Production of Thermostable a-amylase by Bacillus cereus MK in solid state fermentation: Partial purification and characterization of the enzyme

摘要

Thermostable a-amylase production under solid state fermentation was investigated using isolated thermophilic Bacillus cereus MK. Optimization of process parameters followed by leaching parameters for enhanced enzyme yields were carried out. The optimum temperature, pH, incubation period, inoculation size and substrate to moisture ratio were found to be 55 oC, 7.0, 24 h, 15 % (v/w) and 12.5 (w/v), respectively. Among different carbon, nitrogen and trace elements supplemented, glucose, peptone and calcium chloride, respectively enhanced enzyme production. The optimum leaching parameters such as solvents, solvent volume, physical state, solvent temperature and solvent pH for effective extraction of amylase from the fermented bran were found to be sodium acetate buffer (0.1M, pH 5.6), 1:2.5 (w/v), agitation, 50 oC and 7.0, respectively. An overall 14 fold increase in enzyme production was attained by optimization of process conditions and leaching parameters in SSF. The crude amylase showed maximum activity at pH 7.0 and 90 oC. The enzyme was stable at 90 oC for 1h. However in the presence of 4% (w/v) starch, the stability of enzyme was increased to100 oC up to 2h. Introduction Alpha amylases (endo-1,4-α-D-glucan glucanohydrolase, E.C. 3.2.1.1) are extracellular endo enzymes that randomly cleave the 1,4 α-linkage between adjacent glucose units in the linear amylose chain and ultimately generate glucose, maltose and maltotriose units. Among various extracellular enzymes, α-amylase ranks first in terms of commercial exploitation (Babu and Satyanarayana, 1993) and accounts 12% of the sales value of the world market (Baysal et al, 2003). Spectrum of applications of α-amylase has widened in many sectors such as clinical, medicinal and analytical chemistry. Besides their use in starch saccharification, they also find applications in bakery, brewery, detergent, textile, paper and distilling industry (Ramachandran et al, 2004).Alpha amylases has been derived from several fungi, yeasts, bacteria and actinomycetes. However, enzymes from fungal and bacterial sources have dominated applications in industrial sectors (Pandey et al, 2000). Almost all microorganisms of the genus Bacillus synthesize α-amylase, thus this genus has the potential to dominate the enzyme industry (Pretorius et al, 1986). Thermostability is a feature of most of the enzymes sold for bulk industrial usage and thermophilic organisms are therefore of special interest as they could be used for saccharification processes occurring at high temperatures (Peixoto et al, 2003). The advantages of using thermostable amylases in industrial processes include the decreased risk of contamination, cost of external cooling and increased diffusion rate (Lin et al, 1998). Apart from this, they are resistant to denaturing agents, solvents and proteolytic enzymes (Bragger et al, 1989). Amylases with broad pH range have potential applications for starch saccharification in starch and textile industries and also are an ingredient in detergents for automatic dish washers and laundries (Kim et al, 1995). The industrially important Bacillus strains, which are extensively used to produces α-amylase are B. amyloliquefaciens, B. licheniformis (Fogarty and Kelly, 1980), B. stearothermophilus (Wind et al, 1994), B. subtilis (Takasaki, 1985), B. megaterium (Brumm et al, 1981) and B. circulans (Takasaki, 1983). Industrially important enzymes have traditionally been obtained from submerged fermentation (SmF) because of the ease of handling and greater control of environmental factors such as temperature and pH. However, solid-state fermentation (SSF) constitutes an interesting alternative since the metabolites so produced are concentrated and purification procedures are less costly (Pandey, 1992; Nigam and Singh, 1995; Chadha et al, 1997; Pandey et al, 2000). SSF is preferred to SmF because of simple technique, low capital investment, lower levels of catabolic repression and end product inhibition, low