Comparative Characterization and Amylase Activity Assessment of Certain Garden Bacterial Isolates

Munia Islam ^a and Tamanna Zerin ^a*

Screening of soil Bacillus spp. for amylolytic activity

Abstract

Background: Microorganisms are the most important sources of enzymes because of their stability and reduced price in production. The enzyme, amylase has a wide spectrum of application in biotechnology including food, fermentation, textile and laundry, paper and pulp industries. As the use of amylase is increased, it is necessary to search for a new source to produce amylase with better productivity in continuous practice. Method: In our present investigation, amylase producing bacteria were screened by starch hydrolysis test as bacteria are more potent in amylase production than other microorganisms. The bacteria were identified by Bergey’s manual of systematic bacteriology. Enzyme assay and optimization of enzyme activity were performed by the 3,5-dinitrosalicylic acid method. Results: A total of eight bacterial isolates were identified with starch degrading capabilities and they were presumptively placed in the genus, Bacillus due to their characteristic features. All the isolates have potential for amylase activity. At any temperature (25°C, 35°C, 45°C, 50°C and 55°C), pH (6, 7, 8) and starch concentrations (0.5%, 1% and 1.5%), the highest amylase activity was observed by isolate 1A followed by isolate 2C, 1B, 3A, 4B, 3B, 4A, and 4C. Optimum conditions for the highest amylase activity in our laboratory for isolate 1A was 35°C (4.105 U/ml), pH 6 (3.343 U/ml) and 1.5% starch concentration (4.381 U/ml). Conclusion: Our study reveals that the isolates, collected from garden soil, are good amylase producers and they could be exploited in different industries in optimized conditions.

Keywords: Amylase, Enzyme activity, DNS, Soil, Optimization.

Abbreviations: DNS, 3,5 dinitrosalicylic acid; MR, methyl red; VP, voges-proskauer; GPB, glucose phosphate broth; MIU, motility indole urea.

References

Aiba, S., Kitai, K., & Imanaka, T. (1983). Cloning and Expression of Thermostable α-Amylase Gene from Bacillus stearothermophilus in Bacillus stearothermophilus and Bacillus subtilis. Applied and Environmental Microbiology, 46(5), 1059-1065.

Amoozegar, M.A., Malekzadeh. F., & Malik, K.A. (2003). Production of amylase by newly isolated moderate halophile, Halobacillus sp. strain MA-2. Journal of Microbiological Methods, 52(3), 353-9.
https://doi.org/10.1016/S0167-7012(02)00191-4

Bala, J.D., Abioye, O.P., Auta, H.S., Damisa, D., Adabara, N.U., & Tuggen, T.O. (2013). Screening of soil microorganisms for amylase production. BTAIJ, 7(1), 22-27.

Behal, A., Singh, J., Sharma, M.K., Puri, P., & Batra, N. (2006). Characterization of Alkaline α-Amylase from Bacillus sp. AB 04. International journal of agriculture and biology, 8(1), 80-83.

Bernfeld, P. (1955). Amylase α and β. Methods in Enzymology, 1, 149-158. https://doi.org/10.1016/0076-6879(55)01021-5

Cappuccino, J.G. and Sherman, N. (1996) Microbiology: A Laboratory Manual. 4th Edition, p. cm. Includes index. ISBN 0-8053-0573-4.

Coronado, M., Vargas, C., Hofemeister, J., Ventosa, A., & Nieto, J.J. (2000). Production and biochemical characterization of an alpha-amylase from the moderate halophile Halomonas meridiana. FEMS Microbiology Letters, 183(1), 67-71. https://doi.org/10.1111/j.1574-6968.2000.tb08935.x
https://doi.org/10.1016/S0378-1097(99)00628-X

De Souza, P.M., & de Oliveira Magalhães, P. (2010). Application of microbial α-amylase in industry: a review. Brazilian Journal of Microbiology, 41(4), 850-861. https://doi.org/10.1590/S1517-83822010000400004 PMid:24031565 PMCid:PMC3769773

Deutch, C.E. (2002). Characterization of a salt-tolerant extracellular a-amylase from Bacillus dipsosauri. Letters in Applied Microbiology, 35(1), 78-84. https://doi.org/10.1046/j.1472-765X.2002.01142.x
PMid:12081555

Dutta, P., Deb, A., & Sukanta Majumdar, S. (2016). Optimization of the Medium for the Production of Extracellular Amylase by the Pseudomonas stutzeri ISL B5 Isolated from Municipal Solid Waste. International Journal of Microbiology, 2016, 4950743. https://doi.org/10.1155/2016/4950743 
PMid:28096816 PMCid:PMC5206451

Gopinath SC, Anbu P, Arshad MK, Lakshmipriya T, Voon CH, Hashim U & Chinni SV. (2017). Biotechnological Processes in Microbial Amylase Production. Biomed Research International, 2017, 1272193. https://doi.org/10.1155/2017/1272193 PMid:28280725 PMCid:PMC5322433

Gupta, R., Gigras, P., Mohapatra, H., Goswami, V.K., & Chauhan, B. (2003). Microbial α-amylases: a biotechnological perspective. Process Biochemistry, 38(11), 1599-1616. https://doi.org/10.1016/S0032-9592(03)00053-0

Hutcheon, G.W., Vasisht, N., & Bolhuis, A. (2005). Characterisation of a highly stable alpha-amylase from the halophilic archaeon Haloarcula hispanica. Extremophiles, 9(6), 487-95. https://doi.org/10.1007/s00792-005-0471-2 PMid:16075161

Islam, M.R., Mondol, OK , Md. Rahman, M.S., Billah, M.M., Rahman, M.S., & Zohora, U.S. (2016). Screening of α-amylase producing bacteria from tannery wastes of Hazaribag, Bangladesh. Jahangirnagar University Journal of Biological Sciences, 5(2), 1-10. 
https://doi.org/10.3329/jujbs.v5i2.32511

Kandra, L. (2003). α-Amylases of medical and industrial importance. Journal of Molecular Structure (Theochem), 666-667, 487-498.
https://doi.org/10.1016/j.theochem.2003.08.073

Kathiresan, K., & Manivannan, S. (2006). α Amylase production by Penicillium fellutanum isolated from mangrove rhizosphere soil. African Journal of Biotechnology, 5(10), 829-832.

Kaur, A., Kaur, M., Samyal, M.L., & Ahmed, Z. (2012). Isolation, characterization and identification of bacterial strain producing amylase. Journal of Microbiology and Biotechnology Research, 2(4), 573-579.

Mishra, S., & Behera, N. (2008). Amylase activity of a starch degrading bacteria isolated from soil receiving kitchen wastes. African Journal of Biotechnology, 7(18), 3326-3331.

Megahati R.R.P., Mansyurdin, Agustien A., & Tjong, D.H. (2017). Optimization of Bacteria Amylase Activity from Bacillus licheniformis Strain SEM11. International Journal of Current Microbiolology and Applied Sciences, 6(11), 2938-2946.
https://doi.org/10.20546/ijcmas.2017.611.345

Mohamed, S.A., Al-Malki, A.L., & Kumosani, T.A. (2009). Partial purification and characterization of five α-amylases from a wheat local variety (Balady) during germination. Australian Journal of Basic and Applied Sciences, 3(3), 1740-1748.

Pandey, A., Nigam, P., Soccol, C.R., Soccol, V.T., Singh, D., & Mohan, R. (2000). Advances in Microbial Amylases. Biotechnology and Applied Biochemistry, 31(Pt 2), 135-152. https://doi.org/10.1042/BA19990073
PMid:10744959

Patel, G. (2015). Isolation and Characterization of Starch Degrading Bacteria from Garden Soil, Ganpat University, Gujarat, India. Indian Journal of Microbiology Research, 2(2),111-114.

Prakash, B., Vidyasagar, M., Madhukumar, M.S., Muralikrishna, G., & Sreeramulu, K. (2009). Production, purification, and characterization of two extremely halotolerant, thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101. Process Biochemistry, 44(2), 210-215. https://doi.org/10.1016/j.procbio.2008.10.013

Prakash, O., & Jaiswal, N. (2010). alpha-Amylase: an ideal representative of thermostable enzymes. Applied Biochemistry and Biotechnology, 160(8), 2401-14. https://doi.org/10.1007/s12010-009-8735-4
PMid:19763902

Rajagopalan, G. & Krishnan, C. (2008). Alpha-amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate. Bioresource Technology, 99(8), 3044-3050.
https://doi.org/10.1016/j.biortech.2007.06.001 PMid:17644331

Rao, M., Tankasale, A., Ghatge, M., & Desphande, V. (1998). Molecular and biotechnological aspects of microbial proteases. Microbiology and Molecular Biology Reviews, 62(3), 597-634.

Reddy, N.S., Nimmagadda, A., & Sambasiva Rao, K.R.S. (2003). An overview of the microbial α-amylase family. African Journal of Biotechnology, 2(12), 645-648. https://doi.org/10.5897/AJB2003.000-1119

Tanyildizi, M.S., Ozer, D., & Elibol, M. (2005). Optimization of α-amylase production by Bacillus sp. using response surface methodology. Process Biochemistry, 40(7), 2291-2296. 
https://doi.org/10.1016/j.procbio.2004.06.018

Tonkova, A., Manolov, R., & Dobreva, E. (1993). Thermostable α-amylase from derepressed Bacillus licheniformis produced in high yields from glucose. Process Biochemistry, 28(8), 539-542.
https://doi.org/10.1016/0032-9592(93)85015-8

Vaidya, S., & Rathore, P. (2015). Isolation, Screening and Characterization of amylase producing bacteria from soil of potato dump sites from different regions of Madhya Pradesh. Conference article from International conference on recent trends in agriculture, veterinary and life sciences published in Life Science International research journal, ISBN 978-93-84124-26-7.