Isolation and identification of Î±-amylase-producing bacteria from mangrove trees on the west coast of Qeshm Island (Konar siah) and investigation of some biochemical properties of Î±-amylases from Bacillus sp. HR10 and Bacillus sp. HR11

Saleki, Leila; ُShayesteh, Fatemeh

Isolation and identification of Î±-amylase-producing bacteria from mangrove trees on the west coast of Qeshm Island (Konar siah) and investigation of some biochemical properties of Î±-amylases from Bacillus sp. HR10 and Bacillus sp. HR11

Document Type : Original Research

Authors

Leila Saleki ¹

Fatemeh ُShayesteh ²

¹ MSc Student in Marine Biology, Department of Marine Biology, Faculty of Marine Science and Technology, Hormozgan University, Bandar Abbas, Iran

² Assistant Professor of Fisheries Science (Marine), Department of Marine Biology, Faculty of Marine Science and Technology, Hormozgan University, Bandar Abbas

Abstract

The aim of this study was to isolate and identify a-amylase-producing bacteria present in mangrove ecosystems on Qeshm Island, Hormozgan, Iran. Samples of mangrove leaves and roots were screened for a-amylase activity using Lugol’s solution. Crude extracts were prepared of positive samples, and their a-amylase activity was determined by the Bernfeld method. The two strains with the highest activity were identified by molecular analysis of their 16S rRNA genes. a-Amylase production and activity were optimized by varying temperature and pH. 46 bacterial strains were isolated from mangrove tree leaf and root samples. Of these, 28 strains were capable of producing a-amylase. 16S rRNA gene sequence analysis of two strains with the highest enzyme production identified them as Bacillus sp. strain HR10 and Bacillus sp. strain HR11. The optimum temperature for enzyme production was 35 and 30 °C for strains HR10 and HR11, respectively, and the optimum pH was pH 8 for both strains. The highest enzyme activity was observed at 70 °C and 60 °C for the HR10 and HR11 strains, respectively, and the optimum pH was pH 8 for both strains. In conclusion, we have shown that bacteria isolated from mangrove leaf and root samples are potential source of a-amylases, tolerating a wide range of temperature and pH. Such a-amylases may be of interest for use in environmentally friendly industries.

Keywords

α- amylase

Optimum temperature

16s rRNA

Mangrove

Bacillus

Qeshm Island

20.1001.1.23222115.1400.12.4.9.1

Subjects

Microbial biotechnology

1. Parte, S., V. Sirisha, and J. D’Souza, Biotechnological applications of marine enzymes from algae, bacteria, fungi, and sponges, in Advances in food and nutrition research. 2017, Elsevier. p. 75-106.
2. Qamar, M.K., Mangroves of the Active Indus Delta-Changes and their causes. 2009, National College of Buisness Administration & Economics, Lahore. p. p 3.
3. Kasawani, I., J. Kamaruzaman, and M. Nurun-Nadhirah, Biological diversity assessment of Tok Bali mangrove forest, Kelantan, Malaysia. WSEAS Transactions on Environment and Development, 2007. 3(2): p. 30-385.
4. Ghasemi, S., et al., A review of mangrove value and conservation strategy by local communities in Hormozgan province, Iran. Journal of American Science, 2010. 6(10): p. 329-338.
5. Alongi, D.M., Present state and future of the world's mangrove forests. Environmental conservation, 2002: p. 331-349.
6. Sahoo, K. and N. Dhal, Potential microbial diversity in mangrove ecosystems: a review. 2009.
7. Vazquez, P., et al., Phosphate-solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid coastal lagoon. Biology and Fertility of Soils, 2000. 30(5): p. 460-468.
8. Mohanraju, R. and R. Natarajan, Methanogenic bacteria in mangrove sediments, in The Ecology of Mangrove and Related Ecosystems. 1992, Springer. p. 187-193.
9. Harmsen, H., D. Prieur, and C. Jeanthon, Distribution of microorganisms in deep-sea hydrothermal vent chimneys investigated by whole-cell hybridization and enrichment culture of thermophilic subpopulations. Applied and Environmental Microbiology, 1997. 63(7): p. 2876.
10. Farha, A.K., et al., Phylogenetic diversity and biotechnological potentials of marine bacteria from continental slope of eastern Arabian Sea. Journal of Genetic Engineering and Biotechnology, 2018. 16(2): p. 253-258.
11. Gupta, R., et al., Microbial α-amylases: a biotechnological perspective. Process Biochemistry, 2003. 38(11): p. 1599-1616.
12. Nakagawa, Y., et al., Gene cloning and enzymatic characteristics of a novel γ-cyclodextrin-specific cyclodextrinase from alkalophilic Bacillus clarkii 7364. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 2008. 1784(12): p. 2004-2011.
13. Chandra, M.S., et al., Purification and characterization of highly thermostable α-amylase from thermophilic Alicyclobacillus acidocaldarius. Biotechnology and Bioprocess engineering, 2010. 15(3): p. 435-440.
14. Sen, S.K., et al., Characterizing novel thermophilic amylase producing bacteria from Taptapani hot spring, Odisha, India. Jundishapur journal of microbiology, 2014. 7(12).
15. Demirci, A., G. Izmirlioglu, and D. Ercan, Fermentation and enzyme technologies in food processing. Food processing: principles and applications. 2nd ed. New York: Wiley, 2014: p. 107-36.
16. Putri, A.Z. and T. Nakagawa, Microbial α-Amylases in the Industrial Extremozymes. Reviews in Agricultural Science, 2020. 8: p. 158-169.
17. Bhatt, B.M., U.B. Trivedi, and K.C. Patel, Extremophilic amylases: Microbial production and applications, in Microbial enzymes: Roles and applications in industries. 2020, Springer. p. 185-205.
18. Stefanello, C., et al., Starch digestibility, energy utilization, and growth performance of broilers fed corn-soybean basal diets supplemented with enzymes. Poultry science, 2015. 94(10): p. 2472-2479.
19. Gernaey, B., J.O.B. Sorbara, and P.H. Nielsen, Environmental assessment of amylase used as digestibility improvement factor for intensive chicken production in Brazil. Sustainability, 2018. 10(8): p. 2735.
20. Souza, P.M.d., Application of microbial α-amylase in industry-A review. Brazilian journal of microbiology, 2010. 41(4): p. 850-861.
21. Hussain, I., et al., A Review of the Microbiological Aspect of α-amylase Production. International Journal of Agriculture & Biology, 2013. 15(5).
22. Sivaramakrishnan, S., et al., a-Amylases from microbial sources–an overview on recent developments. Food Technol Biotechnol, 2006. 44(2): p. 173-184.
23. Gomathi, V., et al., Endophytic thraustochytrids in Mangrove leaves. Novus International Journal of Biotechnology & Bioscience
2012.
24. Susilowati, R., A. Sabdono, and I. Widowati, Isolation and characterization of bacteria associated with brown algae Sargassum spp. from Panjang Island and their antibacterial activities. Procedia Environmental Sciences, 2015. 23: p. 240-246.
25. Kathiresan, K., et al., Microbial enzyme activity in decomposing leaves of mangroves. Int J Adv Biotechnol Res, 2011. 2(3): p. 382-389.
26. Afrisham, S., et al., Characterization of a thermostable, CaCl2-activated and raw-starch hydrolyzing alpha-amylase from Bacillus licheniformis AT70: Production under solid state fermentation by utilizing agricultural wastes. Journal of Molecular Catalysis B: Enzymatic, 2016. 132: p. 98-106.
27. Ahmadi, A., et al., Purification of α-Amylase from Bacillus sp. GHA1 and its partial characterization. Journal of the Iranian Chemical Society, 2010. 7(2): p. 432-440.
28. Bernfeld, P., Amylases, alpha and beta. Methods in enzymology I, 1955: p. 149-158.
29. Abdelhai, M.H., H.A. Hassanin, and X. Sun, Comparative study of rapid DNA extraction methods of pathogenic bacteria. American Journal of Bioscience and Bioengineering, 2016. 4(1): p. 1.
30. Alimolaei, M. and M. Golchin, An efficient DNA extraction method for Lactobacillus casei, a difficult-to-lyse bacterium. Int J Enteric Pathog, 2016. 4(1): p. 1-6.
31. Boulares, M., et al., Characterisation and identification of spoilage psychotrophic Gram-negative bacteria originating from Tunisian fresh fish. Annals of microbiology, 2013. 63(2): p. 733-744.
32. Tamura, K., et al., MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular biology and evolution, 2007. 24(8): p. 1596-1599.
33. Prakash, B., et al., Production, purification, and characterization of two extremely halotolerant, thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101. Process Biochemistry, 2009. 44(2): p. 210-215.
34. Aullybux, A.A. and D. Puchooa, α-Amylase production on low-cost substrates by Naxibacter sp. isolated from mauritian soils. Microbiology Research Journal International, 2013: p. 478-491.
35. Karakaş, B., M. İnan, and M. Certel, Expression and characterization of Bacillus subtilis PY22 α-amylase in Pichia pastoris. Journal of Molecular Catalysis B: Enzymatic, 2010. 64(3-4): p. 129-134.
36. Ramesh, S. and N. Mathivanan, Screening of marine actinomycetes isolated from the Bay of Bengal, India for antimicrobial activity and industrial enzymes. World Journal of Microbiology and Biotechnology, 2009. 25(12): p. 2103-2111.
37. Toffin, L., et al., Molecular monitoring of culturable bacteria from deep-sea sediment of the Nankai Trough, Leg 190 Ocean Drilling Program. FEMS Microbiology Ecology, 2004. 48(3): p. 357-367.
38. Mansi, K., M. Reddy, and G. Reddy, Polyphasic taxonomic characterization of bacteria isolated from Chilka Lake, Odisha, India. 2013.
39. Du, R., et al., Purification and characterization of novel thermostable and Ca-independent α-amylase produced by Bacillus amyloliquefaciens BH072. International journal of biological macromolecules, 2018. 115: p. 1151-1156.
40. Castro, R.A., et al., Isolation and enzyme bioprospection of endophytic bacteria associated with plants of Brazilian mangrove ecosystem. SpringerPlus, 2014. 3(1): p. 1-9.
41. Mohapatra, B., M. Bapuji, and A. Sree, Production of industrial enzymes (amylase, carboxymethylcellulase and protease) by bacteria isolated from marine sedentary organisms. Acta Biotechnologica, 2003. 23(1): p. 75-84.
42. Sajitha, N., et al., Amylase from an estuarine Bacillus megaterium. Curr. Res. J. Biol. Sci, 2011. 3(2): p. 110-115.
43. Barros, F.F.C., et al., Production of enzymes from agroindustrial wastes by biosurfactant-producing strains of Bacillus subtilis. Biotechnology research international, 2013. 2013.
44. Kim, T.U., et al., Purification and characterization of a maltotetraose-forming alkaline (alpha)-amylase from an alkalophilic Bacillus strain, GM8901. Applied and environmental microbiology, 1995. 61(8): p. 3105.
45. Burhan, A., et al., Enzymatic properties of a novel thermostable, thermophilic, alkaline and chelator resistant amylase from an alkaliphilic Bacillus sp. isolate ANT-6. Process Biochemistry, 2003. 38(10): p. 1397-1403.
46. Lee, S.-P., et al., Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp. strain XAL601. Applied and Environmental Microbiology, 1994. 60(10): p. 3764.
47. Paul, J.S., et al., Parameter’s optimization and kinetics study of α-amylase enzyme of Bacillus sp. MB6 isolated from vegetable waste. Process Biochemistry, 2017. 52: p. 123-129.
48. Chakraborty, K., B. Bhattacharyya, and S. Sen, Purification and characterization of a thermostable α-amylase fromBacillus stearothermophilus. Folia microbiologica, 2000. 45(3): p. 207-210.
49. Deb, P., et al., Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001. SpringerPlus, 2013. 2(1): p. 1-12.
50. Aygan, A., et al., Highly thermostable and alkaline α-amylase from a halotolerant-alkaliphilic Bacillus sp. AB68. Brazilian Journal of Microbiology, 2008. 39(3): p. 547-553.
51. آزاده, ط., et al., خالص سازی و بررسی برخی خصوصیات بیوشیمیایی آنزیم آلفا آمیلاز مقاوم به حرارت باکتری Bacillus cereus.
52. قلندری, س., et al., بررسی خصوصیات ییو شیمیایی آنزیم α- آلفا آمیلاز از یاکتری ترموفیل Geobacillus Stearothermophilus, in علوم جانوران آبزی. 1392.
53. Yang, H., et al., Heterologous expression, biochemical characterization, and overproduction of alkaline α-amylase from Bacillus alcalophilus in Bacillus subtilis. Microbial cell factories, 2011. 10(1): p. 1-9.