Screening and characterization of amylolytic Enterobacter cloacae FZAD and Aeromonas veronii FZD bacterial strains isolated from the fecal sample of spotted deer (Axis axis)
Bacterial amylase is being employed in numerous industries to hydrolyze the glycosidic bonds in starch and similar polysaccharide molecules. Although amylase-producing bacteria have already been isolated from a variety of different sources, a fecal sample from spotted deer (Axis axis) is unexplored. The present study was aimed at identifying and characterizing potential mesophilic amylase-producing bacteria. The spreading of a fecal sample on starch agar plates resulted in the screening of four bacterial isolates based on colony morphological differences. Out of these isolates, AD2 and AD4 showed a high zone of hydrolysis on starch agar plates. Bacterial isolates AD2 and AD4 were identified as Enterobacter cloacae FZAD (OR361741) and Aeromonas veronii FZD (OR226595), respectively, based on the biochemical and molecular characterization. AD2 showed maximum amylase production under optimum culture conditions of pH 6.0 and 45°C after 48 h of incubation, and for AD4, optimum conditions were pH 7.0, 40°C, and 60 h of incubation. The enzymes were characterized, and a maximum amylase activity of 14.13 U/mL for AD2 and 14.92 U/mL for AD4 was observed. The good amylase activity of these bacterial strains can be used in a number of industrial processes such as textiles, paper industries, brewing, and spot removers in dry cleaning.
Shaikh MS, Arab AI, Patel DB, Kumar K. Screening and characterization of amylolytic Enterobacter cloacae FZAD and Aeromonas veronii FZD bacterial strains isolated from the fecal sample of spotted deer (Axis axis). J App Biol Biotech. 2025. Article in Press. http://doi.org/10.7324/JABB.2025.247102
1. Yassin SN, Jiru TM, Indracanti M. Screening and characterization of thermostable amylase-producing bacteria isolated from soil samples of Afdera, Afar region, and molecular detection of amylase-coding gene. Int J Microbiol. 2021;1-14. https://doi.org/10.1155/2021/5592885
2. Eichler J. Biotechnological uses of archaeal extremozymes. Biotechnol Adv. 2001;19(4):261-78. https://doi.org/10.1016/s0734-9750(01)00061-1
3. Nguyen T, Loan T, Van Thuoc D. High amylase production by a novel strain of Bacillus amyloliquefaciens M37 isolated from can Gio mangrove forest, Vietnam. Biointerface Res Appl Chem. 2021;12(4):4675-85. https://doi.org/10.33263/briac124.46754685
4. Silaban S, Marika DB, Simorangkir M. Isolation and characterization of amylase-producing amylolytic bacteria from rice soil samples. J Phys Conf Ser. 2020;1485(1):012006. https://doi.org/10.1088/1742-6596/1485/1/012006
5. Van Der Maarel MJ, Van Der Veen B, Uitdehaag JC, Leemhuis H, Dijkhuizen L. Properties and applications of starch-converting enzymes of the α-amylase family. J Biotechnol. 2002;94(2):137-55. https://doi.org/10.1016/S0168-1656(01)00407-2
6. Joshi N, Andhare P, Marchawala F, Bhattacharya I, Upadhyay D. A study on amylase: Review. Int J Biol Pharm Allied Sci. 2021;10(4):333-40.
7. Benjamin S, Smitha RB, Jisha VN, Pradeep S, Sajith S, Sreedevi S, et al. A monograph on amylases from Bacillus spp. Adv Biosci Biotechnol. 2013;4(2):227-41. https://doi.org/10.4236/abb.2013.42032
8. Alves KJ, Da Silva MC, Cotta SR, Ottoni JR, Van Elsas JD, De Oliveira VM, et al. Mangrove soil as a source for novel xylanase and amylase as determined by cultivation-dependent and cultivation-independent methods. Braz J Microbiol. 2019;51(1):217-28. https://doi.org/10.1007/s42770-019-00162-7
9. Kafilzadeh F, Dehdari F. Amylase activity of aquatic actinomycetes isolated from the sediments of mangrove forests in south of Iran. J Aquat Res. 2015;41(2):197-201. https://doi.org/10.1016/j.ejar.2015.04.003
10. Paul JS, Gupta N, Beliya E, Tiwari S, Jadhav SK. Aspects and recent trends in microbial α-amylase: A review. Appl Biochem Biotechnol. 2021;193:2649-98.
11. Islam T. Isolation of Amylase Producing Bacteria from Soil and Identification by 16S rRNA Gene Sequencing and Characterization of Amylase. Dhaka, Bangladesh: BRAC University; 2016.
12. Ashwini K, Gaurav K, Karthik L, Bhaskara Rao KV. Optimization, production and partial purification of extracellular α-amylase from Bacillus sp. Marini. Arch Appl Sci Res. 2021;3:33-42.
13. Alves PD, de Faria Siqueira F, Facchin S, Horta CR, Victória MN, Kalapothakis E. Survey of microbial enzymes in soil, water, and plant microenvironments. Open Microbiol J. 2014;8:25-31. https://doi.org/10.2174/1874285801408010025
14. Abdullah R, Shaheen, N, Iqtedar M, Naz S, Iftikhar T. Optimization of cultural conditions for the production of α-amylase by Aspergillus niger (BTM-26) in solid state fermentation. Pak J Bot. 2014;46:1071-8.
15. Pandey A. Solid-state fermentation. Biochem Eng J. 2003;13:81-4. https://doi.org/10.1016/j.bej.2013.10.013
16. Saxena R, Singh R. Amylase production by solid-state fermentation of agro-industrial wastes using Bacillus sp. Braz J Microbiol. 2011;42(4):1334- 42. https://doi.org/10.1590/s1517-83822011000400014
17. Asad W, Asif M, Rasool S. Extracellular enzyme production by indigenous thermophilic bacteria: Partial purification and characterization of α-amylase by Bacillus sp. WA21. Pak J Bot. 2011;43:1045-52.
18. Abo-Kamer AM, Abd-El-Salam IS, Mostafa FA, Mustafa AE, Al- Madboly LA. A promising microbial α-amylase production, and purification from Bacillus cereus and its assessment as antibiofilm agent against Pseudomonas aeruginosa pathogen. Microb Cell Fact. 2023;22(1):141.
19. Anto H, Trivedi U, Patel K. α-Amylase production by Bacillus cereus MTCC 1305 using solid-state fermentation. Food Technol Biotechnol. 2006;44(2):241-5.
20. Grubb P. Artiodactyla. In: Species of the World. A Taxonomic and Geographic Reference. 3rd ed. Baltimore, USA: Johns Hopkins University Press; 2005. p. 637-722.
21. Raman TS. Chital Axis axis. In: Mammals of South Asia. Vol. 2. Hyderabad: Universities Press, 2015. p. 192-222.
22. Kanimozhi M, Johny M, Gayathri N, Subashkumar R. Optimization and production of α -amylase from Halophilic Bacillus species isolated from mangrove soil sources. J Appl Environ Microbiol. 2014;2(3):70-3. https://doi.org/10.12691/jaem-2-3-2
23. Ogbonna CN, Okpokwu NM, Okafor CU, Onyia CE. Isolation and screening of amylase producing fungi obtained from garri processing site. Int J Biotechnol Food Sci. 2014;2(5):88-93.
24. Aynadis TH, Tilahun BG, Gulelat DH. Thermostable alpha-amylase from geothermal sites of Ethiopia (Afar Region): Isolation, purification and characterization. Greener J Biol Sci. 2013;3:61-73. https://doi.org/10.15580/gjbs.2013.2.013113421
25. Bernfeld P. Amylases, α and β. Methods Enzymol 1955;1:149-58. https://doi.org/10.1016/0076-6879(55)01021-5
26. Vos P, Garrity G, Jones D, Krieg NR, Ludwig W, Rainey FA, et al. Bergey’s Manual of Systematic Bacteriology. New York: Springer- Verlag; 2011.
27. Bartholomew JW, Mittwer T. The Gram stain. Bacteriol Rev. 1952;16(1):1-29. https://doi.org/10.1128/br.16.1.1-29.1952
28. Biswas S, Saber MA, Tripty IA, Karim MA, Islam MA, Hasan MS, et al. Molecular characterization of cellulolytic (endo- and exoglucanase) bacteria from the largest mangrove forest (Sundarbans), Bangladesh. Ann Microbiol. 2020;70(1):68. https://doi.org/10.1186/s13213-020- 01606-4
29. Tille PM. Bailey and Scott’s Diagnostic Microbiology. Missouri: Mosby; 2021.
30. Roy B, Das T, Bhattacharyya S. Overview on old and new biochemical test for bacterial identification. J Surg Case Rep. 2014;6:23-8. https://doi.org/10.31579/2690-1897/163
31. Winn WC, Allen SC, Janda WM, Koneman EW, Procop GW, Schreckenberger P, et al. Color Atlas and Textbook of Diagnostic Microbiology. Philadelphia, PA: Lippincott Williams and Wilkins; 2006.
32. Swenson JM, Patel JB, Jorgensen JH. Special phenotypic methods for detecting Antibacterial resistance. In: Manual of Clinical Microbiology. Washington, DC: ASM Press; 2011.
33. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007;24(8):1596-9. https://doi.org/10.1093/molbev/msm092
34. Thompson J, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22(22):4673-80. https://doi.org/10.1093/nar/22.22.4673
35. Bozic N, Ruiz J, Lopez-Sant?n J, Vujcic Z. Production and properties of the highly efficient raw starch digesting α- amylase from a Bacillus licheniformis ATCC 9945. Biochem Eng J. 2011;53(2):203- 209. https://doi.org/10.1016/j.bej.2010.10.014
36. Sharif S, Shah AH, Fariq A, Jannat S, Rasheed S, Yasmin A. Optimization of amylase production using response surface methodology from newly isolated thermophilic bacteria. Heliyon. 2023;9(1):e12901. https://doi.org/10.1016/j.heliyon.2023.e12901
37. Wang J, Bao F, Wei H, Zhang Y. Screening of cellulose-degrading bacteria and optimization of cellulase production from Bacillus cereus A49 through response surface methodology. Sci Rep. 2024;14(1):7755. https://doi.org/10.1038/s41598-024-58540-7
38. Singh S, Moholkar VS, Goyal A. Isolation, identification, and characterization of a cellulolytic Bacillus amyloliquefaciens strain SS35 from rhinoceros dung. ISRN Microbiol. 2013;3:1-7.
39. Klinfoong R, Thummakasorn C, Ungwiwatkul S, Boontanom P, Chantarasiri A. Diversity and activity of amylase-producing bacteria isolated from mangrove soil in Thailand. Biodivers J. 2022;23(10):5519-5531. https://doi.org/10.13057/biodiv/d231064
40. Yin LJ, Huang PS, Lin HH. Isolation of cellulase-producing bacteria and characterization of the cellulase from the isolated bacterium Cellulomonas Sp. YJ5. J Agric Food Chem. 2010;58(17):9833-7. https://doi.org/10.1021/jf1019104
41. Miller GL. Use of dinitrosalisylic acid reagent for determination of reducing sugars. Anal Chem. 1959;31(3):426-428. https://doi.org/10.1021/ac60147a030
42. Kumar K, Rajulapati V, Goyal A. In vitro prebiotic potential, digestibility and biocompatibility properties of laminari-oligosaccharides produced from curdlan by β-1,3-endoglucanase from Clostridium thermocellum. 3 Biotech. 2020;10(6):241. https://doi.org/10.1007/s13205-020-02234-0
43. Chakraborty S, Khopade A, Kokare C, Mahadik K, Chopade B. Isolation and characterization of novel α-amylase from marine Streptomyces sp. D1. J Mol Catal B Enzym. 2008;58(1-4):17-23. https://doi.org/10.1016/j.molcatb.2008.10.011
44. Silva TM, De Oliveira M, Somera AF, Jorge JA, Terenzi HF, De Lourdes TM, et al. Thermostable saccharogenic amylase produced under submerged fermentation by filamentous fungus Penicillium purpurogenum. Braz J Microbiol. 2011;42(3):1136-40. https://doi.org/10.1590/s1517-83822011000300035
45. Medda S, Chandra AK. New strains of Bacillus licheniformis and Bacillus coagulans producing Thermostable α?amylase active at alkaline pH. J Appl Bacteriol. 1980;48(1):47-58. https://doi.org/10.1111/j.1365-2672.1980.tb05205.x
46. Suman S, Rani L, Hussain MZ, Kashyap S, Verma R. Biochemical analysis and optimization of Aeromonas and Bacillus species for α-amylase production isolated from soil samples of Ranchi, Jharkhand. Eur Chem Bull. 2022;11:1447-1453. https://doi.org/10.53555/ecb/2022.11.11.133
47. Konkit M, Kim W. Activities of amylase, proteinase, and lipase enzymes from Lactococcus chungangensis and its application in dairy products. J Dairy Sci. 2016;99(7):4999-5007. https://doi.org/10.3168/jds.2016-11002
48. Aiyer PV. Amylases and their applications. Afr J Biotechnol. 2005;4(13):1525-9. https://doi.org/10.5897/ajb2005.000-3267
49. Raul D, Biswas T, Mukhopadhyay S, Das SK, Gupta S. Production and partial purification of alpha amylase from Bacillus subtilis (MTCC 121) using solid state fermentation. Biochem Res Int. 2014;2014:568141. https://doi.org/10.1155/2014/568141
50. Kumar N, Das D. Production and purification of α-amylase from hydrogen producing Enterobacter cloacae IIT-BT 08. Bioprocess Eng. 2000;23(2):205-8. https://doi.org/10.1007/pl00009123
51. Ezebuiro V, Obichi EA, Minimah SO, Ezebuiro NC, Akinido E. Optimization of α-amylase production by Enterobacter cloacae strain D1 isolated from cassava effluent-impacted soil using response surface methodology. Asian J Biotechnol. 2022;8:68-80. https://doi.org/10.9734/ajb2t/2022/v8i4168
52. Uygut MA, Tanyildizi M?. Optimization of alpha-amylase production by Bacillus amyloliquefaciens grown on orange peels. J Sci Technol Trans A Sci. 2016;42(2):443-9. https://doi.org/10.1007/s40995-016- 0077-9
53. Nusrat A, Rahman SR. Comparative studies on the production of extracellular and alpha Amylase by three mesophilic Bacillus isolates. Bangladesh J Microbiol. 1970;24(2):129-32. https://doi.org/10.3329/bjm.v24i2.1257
54. Ikram-Ul-Haq N, Ashraf H, Iqbal J, Qadeer MA. Production of alpha amylase by Bacillus licheniformis using an economical medium. Bioresour Technol. 2002;87(1):57-61. https://doi.org/10.1016/s0960-8524(02)00198-0
55. Arnemo J, Kreeger T. Handbook of Wildlife Chemical Immobilization. 5th ed. United Kingdom: Sunquest Publishing; 2007.
56. Singh S, Bajaj BK. Medium optimization for enhanced production of protease with industrially desirable attributes from Bacillus subtilis K-1. Chem Eng Commun. 2014;202(8):1051-60. https://doi.org/10.1080/00986445.2014.900052
57. Fatokun E, Nwodo U, Okoh A. Classical optimization of cellulase and xylanase production by a marine Streptomyces species. Appl Sci. 2016;6(10):286. https://doi.org/10.3390/app6100286
58. Mojsov K. Application of enzymes in the textile industry: A review. In: II International Congress Engineering, Ecology and Materials in the Processing Industry. 2011. p. 230-9.
59. Dahiya P, Rathi B. Characterization and application of alkaline alpha-amylase from Bacillus licheniformis MTCC1483 as detergent additive. Int Food Res J. 2015;22(3):1293-7.
60. Gupta R, Gigras P, Mohapatra H, Goswami VK, Chauhan B. Microbial α-amylases: A biotechnological perspective. Process Biochem. 2003;38(11):1599-616. https://doi.org/10.1016/s0032-9592(03)00053-0
61. Samie N, Noghabi KA, Gharegozloo Z, Zahiri HS, Ahmadian G, Sharafi H, et al. Psychrophilic α-amylase from Aeromonas veronii NS07 isolated from farm soils. Process Biochem. 2012;47(9):1381- 7. https://doi.org/10.1016/j.procbio.2012.05.007
62. Ndlela LL, Schmidt S. Evaluation of wild herbivore faeces from South Africa as a potential source of hydrolytically active microorganisms. SpringerPlus. 2016;5(1):118. https://doi.org/10.1186/s40064-016- 1739-y
63. Luan L, Jiang Y, Dini-Andreote F, Crowther TW, Li P, Bahram M, et al. Integrating pH into the metabolic theory of ecology to predict bacterial diversity in soil. Proc Nat Acad Sci. 2023;120(3):e2207832120. https://doi.org/10.1073/pnas.2207832120
64. Padhi S, Reddy LK, Priyakumar UD. pH-mediated gating and formate transport mechanism in the Escherichia coli formate channel. Mol Simulat. 2017;43(13-16):1300-6. https://doi.org/10.1080/08927022.2017.1353691
65. Lü W, Du J, Wacker T, Gerbig-Smentek E, Andrade SL, Einsle O. PH-Dependent gating in a FOCA formate channel. Science. 2011;332(6027):352-4. https://doi.org/10.1126/science.1199098
66. Saad WF, Othman AM, Abdel-Fattah M, Ahmad MS. Response surface methodology as an approach for optimization of α-amylase production by the new isolated thermotolerant Bacillus licheniformis WF67 strain in submerged fermentation. Biocatal Agric Biotechnol. 2021;32:101944. https://doi.org/10.1016/j.bcab.2021.101944
67. Ojha SK, Singh PK, Mishra S, Pattnaik R, Dixit S, Verma SK. Response surface methodology-based optimization and scale-up production of amylase from a novel bacterial strain, Bacillus aryabhattai KIIT BE-1. Biotechnol Rep. 2020;27:e00506. https://doi.org/10.1016/j.btre.2020.e00506
68. Liaqat UE, Naz S, Hussain HI. Optimization and production of highly stable amylase isolated from Citrobacter portucalensis using low-cost agro-industrial wastes as substrates: Prospective therapeutics. Kuwait J Sci. 2023;51(1):100156. https://doi.org/10.1016/j.kjs.2023.11.005
69. El-Sayed MH, Gomaa AE, Atta OM, Hassane AM. Characteristics and kinetics of thermophilic actinomycetes? amylase production on agro-wastes and its application for ethanol fermentation. World J Microbiol Biotechnol. 2024;40(8):205. https://doi.org/10.1007/s11274-024-04009-8
70. Hmida BB, Mabrouk SB, Fendri A, Hmida-Sayari A, Sayari A. Optimization of newly isolated Bacillus cereus α-amylase production using orange peels and crab shells and application in wastewater treatment. 3 Biotech. 2024;14(4):119. https://doi.org/10.1007/s13205-024-03962-3
71. Coronado MJ, Vargas C, Hofemeister J, Ventosa A, Nieto JJ. Production and biochemical characterization of an α-amylase from the moderate halophile Halomonas meridiana. FEMS Microbiol Lett. 2000;183(1):67-71. https://doi.org/10.1111/j.1574-6968.2000. tb08935.x
72. Luang-In V, Yotchaisarn M, Saengha W, Udomwong P, Deeseenthum S, Maneewan K. Isolation and identification of amylase-producing bacteria from soil in Nasinuan community forest, Maha Sarakham, Thailand. Biomed Pharmacol J. 2019;12(3):1061-8. https://doi.org/10.13005/bpj/1735
73. Abd-Elaziz AM, Karam EA, Ghanem MM, Moharam ME, Kansoh AL. Production of a novel α-amylase by Bacillus atrophaeus NRC1 isolated from honey: Purification and characterization. Int J Biol Macromol. 2020;148:292-301. https://doi.org/10.1016/j.ijbiomac.2020.01.120
74. Bakare M, Adewale I, Ajayi A, Shonukan O. Purification and characterization of cellulase from the wild-type and two improved mutants of Pseudomonas fluorescens. Afr J Biotechnol. 2005;4(9):898-904. https://doi.org/10.5897/ajb2005.000-3178
75. Kim YJ. Thin layer chromatogram by an extracellular β-amylase of Bacillus sp. KYJ 963 and its amino acid composition. J Life Sci. 2001;11(2):92-3.
76. Kocabay S, Çetinkaya S, Akkaya B, Yenidünya AF. Characterization of thermostable β-amylase isozymes from Lactobacillus fermentum. Int J Biol Macromol. 2016;93:195-202. https://doi.org/10.1016/j.ijbiomac.2016.08.078
Year
Month