The thermodynamic and kinetic properties of solids state raw starch digesting alpha amylase from newly isolated Bacillus licheniformis RT7PE1 strain were studied. The kinetic values , , , and were proved to be best with 15% wheat bran. The molecular weight of purified enzyme was 112？kDa. The apparent and values for starch were 3.4？mg mL？1 and 19.5？IU？mg？1 protein, respectively. The optimum temperature and pH for α-amylase were 55°C, 9.8. The half-life of enzyme at 95°C was 17h. The activation and denaturation activation energies were 45.2 and 41.2？kJ mol？1, respectively. Both enthalpies ( ) and entropies of activation ( ) for denaturation of α-amylase were lower than those reported for other thermostable α-amylases. 1. Introduction Starch is an excellent carbon source in nature and major storage product of many economically important crops. A large-scale starch processing industry has emerged in the last century [1]. Five groups of enzymes play a key role in the hydrolysis of starch. These enzymes comprise about 30% of the world’s enzyme production. Alpha amylases (endo-1,4-a-D-glucan glucanohydrolase, E.C.3.2.1.1) are extracellular endo enzymes that randomly cleave the 1,4-a linkage between adjacent glucose units in the linear amylose chain and ultimately generate glucose, maltose, and maltotriose units [2]. Alpha amylases derived from microbial sources have great success and replaced the chemical hydrolysis of starch in starch processing industries. Alpha amylases have a number of applications, including liquefaction of starch in the alcohol, brewing, and sugar industries and in the textile industry for desizing of fabrics [3]. They also have applications in laundry and detergents or as antisalting agents and baking such as bread making [3], since thermostability and alkaline characteristics are important features for industrial applications of amylase isolated from alkalophilic organisms [4]. The microbial α-amylases for industrial processes are derived mainly from Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis asthey are capable of secreting amylases into the culture supernatant in submerged [5]. The microbial production of alpha-amylase is greatly influenced by the composition of the medium culture and environmental and growth kinetic parameters [5]. However, solid state fermentation provides an interesting alternative for concentrated enzyme production and less purification costs [6]. This work reports the solid state production, kinetic and thermodynamic properties of raw starch-digesting alkaline alpha-amylase from an
References
[1]
P. Nigam and D. Singh, “Enzyme and microbial systems involved in starch processing,” Enzyme and Microbial Technology, vol. 17, no. 9, pp. 770–778, 1995.
[2]
Z. Baysal, F. Uyar, M. Do？ru, and H. Alkan, “Production of extracellular alkaline alpha-amylase by solid state fermentation with a newly isolated Bacillus sp,” Preparative Biochemistry & Biotechnology, vol. 38, no. 2, pp. 184–190, 2008.
[3]
R. R. Singhania, A. K. Patel, C. R. Soccol, and A. Pandey, “Recent advances in solid-state fermentation,” Biochemical Engineering Journal, vol. 44, no. 1, pp. 13–18, 2009.
[4]
M. J. Maarel, V. B. Vander, J. C. Uitdehaag, H. Leemhuis, and L. Dijkhuizen, “Properties and applications of starch-converting enzymes of the α-amylase family,” Journal of Biotechnology, vol. 94, no. 2, pp. 137–155, 2002.
[5]
S. Sivaramakrishnan, D. Gangadharan, K. M. Nampoothiri, C. R. Soccol, and A. Pandey, “α-Amylases from microbial sources—an overview on recent developments,” Food Technology and Biotechnology, vol. 44, no. 2, pp. 173–184, 2006.
[6]
M. Hashemia, S. M. Mousavib, S. H. Razavi, and S. A. Shojaosadati, “Comparison of submerged and solid state fermentation systems effects on the catalytic activity of Bacillus sp. KR-8104 α-amylase at different pH and temperatures,” Industrial Crops and Products, vol. 43, pp. 661–667, 2013.
[7]
P. Bernfield, “Amylases α and β,” in Methods in Enzymology, S. Colowick and N. Keplan, Eds., pp. 149–158, Academic Press, New York, NY, USA, 1951.
[8]
M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976.
[9]
X.-Z. Zhang, S. Xie, X. Wu, F. Jin, and X. Li, “Purification and characterization of thermostable α-amylase II from Bacillus sp-JF2 strain,” Enzyme and Microbial Technology, vol. 16, no. 11, pp. 985–990, 1994.
[10]
M. Hashemi, S. H. Razavi, S. A. Shojaosadati, and S. M. Mousavi, “Evaluation of Ca-independent α-amylase production by Bacillus sp. KR-8104 in submerged and solid state fermentation systems,” Iranian Journal of Biotechnology, vol. 9, no. 3, pp. 188–196, 2011.
[11]
S. Aiba, A. E. Humphrey, and N. F. Millis, Biochemical Engineering Fundamentals, McGraw Hill, New York, NY, USA, 2nd edition, 1973.
[12]
L.-L. Lin, C.-C. Chyau, and W.-H. Hsu, “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, pp. 61–68, 1998.
[13]
H. Anto, U. Trivedi, and K. Patel, “Alpha amylase production by Bacillus cereus MTCC 1305 using solid-state fermentation,” Food Technology and Biotechnology, vol. 44, no. 2, pp. 241–245, 2006.
[14]
Z. Al-Qodah, H. Daghstani, P. Geopel, and W. Lafi, “Determination of kinetic parameters of α-amylase producing thermophile Bacillus sphaericus,” African Journal of Biotechnology, vol. 6, no. 6, pp. 699–706, 2007.
[15]
D. J. Bolton, C. T. Kelly, and W. M. Fogarty, “Purification and characterization of the α-amylase of Bacillus flavothermus,” Enzyme and Microbial Technology, vol. 20, no. 5, pp. 340–343, 1997.
[16]
S. Ito, T. K. Kobayashi, K. Ara, K. Ozaki, S. Kawai, and Y. Hatada, “Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and structures,” Extremophiles, vol. 2, no. 3, pp. 185–190, 1998.
[17]
M. Hashemi, S. H. Razavi, S. A. Shojaosadati, S. M. Mousavi, K. Khajeh, and M. Safari, “Development of a solid-state fermentation process for production of an alpha amylase with potentially interesting properties,” Journal of Bioscience and Bioengineering, vol. 110, no. 3, pp. 333–337, 2010.
[18]
M. R. Swain and R. C. Ray, “Alpha-amylase production by Bacillus subtilis CM3 in solid state fermentation using cassava fibrous residue,” Journal of Basic Microbiology, vol. 47, no. 5, pp. 417–425, 2007.
[19]
D. Gangadharan, K. M. Nampoothiri, S. Sivaramakrishnan, and A. Pandey, “Biochemical characterization of raw-starch-digesting alpha amylase purified from Bacillus amyloliquefacien,” Applied Biochemistry and Biotechnology, vol. 158, no. 3, pp. 653–662, 2009.
[20]
N. Declerck, M. Machius, P. Joyet, G. Wiegand, R. Huber, and C. Gaillardin, “Hyperthermostabilization of Bacillus licheniformis α-amylase and modulation of its stability over a 50°C temperature range,” Protein Engineering, vol. 16, no. 4, pp. 287–293, 2003.
[21]
S. D'Amico, J.-C. Marx, C. Gerday, and G. Feller, “Activity-stability relationships in extremophilic enzymes,” Journal of Biological Chemistry, vol. 278, no. 10, pp. 7891–7896, 2003.
[22]
A. Tanaka and E. Hoshino, “Secondary calcium-binding parameter of Bacillus amyloliquefaciens α-amylase obtained from inhibition kinetics,” Journal of Bioscience and Bioengineering, vol. 96, no. 3, pp. 262–268, 2003.
[23]
R. M. Daniel, “The upper limits of enzyme thermal stability,” Enzyme and Microbial Technology, vol. 19, no. 1, pp. 74–79, 1996.
[24]
H. Chen, U. Bakir, P. J. Reilly, and C. Ford, “Increased thermostability of Asn182？→？Ala mutant Aspergillus awamori glucoamylase,” Biotechnology and Bioengineering, vol. 43, no. 1, pp. 101–105, 1994.
[25]
H. Ohnishi, H. Sakai, Y. Nosoh, and T. Sekiguchi, “Protein engineering for thermotability,” TIBTECH, vol. 19, pp. 16–20, 1996.
