Thermostable α-amylase from Lignocellulosic Residues Using Bacillus amyloliquefaciens


1 Department of Chemical Engineering, Babol Noshirvani University of Technology, Babol, Iran

2 Biochemical & Chemical Engineering, Noshirvani University of Technology, Babol


In this study, thermostable high performance α-amylase was synthesized from lignocellulosic residues using Bacillus amyloliquefaciens. For this purpose, hydrolysates of wheat bran, rice bran and sugarcane bagasse were used as substrate for enzyme production. The maximum enzyme production was achieved in the medium containing hydrolysate of wheat bran. In order to enhance α-amylase production, the medium composition was optimized in terms of supplementary carbon and nitrogen sources. Enzyme activity in the optimized medium (208.94 U mL-1) was considerably higher as compared to non-optimized medium (76.22 U mL-1). The activity and stability of the synthesized enzyme was assessed in various temperature and pH environments. The optimum condition for highest enzyme activity (pH 7 and 70 C) and stability (pH 7 and 50 C) was determined. The effect of various metal ions on the α-amylase activity was investigated. The enzyme activity enhanced in the presence of Mg, Mn,Zn,Na,Cu,Ca ions, while Fe ion hindered the enzyme activity.


1.     Rajagopalan, G. and Krishnan, C., "α-Amylase production from catabolite derepressed Bacillus subtilis KCC103 utilizing sugarcane bagasse hydrolysate", Bioresource Technology,Vol. 99, No. 8, (2008), 3044-3050.

2.     Raplong, H.H., Odeleye, P.O., Hammuel, C., Idoko, M.O., Asanato, J.I. and Odeke, E.H., "Production of Alpha Amylase by Bacillus cereus in Submerged Fermentation", Aceh International Journal of Science and Technology,Vol. 3, No. 3, (2014).

3.     Mojsov, K., "Microbial alpha-amylases and their industrial applications: a review", International Journal of Management, IT and Engineering (IJMIE),Vol. 2, No. 10, (2012), 583-609.

4.     Xie, F., Quan, S., Liu, D., Ma, H., Li, F., Zhou, F. and Chen, G., "Purification and characterization of a novel α-amylase from a newly isolated Bacillus methylotrophicus strain P11-2", Process Biochemistry,Vol. 49, No. 1, (2014), 47-53.

5.     Sharma, A. and Satyanarayana, T., "Microbial acid-stable α-amylases: Characteristics, genetic engineering and applications", Process Biochemistry,Vol. 48, No. 2, (2013), 201-211.

6.     Sivaramakrishnan, S., Gangadharan, D., Nampoothiri, K.M., Soccol, C.R. and Pandey, A., "a-Amylases from microbial sources–an overview on recent developments", Food Technol Biotechnol,Vol. 44, No. 2, (2006), 173-184.

7.     Asgher, M., Asad, M.J., Rahman, S. and Legge, R., "A thermostable α-amylase from a moderately thermophilic Bacillus subtilis strain for starch processing", Journal of Food Engineering,Vol. 79, No. 3, (2007), 950-955.

8.     Ashraf, H., Iqbal, J. and Qadeer, M., "Production of alpha amylase by Bacillus licheniformis using an economical medium", Bioresource Technology,Vol. 87, No. 1, (2003), 57-61.

9.     Rana, N., Walia, A. and Gaur, A., "α-amylases from microbial sources and its potential applications in various industries", National Academy Science Letters,Vol. 36, No. 1, (2013), 9-17.

10.   Kumar, N.M., Karthikeyan, S. and Jayaraman, G., "Thermostable alpha-amylase enzyme production from Bacillus laterosporus: Statistical optimization, purification and characterization", Biocatalysis and Agricultural Biotechnology,Vol. 2, No. 1, (2013), 38-44.

11.   Souza, P.M.d., "Application of microbial α-amylase in industry-A review", Brazilian Journal of Microbiology,Vol. 41, No. 4, (2010), 850-861.

12.   Swargiari, B.N. and Baruah, P.K., "Production of microbial α- amylase by solid state fermentation-an overview", International Journal of Current Research Vol. 4, No. 11, (2012), 350-356.

13.   Hashemi, M., Razavi, S.H., Shojaosadati, S.A. and Mousavi, S.M., "The potential of brewer's spent grain to improve the production of α-amylase by Bacillus sp. KR-8104 in submerged fermentation system", New Biotechnology,Vol. 28, No. 2, (2011), 165-172.

14.   Santos, É.R.d., Teles, Z.N.S., Campos, N.M., Souza, D.A.J.d., Bispo, A.S.d.R. and Nascimento, R.P.d., "Production of α-amylase from Streptomyces sp. SLBA-08 strain using agro-industrial by-products", Brazilian Archives of Biology and Technology,Vol. 55, No. 5, (2012), 793-800.

15.   Tanyildizi, M. and Ozer, D., "An Investigation of α-amylase production in semi solid substrate fermentation by using corn bran with Bacillus amyloliquefaciens", Turkish Journal of Science & Technology,Vol. 6, No. 1, (2011), 47-52.

16.   Goyal, N., Gupta, J. and Soni, S., "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,Vol. 37, No. 7, (2005), 723-734.

17.   Miller, G.L., "Use of dinitrosalicylic acid reagent for determination of reducing sugar", Analytical Chemistry,Vol. 31, No. 3, (1959), 426-428.

18.   Gupta, R., Gigras, P., Mohapatra, H., Goswami, V.K. and Chauhan, B., "Microbial α-amylases: a biotechnological perspective", Process Biochemistry,Vol. 38, No. 11, (2003), 1599-1616.

19.   Ravindar, D.J. and Elangovan, N., "Molecular identification of amylase producing Bacillus subtilis and detection of optimal conditions", Journal of Pharmacy Research,Vol. 6, No. 4, (2013), 426-430.

20.   Passos, M.L. and Ribeiro, C.P., Innovation in food engineering: New techniques and products, CRC Press, (2009).

21.   Gomathi, D., Muthulakshmi, C., Kumar, D.G., Ravikumar, G., Kalaiselvi, M. and Uma, C., "Submerged fermentation of wheat bran by Aspergillus flavus for production and characterization of carboxy methyl cellulase", Asian Pacific Journal of Tropical Biomedicine,Vol. 2, No. 1, (2012), S67-S73.

22.   Lin, L.L., Chyau, C.C. and Hsu, W.H., "Production and properties of a raw-starch-degrading amylase from the thermophilic and alkaliphilic Bacillus sp. TS-23", Biotechnology and Applied Biochemistry,Vol. 28, No. 1, (1998), 61-68.

23.   Deb, P., Talukdar, S.A., Mohsina, K., Sarker, P.K. and Sayem, S., "Production and partial characterization of extracellular amylase enzyme from Bacillus amyloliquefaciens P-001", Springer Plus,Vol. 2, No. 1, (2013), 154-160.

24.   Bano, S., Qader, S.A.U., Aman, A., Syed, M.N. and Azhar, A., "Purification and characterization of novel α-amylase from Bacillus subtilis KIBGE HAS", AAPS PharmSciTech,Vol. 12, No. 1, (2011), 255-261.

25.   Amid, M. and Manap, M.Y.A., "Purification and characterisation of a novel amylase enzyme from red pitaya (Hylocereus polyrhizus) peel", Food Chemistry,Vol. 165, (2014), 412-418.

26.   Sevinc, N. and Demirkan, E., "Production of Protease by Bacillus sp. N-40 isolated from soil and its enzymatic properties", Journal of Biological and Environmental Science,Vol. 5, No. 14, (2011), 95-103.

27.   Alkan, H., Baysal, Z., Uyar, F. and Dogru, M., "Production of lipase by a newly isolated Bacillus coagulans under solid-state fermentation using melon wastes", Applied Biochemistry and Biotechnology,Vol. 136, No. 2, (2007), 183-192.

28.   Fincan, S.A., Enez, B., Özdemir, S. and Bekler, F.M., "Purification and characterization of thermostable α-amylase from thermophilic Anoxybacillus flavithermus", Carbohydrate Polymers,Vol. 102, (2014), 144-150.

29.   Rezaei, P.S., Darzi, G.N. and Shafaghat, H., "Optimization of the fermentation conditions and partial characterization for acido-thermophilic α-amylase from Aspergillus niger NCIM 548", Korean Journal of Chemical Engineering,Vol. 27, No. 3, (2010), 919-924.