Amylases are widely used in many industrial processes, such as sugar, brewing, alcohol, textile and paper industries (Fogarty et al. 1983; Emanuilova & Toda 1984; Plant et al. 1987). In all these processes, thinning and liquification of starch is a prerequisite and is carried out at elevated temperatures using thermostable amylases (Weber et al. 1990; Crabb & Mitchinson 1997). The main source of commercial thermostable amylases is strains of the mesophilic bacteria, Bacillus licheniformis and B. amyloliquefaciens (Kelly et al. 1990). The need for enzymes with improved properties has initiated a continuous search for micro-organisms producing novel amylases for industrial application (Ng & Kenealy 1986; Kochhar & Dua 1990). This is especially important as so far, only a very small percentage of the earth's microbial population has been properly examined (Cheetham 1987; Bull et al. 1992). Thermophilic micro-organisms are believed to be potentially good alternative sources of thermostable enzymes (Brock 1985; Kristjansson 1989; Antranikian 1990; Wind et al. 1994; Bolton et al. 1997; Busch & Stutzenberger 1997; Egas et al. 1998). Thermophilic bacteria can be isolated from natural high temperature environments distributed throughout the world and found in association with tectonically active sites (Brock 1985). In this paper, the isolation of amylolytic Bacillus sp. from a hot spring in Ethiopia, and some properties of the partially purified enzyme, is reported.
A thermostable amylase-producing Bacillus sp. WN11 was isolated from Wondo Genet hot spring in Ethiopia. The enzyme had a temperature optimum of 75–80 °C. Over 80% of its peak activity was in the pH range of 5–8, with an optimum at 5·5. Thermal stability of the enzyme at 105 °C was higher with the addition of starch. The stabilizing effect of starch was concentration-dependent, showing better stability with increasing concentration of starch. At liquefying temperature (105 °C), addition of Ca2+ did not result in further improvement of the stabilizing effect of starch. This indicates that in the presence of starch, WN11 amylase does not require Ca2+ as a stabilizer at liquefying temperatures as high as 105 °C.
Materials and methods
Isolation and characterization
Sample collected from Wondo Genet hot spring, Southern Ethiopia, was enriched at 65 °C in nutrient broth supplemented with 0·5% (w/v) starch. Amylolytic isolates were selected by flooding the agar plates with Gram's iodine solution. Isolates having a higher ratio of clearing zone to colony size were grown in liquid culture and the level of amylase production was determined from cell-free culture supernatant fluid. Characterization and identification of the isolate was made following Bergey's Manual of Systematic Bacteriology (Sneath 1986).
The medium for enzyme production comprised (g l−1): starch, 10; bacteriological peptone, 5; NaCl, 1; K2HPO4, 2; CaCl2, 0·1; MgSO4, 0·1; and trace mineral solution, 20 ml l−1. This medium (100 ml) was inoculated with 4 ml of an overnight culture and incubated at 65 °C. After 18 h of incubation, the culture was centrifuged and the cell-free culture supernatant fluid used as the enzyme source.
The cell-free culture supernatant fluid was precipitated using solid ammonium sulphate to 60% saturation. The precipitate was recovered by centrifugation and dissolved in a minimum volume of 50 mmol l−1 phosphate buffer, pH 7, and dialysed overnight against the same buffer. The dialysate was used for all enzyme characterization studies.
Thin layer chromatography
Thin layer chromatography (TLC) analysis of end products was carried out following the methods of Kimura & Horikoshi (1989).
Amylase activity was determined as described previously (Mamo & Gessesse 1997). One unit of amylase activity was defined as the amount of enzyme that released one micromole of reducing sugar equivalent to glucose per minute under the assay condition.
Isolation and characterization
On the basis of level of productivity and thermostability of the amylase produced, one strain, designated as WN11, was selected for subsequent studies. This isolate was rod-shaped, aerobic, terminal endospore-forming, catalase-positive and Gram-variable. It grew optimally at 65 °C. Based on these biochemical and cultural properties, it is identified as a thermophilic strain of the genus Bacillus.
Growth and amylase production
Isolate WN11 entered stationary phase after 18 h and the culture pH was dropped from 7·0 to 5·1 within 12 h. Amylase production was detected shortly after inoculation (at 3 h) and reached a maximum (7·3 U ml−1) after 18 h (Fig. 1).
Temperature and pH profile
The temperature profile of the enzyme was determined by assaying the enzyme at different temperatures in the presence and absence of Ca2+. The enzyme had an optimum temperature of 75–80 °C, with 93% of its maximum activity retained at 85 °C (Fig. 2). Up to 85 °C, addition of 5 mmol l−1 Ca2+ did not alter the temperature profile of the enzyme. However, at 90 °C, a slight increase in activity was observed. The pH profile of WN11 amylase was investigated using different buffers with various pH values at 70 °C. The enzyme had an optimum pH of 5·5. Over 80% of its peak activity was maintained between pH 5 and 8. Moreover, it was stable within this pH range.
Stability at 105 °C was determined by incubating the enzyme in the presence of 2% starch and 5 mmol l−1 Ca2+. The data presented in Fig. 3 show that starch was a better stabilizer than Ca2+. Incubation of the enzyme in the presence of both starch and Ca2+ did not further improve the stability of the enzyme. The effect of starch concentration on thermal stability was determined at 100 °C in the presence of 5, 10, 15 and 20% (w/v) starch. Enzyme stability was higher with increasing starch concentration (Fig. 4).
End product profile of starch hydrolysis
The chromatogram (Fig. 5) indicates the formation of a range of oligosaccharides from soluble starch. This suggests random degradation and that the enzyme was essentially an α- amylase.
Amylase production by Bacillus sp. WN11 was growth-associated, reaching a maximum around 18 h. This may be an interesting property because it could allow harvesting of the enzyme in a shorter time. The mesophilic B. licheniformis and B. amyloliquefaciens, which are the main sources of commercial amylase at present, are reported to require a batch time of 48–72 h for optimum production (Roychudhury et al. 1988; Kochhar & Dua 1990; Tonkova et al. 1993).
The enzyme was optimally active and stable at pH 5·5. The use of liquefying amylases that are active at relatively lower pH could reduce the amount of acid used to lower the pH from a liquefying to a saccharifying range. This again reduces the cost of ion exchange media and chemicals required for downstream processing (Wind et al. 1994; Crabb & Mitchinson 1997), thus increasing the cost efficiency of starch processing. Moreover, such enzymes may enable the development of a continuous system for the conversion of starch to glucose (Ng & Kenealy 1986). Hence, the amylase from Bacillus sp. WN11 may offer some technical advantage when potential industrial application is considered.
The other important feature of isolate WN11 amylase is its high thermal stability. Very few strains of thermophilic Bacillus have been reported so far that produce highly thermostable amylases (Vihinen & Mantsala 1990; Wind et al. 1994). At liquefying temperatures, commercially used amylases require Ca2+ as a stabilizer (Kumar et al. 1990). WN11 amylase exhibited a high degree of thermal stability at liquefying temperatures when incubated in the presence of starch alone. Thermal stability of this enzyme was higher with increasing starch concentration. Hence, at the level of industrial liquefication, which is normally 35–40% (w/v), the enzyme from Bacillus sp. WN11 is expected to have better stability without the addition of Ca2+. From the application point of view, this may be a very important property, especially in the production of starch-based sweeteners. Calcium is known to inhibit glucose isomerase, the enzyme used for the isomerization of glucose to fructose (Takasaki 1966; Chen 1980; Kumar et al. 1990; Lehmacher & Bisswanger 1990; Smith et al. 1991). Therefore, amylases with little or no requirement for Ca2+ at liquefying temperatures would improve the efficiency of starch conversion processes.
In conclusion, the ability of Bacillus sp. WN11 amylase to withstand a temperature of up to 105 °C in the absence of Ca2+, and its slightly acidic pH optimum for activity and stability, could suggest potential for this enzyme in industrial starch liquefication.
The financial support of the Swedish International Development Agency (SIDA) administered through the Ethiopian Science and Technology Commission is gratefully acknowledged.
Present address: Department of Biological Sciences, University of Botswana, Private Bag 0022, Gaborone, Botswana.