey, A. (2006). α-Amylases from microbial sources–An overview on recent developments. Food Science and Biotechnology, 44, 173–184.

    CAS  Google Scholar 

  42. Song, J., Shen, X., Liu, F., Zhao, X., Wang, Y., Wang, S., Wang, P., Wang, J., Su, F., & Li, S. (2023). Ca2+-based metal-organic framework as enzyme preparation to promote the catalytic activity of amylase. Materials Today Chemistry, 30, 101522.

    Article  CAS  Google Scholar 

  43. Fan, Q., Zhang, L., Dong, C., Zhong, L., Fang, X., Huan, M., Ye, X., Huang, Y., Li, Z., & Cui, Z. (2021). Novel malto-oligosaccharide‐producing amylase AmyAc from Archangium sp. strain AC19 and its catalytic properties. Starch‐Stärke, 73, 2100114.

    Article  CAS  Google Scholar 

  44. Wang, J., Zhang, L., Wang, P., Lei, J., Zhong, L., Zhan, L., Ye, X., Huang, Y., Luo, X., & Cui, Z. (2023). Identification and characterization of novel malto-oligosaccharide-forming amylase AmyCf from Cystobacter sp. Strain CF23. Foods, 12, 3487.

  45. Wu, J., **a, B., Li, Z., Ye, X., Chen, Q., Dong, W., Zhou, J., Huang, Y., & Cui, Z. (2015). Molecular cloning and characterization of a novel GH13 saccharifying α-amylase AmyC from Corallococcus sp. EGB. Starch‐Stärke, 67, 810–819.

    Article  CAS  Google Scholar 

  46. Li, S., Zuo, Z., Niu, D., Singh, S., Permaul, K., Prior, B. A., Shi, G., & Wang, Z. (2011). Gene cloning, heterologous expression, and characterization of a high maltose-producing α-amylase of Rhizopus oryzae. Applied Biochemistry Biotechnology, 164, 581–592.

    Article  CAS  PubMed  Google Scholar 

  47. Dauter, Z., Dauter, M., Brzozowski, A. M., Christensen, S., Borchert, T. V., Beier, L., Wilson, K. S., & Davies, G. J. (1999). X-ray structure of Novamyl, the five-domain maltogenic α-amylase from Bacillus stearothermophilus: Maltose and acarbose complexes at 1.7 Å resolution. Biochemistry, 38, 8385–8392.

    Article  CAS  PubMed  Google Scholar 

  48. Kim, J. S., Cha, S. S., Kim, H. J., Kim, T. J., Ha, N. C., Oh, S. T., Cho, H. S., Cho, M. J., Kim, M. J., & Lee, H. S. (1999). Crystal structure of a maltogenic amylase provides insights into a catalytic versatility. Journal of Biological Chemistry, 274, 26279–26286.

    Article  CAS  PubMed  Google Scholar 

  49. Qi, Y., Geib, T., & Volmer, D. A. (2015). Determining the binding sites of β-cyclodextrin and peptides by electron-capture dissociation high resolution tandem mass spectrometry. Journal of the American Society for Mass Spectrometry, 26, 1143–1149.

    Article  ADS  CAS  PubMed  Google Scholar 

  50. An, Y., Tran, P. L., Yoo, M. J., Song, H. N., Park, K. H., Kim, T. J., Park, J. T., & Woo, E. J. (2023). The distinctive permutated domain structure of periplasmic α-amylase (MalS) from glycoside hydrolase family 13 subfamily 19. Molecules, 28, 3972.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Chen, X., Zhang, L., Li, X., Qiao, Y., Zhang, Y., Zhao, Y., Chen, J., Ye, X., Huang, Y., & Li, Z. (2020). Impact of maltogenic α-amylase on the structure of potato starch and its retrogradation properties. International Journal of Biological Macromolecules, 145, 325–331.

    Article  CAS  PubMed  Google Scholar 

  52. Zhang, L., Zhong, L., Wang, P., Zhan, L., Yangzong, Y., He, T., Liu, Y., Mao, D., Ye, X., & Cui, Z. (2023). Structural and functional properties of porous corn starch obtained by treating raw starch with AmyM. Foods, 12, 3157.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Zhang, L., Li, Z., Qiao, Y., Zhang, Y., Zheng, W., Zhao, Y., Huang, Y., & Cui, Z. (2019). Improvement of the quality and shelf life of wheat bread by a maltohexaose producing α-amylase. Journal of Cereal Science, 87, 165–171.

    Article  CAS  Google Scholar 

  54. Yang, T., Zhong, L., Jiang, G., Liu, L., Wang, P., Zhong, Y., Yue, Q., Ouyang, L., Zhang, A., & Li, Z. (2022). Comparative study on bread quality and starch digestibility of normal and waxy wheat (Triticum aestivum L.) modified by maltohexaose producing α-amylases. Food Research International, 162, 112034.

    Article  CAS  PubMed  Google Scholar 

  55. Kelly, C. T., Collins, B. S., Fogarty, W. N., & Doyle, E. M. (1993). Mechanisms of action of the α-amylase of Micromonospora melanosporea. Applied Microbiology Biotechnology, 39, 599–603.

    Article  CAS  Google Scholar 

  56. Collins, B. S., Kelly, C. T., Fogarty, W. M., & Doyle. (1993). The high maltose-producing α-amylase of the thermophilic actinomycete, Thermomonospora curvata. Applied Microbiology and Biotechnology, 39, 31–35.

    Article  CAS  PubMed  Google Scholar 

  57. Doyle, E. M., Kelly, C. T., & Fogarty, W. M. (1989). The high maltose-producing α-amylase of Penicillium Expansum Applied Microbiology Biotechnology, 30, 492–496.

    Article  CAS  Google Scholar 

  58. McMahon, H. E., Kelly, C. T., & Fogarty, W. M. (1999). High maltose-producing amylolytic system of a Streptomyces Sp. Biotechnology Letters, 21, 23–26.

    Article  CAS  Google Scholar 

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Funding

This work was supported by the Natural Science Foundation of China (32370119) and Liaocheng University (No 318052291).

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Authors and Affiliations

  1. College of Life Sciences of Liaocheng University, Liaocheng, 252000, People’s Republic of China

    Yanxin Wang, Huairen Xue & Zhensong Zhao

  2. Key Laboratory of Agricultural Environmental Microbiology, Ministry of Agriculture and Rural Affairs, College of Life Sciences of Nan**g Agricultural University, Nan**g, 210095, People’s Republic of China

    Yanxin Wang, Tingting **e, Guanhua Yan & **anfeng Ye

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  1. Yanxin Wang
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  2. Tingting **e
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Contributions

YX Wang and XF Ye designed the experiments and wrote the manuscript. YX Wang carried out the experiments. HR Xue and ZS Zhao provided help in the construction of the plasmid. TT **e and GH Yan helped to finish the experiments. XF Ye provided valuable suggestions for improving the experimental methods. All authors read and approved the final manuscript.

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Correspondence to **anfeng Ye.

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Wang, Y., **e, T., Yan, G. et al. Heterologous Expression and Characterization of a Novel Mesophilic Maltogenic α-Amylase AmyFlA from Flavobacterium sp. NAU1659. Appl Biochem Biotechnol (2024). https://doi.org/10.1007/s12010-024-04874-x

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