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Production and properties of the highly efficient raw starch digesting α-amylase from a Bacillus licheniformis ATCC 9945a
Biochemical Engineering Journal 53 (2011) 203–209
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Production and properties of the highly efficient raw starch digesting ␣-amylase from a Bacillus licheniformis ATCC 9945a Nataˇsa Boˇzic´ a,∗ , Jordi Ruiz b , Josep López-Santín b , Zoran Vujˇcic´ c a
Centre of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia Departament d’Enginyeria Química, Escola d’Enginyeria, Unitat de Biocatàlisi Aplicada Associada al IIQA (CSIC), Universitat Autònoma de Barcelona, Edifici Q, 08193 Bellaterra, Spain c Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Article history: Received 23 June 2010 Received in revised form 14 September 2010 Accepted 23 October 2010
Keywords: ␣-Amylase Bacillus licheniformis Raw starch hydrolysis Thermostable Purification
a b s t r a c t Highly efficient raw starch digesting ␣-amylase was produced after 24 h of batch fermentation of Bacillus licheniformis ATCC 9945a in laboratory bioreactor at 37 ◦ C. The enzyme was purified by gel filtration chromatographies with 6-fold increase of specific activity and 38% recovery and showed a molecular mass of 31 kDa by SDS-PAGE. The purified enzyme had an optimum pH of 6.5 and optimum temperature of 90 ◦ C. The purified ␣-amylase in the presence of CaCl2 retained 55% of its activity after 6 h of incubation at 70 ◦ C. Co2+ , Ni2+ and Ca2+ slightly stimulated, while Hg2+ completely inhibited ␣-amylase activity. Hydrolysis rates of raw triticale, wheat, potato, horseradish and corn starches, at 1% concentration were 63, 60, 59, 52 and 37%, respectively, in a period of 4 h. The properties of the purified enzyme proved its high efficacy for digesting diverse raw starches below gelatinization temperature and, hence, its potential commercial value to use as an industrial enzyme. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Starch is the most important carbon and energy source among plant carbohydrates, and it is the second following cellulose in total biosynthesis [1]. It represents an inexpensive source for production of glucose, fructose and maltose syrups which are widely used in food industries [2] and for obtaining the products of their fermentation such as fuel ethanol. Besides agricultural crops, starch is a significant component of domestic and commercial wastes and these could become useful resources to be converted into ethanol. Conventionally, conversion of starch to glucose is a high temperature, liquid-phase enzymatic hydrolysis process which requires a high-energy input resulting in increased production cost of starch-based products [3]. An alternative, cold hydrolysis approach represents a step towards a “green” method for the production of fuel ethanol. Regarding energy costs, effective utilization of natural resources, minimization of the formation of pollutants and viscosity (handling) problems, use of raw starch digesting enzymes that can perform direct hydrolysis of raw starch below gelatinization temperature is desirable [4]. The removal of the cooking stage also has
∗ Corresponding author. Tel.: +381 113282393; fax: +381 112636061. ´ [email protected] E-mail addresses: [email protected] (N. Boˇzic), ´ (J. Ruiz), [email protected] (J. López-Santín), [email protected] (Z. Vujˇcic). 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.10.014
the potential to increase the value of the co-products since valuable proteins would undergo less thermal stress [5]. In spite of the wide distribution of raw starch digesting enzymes, microbial sources have many advantages for the industrial production such as cost effectiveness, consistency, less time and space required for production and ease of process modification and optimization [6]. Since many of the commercially available amylases do not withstand industrial reaction conditions, isolation and characterization of novel amylases with desirable properties is very important [7]. Several raw starch digesting alpha amylases which can directly hydrolyze the raw starch in a single step at temperatures below the gelatinization temperature of starch has been reported [8]. Raw starch digesting amylases from Bacillus sp. usually need prolonged time of incubation for efficient raw starch hydrolysis and are not able to digest all types of starch granules with same efficiency [9]. Often, better results were obtained with thermostable raw starch digesting amylases at temperatures between 60 and 70 ◦ C [8]. Raw cereal starches are more completely and rapidly hydrolyzed than those from tubers or roots when digested by single, purified enzymes [10]. Moreover most raw starch digesting alpha amylase reported to date hardly digest potato starch [8,10]. Since corn, wheat and potato are the most important sources of starch in EU [1], enzymes that are capable of digesting all these types of raw starches efficiently are economically attractive.
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The aim of this study was to produce and characterize raw starch digesting ␣-amylase from Bacillus licheniformis ATCC 9945a capable for direct hydrolysis of cereal, tuber and root raw starches below the gelatinization temperature of starch with same efficiency in short duration of time. 2. Materials and methods 2.1. Chemicals All reagents and solvents used were of the highest available purity and at least of analytical grade. They were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Raw potato, corn, triticale, wheat and horseradish starches were isolated in our laboratory according to standard recommended procedure.
2.4. Zymogram and isoelectric point of crude ˛-amylase Isoelectric focusing was performed using Multiphor II electrophoresis system (Pharmacia-LKB Biotechnology) according to manufacturer’s instruction. Focusing was carried out on 7.5% actylamide gel with ampholytes in a pH range 3.0–10.0, at 7 W constant powers for 1.5 h at 10 ◦ C. Broad pI kit (GE Healthcare) was used as isoelectric point (pI) markers. After the run, ␣-amylases were detected using in-gel activity staining with I2 /KI staining solution according to a previously published method [12]. ␣-Amylase activity appeared as clear bands on a dark background. 2.5. Determination of protein concentration Protein concentration was determined by the Bradford method [13] using bovine serum albumin as the protein standard.
2.2. Media composition and cultivation conditions
2.6. Enzyme purification
Frozen stock aliquots containing glycerol prepared from exponential phase Bacillus licheniformis ATCC 9945a cultures grown in Luria–Bertani media (LB) were stored at −80 ◦ C. Precultures were routinely grown in Luria–Bertani (LB) broth medium composed of (g/L): peptone, 10.0; yeast extract, 5.0; NaCl, 10.0. Preinoculum cultures were grown from glycerol stocks in a 100 mL shake flask containing 15 mL LB media and incubated overnight at 37 ◦ C in a rotary shaker at 150 rpm. For shake flasks experiments 5 mL of preinoculum cultures were transferred aseptically to a 500 mL baffled shake flask containing 100 mL of semicomplex growth medium. Unless otherwise stated, the growth medium used for ␣-amylase production was composed of (g/L): soluble starch, 10; tryptone, 5; MgSO4 ·7H2 O, 0.5; K2 HPO4 , 2; Na2 HPO4 , 5; NaCl, 2; (NH4 )2 SO4 , 4; FeCl3 , 0.05; CaCl2 ·2H2 O, 0.05. The medium was adjusted to pH 6.5. Cultivations were maintained at 37 ◦ C with agitation at 150 rpm for 72 h. The effect of different levels of tryptone (0.1–1.5%) and different levels of starch (0.4–1.2%) in the basal medium on amylase production were examined. Growth was followed by optical density measurements at 600 nm (OD600 ). Samples were diluted in deionised water and adjusted at values between 0.2 and 0.8 OD600 . For bioreactor experiments, 100 mL of preinoculum culture (grown in LB medium) were transferred to the bioreactor containing 900 mL of semicomplex growth medium. All growths were carried out using a Biostat B bioreactor (Sartorius) equipped with a 2 L fermentation vessel. The pH was maintained at 6.50 ± 0.05 by adding 30% H3 PO4 solution to the reactor. The temperature was kept at 37 ◦ C. The pO2 value was maintained at 50% of air saturation by adapting the stirrer speed between 450 and 900 rpm and supplying air (enriched with pure oxygen when necessary) at a space velocity of 2 vvm. The end of the batch phase was identified by a reduction in the oxygen consumption rate and an increase in the acid addition rate. The cultures were centrifuged at 10,000 rpm for 20 min at 4 ◦ C using a Beckman J2-21 M/E centrifuge and the cell-free supernatants were used as a crude enzyme preparation.
A total amount of 900 mL fermentation broth was lyophilized and concentrated 10 times in order to recover amylase activities. 90 mL of broth (86.9 g protein/mL, 51.4 U/mL) was loaded onto Sephadex G-100 column (50/100, Pharmacia, Uppsala, Sweden) equilibrated with 0.15 M NaCl in 20 mM acetate buffer, pH 6.0 at a flow rate of 100 mL/h. Fractions of 20 mL were collected and assayed for enzyme activity. Fractions containing amylase activity were pooled and concentrated by lyophilization. 2 mL of obtained amylase (0.18 mg/mL, 308.5 U/mL) was subjected to several gel filtrations on Superose 12 column (16/60 HR) on a fast protein liquid chromatography (FPLC) system (Pharmacia, Uppsala). The column was previously equilibrated with 0.9% NaCl, 20 mM acetate buffer, pH 6.0 at a flow rate of 0.5 mL/min. 0.5 mL fractions were collected and assayed for amylase activity.
2.3. ˛-Amylase activity assay
2.7.2. Optimum pH studies To determine the optimum pH of ␣-amylase activity against soluble starch, 50 L of the purified enzyme and a series of 50 mM buffers in the pH range from 4.0 to 10.5 were used (acetate, pH 4.0–6.0; phosphate, pH 6.5–8.5; carbonate/bicarbonate, pH 9.0–10.5) and the residual activity was determined under the enzyme assay conditions.
␣-Amylase activity was determined by measuring the formation of reducing sugars released during starch hydrolysis. The reaction mixture containing 0.05 mL of appropriately diluted enzyme and 0.45 mL of 1.0% (w/v) soluble starch (Merck) in 50 mM phosphate buffer (pH 6.5) was incubated at 75 ◦ C for 15 min. The amount of liberated reducing sugar was determined by the dinitrosalicylic (DNS) acid method [11]. One unit of amylase activity was defined as the amount of enzyme that released 1 mol of reducing end groups per minute at 75 ◦ C. D-Glucose was used to construct a standard curve.
2.7. Enzyme characterization 2.7.1. Molecular mass The apparent molecular mass of ␣-amylase subunits was determined by sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) by comparison with standards. Sample was mixed with reducing sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 5% beta-mercapthoethanol, 10% glycerol and 0.002% bromophenol blue) and heated for 3 min in boiling water bath. Electrophoresis was carried out according to Laemmli [14] using 10% acrylamide. LMW-SDS marker kit (GE Healthcare) was used as molecular mass standards. After the run gel was silver stained [15]. The molecular mass of native ␣-amylase was determined by gel filtration on FPLC Superose 12 column (10 mm × 300 mm). The column was previously equilibrated with 0.9% NaCl, 20 mM acetate buffer, pH 6.0 at a flow rate of 36 mL/h. 150 L fractions were collected and assayed for amylase activity. The column was calibrated using bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and lysozyme (14.4 kDa) (GE Healthcare).
2.7.3. Effect of temperature on activity and stability The effect of temperature on ␣-amylase activity was studied from 40 to 100 ◦ C. Thermal inactivation of the purified enzyme
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Fig. 1. Effect of (A) starch and (B) tryptone concentration on ␣-amylase production during 72 h of submerged fermentation of B. licheniformis ATCC 9945a at 37 ◦ C in shaking flasks. Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).
was also examined by incubating the enzyme preparation 1–6 h at different temperatures. Aliquots were withdrawn at desired time intervals and the remaining activity was measured under enzyme assay conditions. The non-heated enzyme was considered as control (100%). 2.7.4. Effects of divalent metal ions The effect of metal ions on amylolytic activity was determined by adding 2 mM of each ion chloride to the standard assay. Various concentrations of the cations were first incubated with ␣-amylase at 30 ◦ C for 10 min before starch was added to the reaction mixture. Relative activities were expressed as a percentage of the activity of the untreated control taken as 100%. 2.7.5. Raw starch degradation and adsorbability Raw starch grains isolated from wheat (Triticum sp.), potato (Solanum tuberosum), horseradish (Armoracia rusticana), corn (Zea mays) and triticale (Triticale hexaploide Lart.) were washed 5 times with Milli-Q water to remove substances which may interfere with ␣-amylase activity. The grains were desiccated and 1% of each starch grain was suspended in 980 L of 50 mM phosphate buffer pH 6.5. The reaction was started by adding 10 L of enzyme solution (0.7 units). Mixtures of enzyme and raw starches were incubated at 65 ◦ C for 1, 2, 3 and 4 h, then centrifuged and supernatants used
to determine released sugars with a DNS acid method with maltose as the standard. The extent of hydrolysis of raw starch (Rh ) was defined by following formula: Rh (%) = (A1 /A0 ) × 100, where A1 was the amount of sugar in the supernatant after the reaction, and A0 was the amount of raw starch before the reaction [16]. After 4 h of hydrolysis all starch grains were washed two times with pure ethanol and examined under light microscopy using Leitz Diaplan microscope with 400 times magnification. Pictures were taken using digital CCD Nikon camera. To detect the adsorption of enzyme onto raw starch 1% and 10% of each starch grain was suspended in 980 L and 890 L of 50 mM phosphate buffer pH 6.5 with 10 L of enzyme solution (0.7 units) and left for 10 min at 65 ◦ C with occasional shaking [17]. After centrifugation at 14,000 × g for 2 min, the amylase activity of the supernatant (B) was measured and compared to the original amylase activity (A). The percentage of adsorption was defined by the following equation: (%) = (A − B)/A × 100 [17]. 2.8. Statistical analysis Each data point represents the mean of three independent assays. The data in Figs. 1, 2A, 3–5 and Tables 2 and 3 are presented as the mean ± standard error of the mean (SEM). The data in the Tables 2 and 3 are presented as percentages, taking the control value as 100%.
Fig. 2. (A) Pattern of growth and extracellular ␣-amylase production during 24 h of submerged fermentation of B. licheniformis ATCC 9945a at 37 ◦ C in laboratory fermenter. – enzyme activity, 䊉 – OD (600 nm). Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means). (B) Zymogram of ␣-amylases after isoelectric focusing. (1) Crude enzyme preparation (pI) positions of standard proteins pI values. (C) SDS-PAGE of ␣-amylase. (kDa) molecular masses of standard proteins, (1) the purified enzyme. Arrow indicates position of the band referred to ␣-amylase.
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Fig. 3. Effect of (A) pH and (B) temperature on ␣-amylase activity. Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).
3. Results and discussion 3.1. ˛-Amylase production A semisynthetic medium for maximal production of extracellular amylase by a B. licheniformis ATCC 9945a strain was formulated. Levels of amylase in crude culture supernatant varied greatly in response to the carbon and nitrogen source used for growth of the strain. On the basis of amylase productivity level in shake flask cultures after 72 h of growing (results not shown), growth medium containing starch and tryptone was selected as the best medium. Tryptone was the only nitrogen source that induced ␣amylase productivity. Moreover, ␣-amylase biosynthesis appeared to be independent of starch availability, since enzyme activity was detected in culture broth in the presence of tryptone as a single carbon source. This was also the case with amylase from B. subtilis [18]. Nevertheless addition of starch in growth medium increased the level of secreted enzyme. These results were in agreement with previously reported maximum ␣-amylase production when starch was used as a carbon source [19,20]. Experiments carried out at different initial concentration of starch and tryptone showed that 1% starch and 0.5% tryptone were necessary for maximum ␣-amylase production, inducing 5.2 IU/mL of amylase to be secreted after 72 h of cultivation (Fig. 1). The results on the time profiles for both ␣-amylase production and biomass of B. licheniformis ATCC 9945a strain grown in growth medium in the fermenter are shown in Fig. 2A. When the strain was cultivated in a laboratory fermenter ␣-amylase production peaked (5.2 U/mL) at 24 h and was found to remain constant during next 16 h. It was observed that maximum ␣-amylase production
Fig. 4. Effect of temperature on the stability of ␣-amylase at pH 6.5. (A) 70 ◦ C, (B) 80 ◦ C, (C) 90 ◦ C. – without CaCl2 , 䊉 – with CaCl2 . Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).
Fig. 5. Hydrolysis of raw starches by ␣-amylase. – raw corn starch, 䊉 – raw potato starch, – raw horseradish starch, – raw triticale starch, ♦ – raw wheat starch.
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Table 1 Purification of ␣-amylase from Bacillus licheniformis ATCC 9945a strain. Purification stage Crude midgut extract Sephadex G-100 FPLC Superose 12
Total protein (g) 7821 1071 486
Total activity (U)
Specific activity (U/mg)
Purification (-fold)
Yield (%)
4626 1851 1782
591.5 1728.3 3666.7
1 2.9 6.2
100 40 38
occurred when optical density started to decrease after it reached peak. Amylase production by this strain was found to be growth independent as maximum enzyme production was observed during the end of stationary phase, when the OD was starting to decrease. Similar result was obtained with amylase from Bacillus thermooleovorans [21] and amylase from B. subtilis and Bacillus amyloliquefaciens [22]. ␣-Amylase activity from the fermentation broth obtained in batch laboratory fermenter was detected after isoelectric focusing using in-gel activity staining in the slightly alkaline region (Fig. 2B). Totally six isoforms in the crude extract were detected. After comparing with standard protein pI values it can be seen that all isoforms were ranging from 6.20 to 8.15. pI for two most dominant isoforms of amylase were calculated to be 6.80 and 7.11. 3.2. ˛-Amylase purification The isolation and purification of the B. licheniformis ATCC 9945a ␣-amylase was monitored by ability of enzyme to hydrolyse the soluble starch. The purification procedure consisted of two gel filtration steps: gel filtration on Sephadex G-100 and on Superose 12 column. The results of the purification are summarized in Table 1. ␣-Amylase was purified 6-fold with a yield of 38% and was homogenous according to SDS PAGE (Fig. 2C, lane 1). The purification procedure yielded a ␣-amylase with a very high specific activity of 3666.7 U/mg. 3.3. ˛-Amylase characterization The apparent MM of the enzyme was estimated to be 31 kDa according to SDS-PAGE (Fig. 2C). Purified ␣-amylase rechromatographed on the FPLC Superose 12 column yielded a single peak of activity eluting at 15 mL. The molecular mass of ␣-amylase was calculated from the plot of log MM versus Ve using standard proteins as markers after elution from a Superose 12 FPLC column (results not shown). The molecular mass of ␣-amylase was estimated to be 32 kDa. These data suggest that B. licheniformis ATCC 9945a ␣-amylase is a monomer. Different molecular masses of the ␣-amylases from various Bacillus sp., including B. licheniformis, ranging from 42 to 150 kDa [23] and from 23 to 50 kDa [24] have been reported. Maximum ␣-amylase activity using soluble starch as a substrate was observed at pH 6.5 in phosphate buffer (Fig. 3A). Between pH 5.0 and 9.0 there were still 50% of activity remaining. Similar pH optimum has been found for previously reported B. licheniformis ␣-amylases [23]. Temperature-dependant ␣-amylase activity was determined within a temperature range from 40 to 100 ◦ C. ␣-Amylase showed maximal activity at 90 ◦ C while at 100 ◦ C started to denature (Fig. 3B). Optimum temperature of 90 ◦ C was also found for B. licheniformis isolated from cassava steep water and activated sludge [25,26]. The thermal stability of ␣-amylase was determined at 70 ◦ C, ◦ 80 C and 90 ◦ C in the presence and absence of CaCl2 . The presence of Ca2+ had positive effect on enzyme thermostability (Fig. 4). At 70 ◦ C the activity of ␣-amylase dropped to 35% after 6 h while in the presence of CaCl2 enzyme retained 55% of its activity for the same period of time. At 90 ◦ C the activity of ␣-amylase dropped to
55% after 20 min while in the presence of CaCl2 enzyme retained 85% of its activity for the same period of time. This effect has been recognized for other amylolytic enzymes previously [17]. The improvement in ␣-amylase thermostability against thermal inactivation in the presence of Ca2+ has been correlated with the formation of a calcium–sodium–calcium metal triad in the main Ca2+ -binding site of the enzyme [27]. External factors such as cations and additives have been known to affect the activity of enzyme. Among the divalent cation tested 2 mM Hg2+ completely inhibited ␣-amylase activity (Table 2). Inhibition of Bacillus sp. ␣-amylase with heavy metals like Hg2+ has been well recognized [8,28]. Fe2+ inhibited 40% of amylase activity; Zn2+ and Cu2+ between 15 and 25%, while Cd2+ , Mg2+ and Mn2+ had little effect on ␣-amylase activity. Other cations, especially Co2+ , showed slight stimulation of ␣-amylase activity. EDTA inhibited 30% of enzyme activity. These results are in agreement with the work of Adeyanju et al. [25] and Lin et al. [17] who also reported that EDTA, Cu2+ and Zn2+ had inhibitory effect on amylase activity. Activation with Co2+ was reported for ␣-amylase [28,29] and with Co2+ and Ni2+ for B. licheniformis ␣-amylase [25]. The potential raw starch digestion ability of B. licheniformis ATCC 9945a ␣-amylase was investigated by monitoring the extent of hydrolysis of various raw starch granules in a short duration of time (Fig. 5). Hydrolysis rates of raw triticale, wheat, potato, horseradish and corn starches, at 1% concentration were 63, 60, 59, 52 and 37%, respectively, in a period of 4 h. Comparison to other Bacillus sp amylase [16,30,31] due to the different reaction conditions that were used for every particular enzyme is not quite possible, however
Table 2 The effect of metal divalent cations on ␣-amylase from Bacillus licheniformis ATCC 9945a. Each value represents the mean of three independent assays ± standard error value (the standard errors were less than 5% of the means). Compound
Residual activity (%)
␣-amylase MgSO4 × 7H2 O CaCl2 CoCl2 × 6H2 O NiSO4 × 7H2 O CdCl2 MnCl2 × 4H2 O ZnSO4 CuCl2 × 2H2 O EDTA FeSO4 × 7H2 O HgCl2
100.0 103.7 111.6 124.9 118.6 91.6 91.2 86.9 77.1 69.6 60.9 0.1
± ± ± ± ± ± ± ± ± ± ± ±
1.0 2.1 2.2 3.1 2.7 2.9 1.3 1.9 0.7 0.9 2.1 0.0
Table 3 Percentage adsorption of B. licheniformis ATCC 9945a raw starch digesting ␣-amylase on starch granules from different sources. Each value represents the mean of three independent assays ± standard error value (the standard errors were less than 5% of the means). Starch
Corn Potato Horseradish Triticale Wheat
Adsorption (%) 1% starch
10% starch
31.5 ± 1.1 35.7 ± 0.6 31.2 ± 2.2 36.4 ± 1.3 30.1 ± 2.7
24.5 ± 3.9 31.2 ± 2.7 62.1 ± 1.4 50.6 ± 2.5 52.4 ± 2.1
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amylase (0.16 U/mg of starch) hydrolyzed 0.63% of raw corn, sweet potato and wheat starch by 36%, 35% and 32%, respectively after five days at 30 ◦ C [32]. At the same time raw starch digesting ␣-amylase showed approximately 30–36% adsorbability onto 1% of raw starches used, while adsorbability onto 10% of raw starches varied more, Table 3. The highest adsorption (62%) was observed for the smallest (horseradish) starch granules. Strong correlation between the hydrolysis of raw starch and the adsorption to raw starch has been found in bacterial ␣-amylase [16,17]. Hamilton et al. [33] classified bacterial ␣-amylases into two groups; raw starch digesting and raw starch adsorbing ␣-amylases and raw starch digesting non-raw starch adsorbing ␣-amylases. Adsorption of ␣-amylases to raw starch and the subsequent hydrolysis of raw starch are associated with the presence of a starch-binding domain, while amylase in the second group is thought to have certain affinity sites, which are involved in the interaction between the enzyme and raw starch. Since the amount of B. licheniformis ATCC 9945a raw ␣-amylase adsorbed did not correlate with the hydrolysis rate of 1% raw starches, further studies are necessary to reveal the correlation between the hydrolysis and adsorption rate for this enzyme. Enzymes capable of digesting various raw starch granules are economically attractive as they can increase the range of starch sources for direct hydrolysis. As observed by light microscopy (Fig. 6), after 4 h of hydrolysis the number of raw starch granules was significantly reduced while the structure of visible starch granules was more or less completely damaged. High efficiency of B. licheniformis ATCC 9945a raw starch digesting ␣-amylase was demonstrated by the reduction of numbers of treated raw corn starch granules (Fig. 6, panel 1) and raw horseradish starch granules (Fig. 6, panel 3) as well as by the reduction of number and the existence of holes in the hydrolyzed raw starch potato granules (Fig. 6, panel 2), raw starch triticale granules (Fig. 6, panel 4) and raw starch wheat granules (Fig. 6, panel 5). Since B. licheniformis ATCC 9945a raw starch digesting ␣-amylase showed no complete raw starch adsorbability, raw starch hydrolysis depended on the thrust between the enzyme and specific starch granule. Because of that digested starch showed random style. The potential raw starch digestion ability of B. licheniformis ATCC 9945a ␣-amylase was confirmed by monitoring the hydrolysis of raw starches directly from the dry-milled corn, wheat and triticale flour. After 8 h of incubation, raw starches could not be detected with iodine staining solution (results not shown).
4. Conclusion
Fig. 6. Light microscopies of (A) untreated and (B) treated raw starch granules with ␣-amylase. (1) Raw corn starch, (2) raw potato starch, (3) raw horseradish starch, (4) raw triticale starch, (5) raw wheat starch.
concerning very low units of B. licheniformis ATCC 9945a ␣-amylase applied it can be concluded that this enzyme is highly efficient in raw starch hydrolysis in a short duration of time. Mitsuiki et al. [16] have used 8 U/mL of enzyme for the hydrolysis of 0.6% starch at 40 ◦ C for 5 days to obtain 50% hydrolysis in the best case. Liu and Xu [30] obtained 57.5%, 53% and 45.1% hydrolysis of 1% raw corn, wheat and potato starches, respectively, after 8 h by employing Bacillus sp. YX-1 amylase (1 U/mg of starch). Bacillus subtilis 65
The present study has yielded a highly efficient, thermostable, diverse raw starch digesting ␣-amylase by submerged fermentation from a B. licheniformis ATCC 9945a. The advantages of this amylase compared to previously reported ones are related to a high hydrolytic affinity of this enzyme towards different types of raw starch granules; cereals, tubers and roots. Since the most raw starch digesting alpha amylase reported to date hardly digest potato starch as well as the starch from cereals and tubers with the same high efficiency and having in mind that corn, wheat and potato are the most important sources of starch in EU, B. licheniformis ATCC 9945a raw starch digesting ␣-amylase capable of digesting all these types of raw starches efficiently is economically attractive. Enzyme appears to be a good candidate for the direct hydrolysis of diverse raw starches, using very low doses (0.07 U/mg of starch) and omitting energy intensive and expensive gelatinization step. Due to the importance of these findings, further studies will be carried on in order to commercialize the production process after necessary optimization for enhanced enzyme production.
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Acknowledgements This work was supported by the Serbian Ministry of Science and Technological Development, project grant number 142026B and by the Spanish MICINN, project number CTQ2008-00578. N.B. acknowledges Serbian Ministry of Science and Technological Development for postdoctoral grant. The UAB group constitutes the Research group 2009SGR281 and Biochemical Engineering Unit of the Reference Network in Biotechnology (XRB), Generalitat de Catalunya. References [1] K. Buchholz, J. Seibel, Industrial carbohydrate biotransformations, Carbohydr. Res. 343 (2008) 1966–1979. [2] I. Roy, M.N. Gupta, Hydrolysis of starch by a mixture of glucoamylase and pullulanase entrapped individually in calcium alginate beads, Enzyme Microb. Technol. 34 (2004) 26–32. [3] H. Sun, P. Zhao, X. Ge, Y. Xia, Z. Hao, J. Liu, M. Peng, Recent advances in microbial raw starch degrading enzymes, Appl. Biochem. Biotechnol. 160 (2010) 988–1003. [4] G.H. Robertson, D.W.S. Wong, C.C. Lee, K. Wagschal, M.R. Smith, W.J. Orts, Native or raw starch digestion: a key step in energy efficient biorefining of grain, J. Agric. Food Chem. 54 (2006) 353–365. [5] N. Bawa, D. Bear, G. Hill, C. Niu, W. Roesler, Fermentation of glucose and starch particles using an inexpensive medium, J. Chem. Technol. Biotechnol. 85 (2010) 441–446. [6] H. Sun, X. Ge, L. Wang, P. Zhao, M. Peng, Microbial production of raw starch digesting enzymes, Afr. J. Biotechnol. 8 (2009) 1734–1739. [7] M. Shafiei, A.-A. Ziaee, M.A. Amoozegar, Purification and biochemical characterization of a novel SDS and surfactant stable, raw starch digesting, and halophilic ␣-amylase from a moderately halophilic bacterium, Nesterenkonia sp. strain F, Process. Biochem. 45 (2010) 694–699. [8] N. Goyal, J.K. Gupta, S.K. Soni, A novel raw starch digesting thermostable ␣amylase from Bacillus sp. I-3 and its use in the direct hydrolysis of raw potato starch, Enzyme Microb. Technol. 37 (2005) 723–734. [9] B.G. Dettori, F.G. Priest, J.R. Stark, Hydrolysis of starch granules by the amylase from Bacillus stearothermophilus NCA 26, Process Biochem. 27 (1992) 17–21. [10] C.G. Qates, Towards an understanding of starch granule structure and hydrolysis, Trends Food Sci. Technol. 8 (1997) 375–382. [11] P. Bernfeld, Amylases, ␣ and , in: P. De Murray (Ed.), Methods in Enzymology, vol. I, Deutcher Academic Press Inc., San Diego, California, 1955, pp. 149–158. ´ V. Nenadovic, ´ J. Ivanovic, ´ Z. Vujˇcic, ´ Purification and [12] B. Dojnov, N. Boˇzic, properties of midgut ␣-amylase isolated from Morimus funereus (Coleoptera: cerambycidae) larvae, Comp. Biochem. Physiol. 149B (2008) 153–160. [13] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [14] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Production and properties of the highly efficient raw starch digesting ␣-amylase from a Bacillus licheniformis ATCC 9945a Nataˇsa Boˇzic´ a,∗ , Jordi Ruiz b , Josep López-Santín b , Zoran Vujˇcic´ c a
Centre of Chemistry, Institute of Chemistry, Technology and Metallurgy, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia Departament d’Enginyeria Química, Escola d’Enginyeria, Unitat de Biocatàlisi Aplicada Associada al IIQA (CSIC), Universitat Autònoma de Barcelona, Edifici Q, 08193 Bellaterra, Spain c Department of Biochemistry, Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbia b
a r t i c l e
i n f o
Article history: Received 23 June 2010 Received in revised form 14 September 2010 Accepted 23 October 2010
Keywords: ␣-Amylase Bacillus licheniformis Raw starch hydrolysis Thermostable Purification
a b s t r a c t Highly efficient raw starch digesting ␣-amylase was produced after 24 h of batch fermentation of Bacillus licheniformis ATCC 9945a in laboratory bioreactor at 37 ◦ C. The enzyme was purified by gel filtration chromatographies with 6-fold increase of specific activity and 38% recovery and showed a molecular mass of 31 kDa by SDS-PAGE. The purified enzyme had an optimum pH of 6.5 and optimum temperature of 90 ◦ C. The purified ␣-amylase in the presence of CaCl2 retained 55% of its activity after 6 h of incubation at 70 ◦ C. Co2+ , Ni2+ and Ca2+ slightly stimulated, while Hg2+ completely inhibited ␣-amylase activity. Hydrolysis rates of raw triticale, wheat, potato, horseradish and corn starches, at 1% concentration were 63, 60, 59, 52 and 37%, respectively, in a period of 4 h. The properties of the purified enzyme proved its high efficacy for digesting diverse raw starches below gelatinization temperature and, hence, its potential commercial value to use as an industrial enzyme. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Starch is the most important carbon and energy source among plant carbohydrates, and it is the second following cellulose in total biosynthesis [1]. It represents an inexpensive source for production of glucose, fructose and maltose syrups which are widely used in food industries [2] and for obtaining the products of their fermentation such as fuel ethanol. Besides agricultural crops, starch is a significant component of domestic and commercial wastes and these could become useful resources to be converted into ethanol. Conventionally, conversion of starch to glucose is a high temperature, liquid-phase enzymatic hydrolysis process which requires a high-energy input resulting in increased production cost of starch-based products [3]. An alternative, cold hydrolysis approach represents a step towards a “green” method for the production of fuel ethanol. Regarding energy costs, effective utilization of natural resources, minimization of the formation of pollutants and viscosity (handling) problems, use of raw starch digesting enzymes that can perform direct hydrolysis of raw starch below gelatinization temperature is desirable [4]. The removal of the cooking stage also has
∗ Corresponding author. Tel.: +381 113282393; fax: +381 112636061. ´ [email protected] E-mail addresses: [email protected] (N. Boˇzic), ´ (J. Ruiz), [email protected] (J. López-Santín), [email protected] (Z. Vujˇcic). 1369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.10.014
the potential to increase the value of the co-products since valuable proteins would undergo less thermal stress [5]. In spite of the wide distribution of raw starch digesting enzymes, microbial sources have many advantages for the industrial production such as cost effectiveness, consistency, less time and space required for production and ease of process modification and optimization [6]. Since many of the commercially available amylases do not withstand industrial reaction conditions, isolation and characterization of novel amylases with desirable properties is very important [7]. Several raw starch digesting alpha amylases which can directly hydrolyze the raw starch in a single step at temperatures below the gelatinization temperature of starch has been reported [8]. Raw starch digesting amylases from Bacillus sp. usually need prolonged time of incubation for efficient raw starch hydrolysis and are not able to digest all types of starch granules with same efficiency [9]. Often, better results were obtained with thermostable raw starch digesting amylases at temperatures between 60 and 70 ◦ C [8]. Raw cereal starches are more completely and rapidly hydrolyzed than those from tubers or roots when digested by single, purified enzymes [10]. Moreover most raw starch digesting alpha amylase reported to date hardly digest potato starch [8,10]. Since corn, wheat and potato are the most important sources of starch in EU [1], enzymes that are capable of digesting all these types of raw starches efficiently are economically attractive.
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The aim of this study was to produce and characterize raw starch digesting ␣-amylase from Bacillus licheniformis ATCC 9945a capable for direct hydrolysis of cereal, tuber and root raw starches below the gelatinization temperature of starch with same efficiency in short duration of time. 2. Materials and methods 2.1. Chemicals All reagents and solvents used were of the highest available purity and at least of analytical grade. They were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated. Raw potato, corn, triticale, wheat and horseradish starches were isolated in our laboratory according to standard recommended procedure.
2.4. Zymogram and isoelectric point of crude ˛-amylase Isoelectric focusing was performed using Multiphor II electrophoresis system (Pharmacia-LKB Biotechnology) according to manufacturer’s instruction. Focusing was carried out on 7.5% actylamide gel with ampholytes in a pH range 3.0–10.0, at 7 W constant powers for 1.5 h at 10 ◦ C. Broad pI kit (GE Healthcare) was used as isoelectric point (pI) markers. After the run, ␣-amylases were detected using in-gel activity staining with I2 /KI staining solution according to a previously published method [12]. ␣-Amylase activity appeared as clear bands on a dark background. 2.5. Determination of protein concentration Protein concentration was determined by the Bradford method [13] using bovine serum albumin as the protein standard.
2.2. Media composition and cultivation conditions
2.6. Enzyme purification
Frozen stock aliquots containing glycerol prepared from exponential phase Bacillus licheniformis ATCC 9945a cultures grown in Luria–Bertani media (LB) were stored at −80 ◦ C. Precultures were routinely grown in Luria–Bertani (LB) broth medium composed of (g/L): peptone, 10.0; yeast extract, 5.0; NaCl, 10.0. Preinoculum cultures were grown from glycerol stocks in a 100 mL shake flask containing 15 mL LB media and incubated overnight at 37 ◦ C in a rotary shaker at 150 rpm. For shake flasks experiments 5 mL of preinoculum cultures were transferred aseptically to a 500 mL baffled shake flask containing 100 mL of semicomplex growth medium. Unless otherwise stated, the growth medium used for ␣-amylase production was composed of (g/L): soluble starch, 10; tryptone, 5; MgSO4 ·7H2 O, 0.5; K2 HPO4 , 2; Na2 HPO4 , 5; NaCl, 2; (NH4 )2 SO4 , 4; FeCl3 , 0.05; CaCl2 ·2H2 O, 0.05. The medium was adjusted to pH 6.5. Cultivations were maintained at 37 ◦ C with agitation at 150 rpm for 72 h. The effect of different levels of tryptone (0.1–1.5%) and different levels of starch (0.4–1.2%) in the basal medium on amylase production were examined. Growth was followed by optical density measurements at 600 nm (OD600 ). Samples were diluted in deionised water and adjusted at values between 0.2 and 0.8 OD600 . For bioreactor experiments, 100 mL of preinoculum culture (grown in LB medium) were transferred to the bioreactor containing 900 mL of semicomplex growth medium. All growths were carried out using a Biostat B bioreactor (Sartorius) equipped with a 2 L fermentation vessel. The pH was maintained at 6.50 ± 0.05 by adding 30% H3 PO4 solution to the reactor. The temperature was kept at 37 ◦ C. The pO2 value was maintained at 50% of air saturation by adapting the stirrer speed between 450 and 900 rpm and supplying air (enriched with pure oxygen when necessary) at a space velocity of 2 vvm. The end of the batch phase was identified by a reduction in the oxygen consumption rate and an increase in the acid addition rate. The cultures were centrifuged at 10,000 rpm for 20 min at 4 ◦ C using a Beckman J2-21 M/E centrifuge and the cell-free supernatants were used as a crude enzyme preparation.
A total amount of 900 mL fermentation broth was lyophilized and concentrated 10 times in order to recover amylase activities. 90 mL of broth (86.9 g protein/mL, 51.4 U/mL) was loaded onto Sephadex G-100 column (50/100, Pharmacia, Uppsala, Sweden) equilibrated with 0.15 M NaCl in 20 mM acetate buffer, pH 6.0 at a flow rate of 100 mL/h. Fractions of 20 mL were collected and assayed for enzyme activity. Fractions containing amylase activity were pooled and concentrated by lyophilization. 2 mL of obtained amylase (0.18 mg/mL, 308.5 U/mL) was subjected to several gel filtrations on Superose 12 column (16/60 HR) on a fast protein liquid chromatography (FPLC) system (Pharmacia, Uppsala). The column was previously equilibrated with 0.9% NaCl, 20 mM acetate buffer, pH 6.0 at a flow rate of 0.5 mL/min. 0.5 mL fractions were collected and assayed for amylase activity.
2.3. ˛-Amylase activity assay
2.7.2. Optimum pH studies To determine the optimum pH of ␣-amylase activity against soluble starch, 50 L of the purified enzyme and a series of 50 mM buffers in the pH range from 4.0 to 10.5 were used (acetate, pH 4.0–6.0; phosphate, pH 6.5–8.5; carbonate/bicarbonate, pH 9.0–10.5) and the residual activity was determined under the enzyme assay conditions.
␣-Amylase activity was determined by measuring the formation of reducing sugars released during starch hydrolysis. The reaction mixture containing 0.05 mL of appropriately diluted enzyme and 0.45 mL of 1.0% (w/v) soluble starch (Merck) in 50 mM phosphate buffer (pH 6.5) was incubated at 75 ◦ C for 15 min. The amount of liberated reducing sugar was determined by the dinitrosalicylic (DNS) acid method [11]. One unit of amylase activity was defined as the amount of enzyme that released 1 mol of reducing end groups per minute at 75 ◦ C. D-Glucose was used to construct a standard curve.
2.7. Enzyme characterization 2.7.1. Molecular mass The apparent molecular mass of ␣-amylase subunits was determined by sodium-dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) by comparison with standards. Sample was mixed with reducing sample buffer (62.5 mM Tris pH 6.8, 2% SDS, 5% beta-mercapthoethanol, 10% glycerol and 0.002% bromophenol blue) and heated for 3 min in boiling water bath. Electrophoresis was carried out according to Laemmli [14] using 10% acrylamide. LMW-SDS marker kit (GE Healthcare) was used as molecular mass standards. After the run gel was silver stained [15]. The molecular mass of native ␣-amylase was determined by gel filtration on FPLC Superose 12 column (10 mm × 300 mm). The column was previously equilibrated with 0.9% NaCl, 20 mM acetate buffer, pH 6.0 at a flow rate of 36 mL/h. 150 L fractions were collected and assayed for amylase activity. The column was calibrated using bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa) and lysozyme (14.4 kDa) (GE Healthcare).
2.7.3. Effect of temperature on activity and stability The effect of temperature on ␣-amylase activity was studied from 40 to 100 ◦ C. Thermal inactivation of the purified enzyme
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Fig. 1. Effect of (A) starch and (B) tryptone concentration on ␣-amylase production during 72 h of submerged fermentation of B. licheniformis ATCC 9945a at 37 ◦ C in shaking flasks. Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).
was also examined by incubating the enzyme preparation 1–6 h at different temperatures. Aliquots were withdrawn at desired time intervals and the remaining activity was measured under enzyme assay conditions. The non-heated enzyme was considered as control (100%). 2.7.4. Effects of divalent metal ions The effect of metal ions on amylolytic activity was determined by adding 2 mM of each ion chloride to the standard assay. Various concentrations of the cations were first incubated with ␣-amylase at 30 ◦ C for 10 min before starch was added to the reaction mixture. Relative activities were expressed as a percentage of the activity of the untreated control taken as 100%. 2.7.5. Raw starch degradation and adsorbability Raw starch grains isolated from wheat (Triticum sp.), potato (Solanum tuberosum), horseradish (Armoracia rusticana), corn (Zea mays) and triticale (Triticale hexaploide Lart.) were washed 5 times with Milli-Q water to remove substances which may interfere with ␣-amylase activity. The grains were desiccated and 1% of each starch grain was suspended in 980 L of 50 mM phosphate buffer pH 6.5. The reaction was started by adding 10 L of enzyme solution (0.7 units). Mixtures of enzyme and raw starches were incubated at 65 ◦ C for 1, 2, 3 and 4 h, then centrifuged and supernatants used
to determine released sugars with a DNS acid method with maltose as the standard. The extent of hydrolysis of raw starch (Rh ) was defined by following formula: Rh (%) = (A1 /A0 ) × 100, where A1 was the amount of sugar in the supernatant after the reaction, and A0 was the amount of raw starch before the reaction [16]. After 4 h of hydrolysis all starch grains were washed two times with pure ethanol and examined under light microscopy using Leitz Diaplan microscope with 400 times magnification. Pictures were taken using digital CCD Nikon camera. To detect the adsorption of enzyme onto raw starch 1% and 10% of each starch grain was suspended in 980 L and 890 L of 50 mM phosphate buffer pH 6.5 with 10 L of enzyme solution (0.7 units) and left for 10 min at 65 ◦ C with occasional shaking [17]. After centrifugation at 14,000 × g for 2 min, the amylase activity of the supernatant (B) was measured and compared to the original amylase activity (A). The percentage of adsorption was defined by the following equation: (%) = (A − B)/A × 100 [17]. 2.8. Statistical analysis Each data point represents the mean of three independent assays. The data in Figs. 1, 2A, 3–5 and Tables 2 and 3 are presented as the mean ± standard error of the mean (SEM). The data in the Tables 2 and 3 are presented as percentages, taking the control value as 100%.
Fig. 2. (A) Pattern of growth and extracellular ␣-amylase production during 24 h of submerged fermentation of B. licheniformis ATCC 9945a at 37 ◦ C in laboratory fermenter. – enzyme activity, 䊉 – OD (600 nm). Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means). (B) Zymogram of ␣-amylases after isoelectric focusing. (1) Crude enzyme preparation (pI) positions of standard proteins pI values. (C) SDS-PAGE of ␣-amylase. (kDa) molecular masses of standard proteins, (1) the purified enzyme. Arrow indicates position of the band referred to ␣-amylase.
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Fig. 3. Effect of (A) pH and (B) temperature on ␣-amylase activity. Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).
3. Results and discussion 3.1. ˛-Amylase production A semisynthetic medium for maximal production of extracellular amylase by a B. licheniformis ATCC 9945a strain was formulated. Levels of amylase in crude culture supernatant varied greatly in response to the carbon and nitrogen source used for growth of the strain. On the basis of amylase productivity level in shake flask cultures after 72 h of growing (results not shown), growth medium containing starch and tryptone was selected as the best medium. Tryptone was the only nitrogen source that induced ␣amylase productivity. Moreover, ␣-amylase biosynthesis appeared to be independent of starch availability, since enzyme activity was detected in culture broth in the presence of tryptone as a single carbon source. This was also the case with amylase from B. subtilis [18]. Nevertheless addition of starch in growth medium increased the level of secreted enzyme. These results were in agreement with previously reported maximum ␣-amylase production when starch was used as a carbon source [19,20]. Experiments carried out at different initial concentration of starch and tryptone showed that 1% starch and 0.5% tryptone were necessary for maximum ␣-amylase production, inducing 5.2 IU/mL of amylase to be secreted after 72 h of cultivation (Fig. 1). The results on the time profiles for both ␣-amylase production and biomass of B. licheniformis ATCC 9945a strain grown in growth medium in the fermenter are shown in Fig. 2A. When the strain was cultivated in a laboratory fermenter ␣-amylase production peaked (5.2 U/mL) at 24 h and was found to remain constant during next 16 h. It was observed that maximum ␣-amylase production
Fig. 4. Effect of temperature on the stability of ␣-amylase at pH 6.5. (A) 70 ◦ C, (B) 80 ◦ C, (C) 90 ◦ C. – without CaCl2 , 䊉 – with CaCl2 . Each data point represents the mean of three independent assays (the standard errors were less than 5% of the means).
Fig. 5. Hydrolysis of raw starches by ␣-amylase. – raw corn starch, 䊉 – raw potato starch, – raw horseradish starch, – raw triticale starch, ♦ – raw wheat starch.
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Table 1 Purification of ␣-amylase from Bacillus licheniformis ATCC 9945a strain. Purification stage Crude midgut extract Sephadex G-100 FPLC Superose 12
Total protein (g) 7821 1071 486
Total activity (U)
Specific activity (U/mg)
Purification (-fold)
Yield (%)
4626 1851 1782
591.5 1728.3 3666.7
1 2.9 6.2
100 40 38
occurred when optical density started to decrease after it reached peak. Amylase production by this strain was found to be growth independent as maximum enzyme production was observed during the end of stationary phase, when the OD was starting to decrease. Similar result was obtained with amylase from Bacillus thermooleovorans [21] and amylase from B. subtilis and Bacillus amyloliquefaciens [22]. ␣-Amylase activity from the fermentation broth obtained in batch laboratory fermenter was detected after isoelectric focusing using in-gel activity staining in the slightly alkaline region (Fig. 2B). Totally six isoforms in the crude extract were detected. After comparing with standard protein pI values it can be seen that all isoforms were ranging from 6.20 to 8.15. pI for two most dominant isoforms of amylase were calculated to be 6.80 and 7.11. 3.2. ˛-Amylase purification The isolation and purification of the B. licheniformis ATCC 9945a ␣-amylase was monitored by ability of enzyme to hydrolyse the soluble starch. The purification procedure consisted of two gel filtration steps: gel filtration on Sephadex G-100 and on Superose 12 column. The results of the purification are summarized in Table 1. ␣-Amylase was purified 6-fold with a yield of 38% and was homogenous according to SDS PAGE (Fig. 2C, lane 1). The purification procedure yielded a ␣-amylase with a very high specific activity of 3666.7 U/mg. 3.3. ˛-Amylase characterization The apparent MM of the enzyme was estimated to be 31 kDa according to SDS-PAGE (Fig. 2C). Purified ␣-amylase rechromatographed on the FPLC Superose 12 column yielded a single peak of activity eluting at 15 mL. The molecular mass of ␣-amylase was calculated from the plot of log MM versus Ve using standard proteins as markers after elution from a Superose 12 FPLC column (results not shown). The molecular mass of ␣-amylase was estimated to be 32 kDa. These data suggest that B. licheniformis ATCC 9945a ␣-amylase is a monomer. Different molecular masses of the ␣-amylases from various Bacillus sp., including B. licheniformis, ranging from 42 to 150 kDa [23] and from 23 to 50 kDa [24] have been reported. Maximum ␣-amylase activity using soluble starch as a substrate was observed at pH 6.5 in phosphate buffer (Fig. 3A). Between pH 5.0 and 9.0 there were still 50% of activity remaining. Similar pH optimum has been found for previously reported B. licheniformis ␣-amylases [23]. Temperature-dependant ␣-amylase activity was determined within a temperature range from 40 to 100 ◦ C. ␣-Amylase showed maximal activity at 90 ◦ C while at 100 ◦ C started to denature (Fig. 3B). Optimum temperature of 90 ◦ C was also found for B. licheniformis isolated from cassava steep water and activated sludge [25,26]. The thermal stability of ␣-amylase was determined at 70 ◦ C, ◦ 80 C and 90 ◦ C in the presence and absence of CaCl2 . The presence of Ca2+ had positive effect on enzyme thermostability (Fig. 4). At 70 ◦ C the activity of ␣-amylase dropped to 35% after 6 h while in the presence of CaCl2 enzyme retained 55% of its activity for the same period of time. At 90 ◦ C the activity of ␣-amylase dropped to
55% after 20 min while in the presence of CaCl2 enzyme retained 85% of its activity for the same period of time. This effect has been recognized for other amylolytic enzymes previously [17]. The improvement in ␣-amylase thermostability against thermal inactivation in the presence of Ca2+ has been correlated with the formation of a calcium–sodium–calcium metal triad in the main Ca2+ -binding site of the enzyme [27]. External factors such as cations and additives have been known to affect the activity of enzyme. Among the divalent cation tested 2 mM Hg2+ completely inhibited ␣-amylase activity (Table 2). Inhibition of Bacillus sp. ␣-amylase with heavy metals like Hg2+ has been well recognized [8,28]. Fe2+ inhibited 40% of amylase activity; Zn2+ and Cu2+ between 15 and 25%, while Cd2+ , Mg2+ and Mn2+ had little effect on ␣-amylase activity. Other cations, especially Co2+ , showed slight stimulation of ␣-amylase activity. EDTA inhibited 30% of enzyme activity. These results are in agreement with the work of Adeyanju et al. [25] and Lin et al. [17] who also reported that EDTA, Cu2+ and Zn2+ had inhibitory effect on amylase activity. Activation with Co2+ was reported for ␣-amylase [28,29] and with Co2+ and Ni2+ for B. licheniformis ␣-amylase [25]. The potential raw starch digestion ability of B. licheniformis ATCC 9945a ␣-amylase was investigated by monitoring the extent of hydrolysis of various raw starch granules in a short duration of time (Fig. 5). Hydrolysis rates of raw triticale, wheat, potato, horseradish and corn starches, at 1% concentration were 63, 60, 59, 52 and 37%, respectively, in a period of 4 h. Comparison to other Bacillus sp amylase [16,30,31] due to the different reaction conditions that were used for every particular enzyme is not quite possible, however
Table 2 The effect of metal divalent cations on ␣-amylase from Bacillus licheniformis ATCC 9945a. Each value represents the mean of three independent assays ± standard error value (the standard errors were less than 5% of the means). Compound
Residual activity (%)
␣-amylase MgSO4 × 7H2 O CaCl2 CoCl2 × 6H2 O NiSO4 × 7H2 O CdCl2 MnCl2 × 4H2 O ZnSO4 CuCl2 × 2H2 O EDTA FeSO4 × 7H2 O HgCl2
100.0 103.7 111.6 124.9 118.6 91.6 91.2 86.9 77.1 69.6 60.9 0.1
± ± ± ± ± ± ± ± ± ± ± ±
1.0 2.1 2.2 3.1 2.7 2.9 1.3 1.9 0.7 0.9 2.1 0.0
Table 3 Percentage adsorption of B. licheniformis ATCC 9945a raw starch digesting ␣-amylase on starch granules from different sources. Each value represents the mean of three independent assays ± standard error value (the standard errors were less than 5% of the means). Starch
Corn Potato Horseradish Triticale Wheat
Adsorption (%) 1% starch
10% starch
31.5 ± 1.1 35.7 ± 0.6 31.2 ± 2.2 36.4 ± 1.3 30.1 ± 2.7
24.5 ± 3.9 31.2 ± 2.7 62.1 ± 1.4 50.6 ± 2.5 52.4 ± 2.1
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amylase (0.16 U/mg of starch) hydrolyzed 0.63% of raw corn, sweet potato and wheat starch by 36%, 35% and 32%, respectively after five days at 30 ◦ C [32]. At the same time raw starch digesting ␣-amylase showed approximately 30–36% adsorbability onto 1% of raw starches used, while adsorbability onto 10% of raw starches varied more, Table 3. The highest adsorption (62%) was observed for the smallest (horseradish) starch granules. Strong correlation between the hydrolysis of raw starch and the adsorption to raw starch has been found in bacterial ␣-amylase [16,17]. Hamilton et al. [33] classified bacterial ␣-amylases into two groups; raw starch digesting and raw starch adsorbing ␣-amylases and raw starch digesting non-raw starch adsorbing ␣-amylases. Adsorption of ␣-amylases to raw starch and the subsequent hydrolysis of raw starch are associated with the presence of a starch-binding domain, while amylase in the second group is thought to have certain affinity sites, which are involved in the interaction between the enzyme and raw starch. Since the amount of B. licheniformis ATCC 9945a raw ␣-amylase adsorbed did not correlate with the hydrolysis rate of 1% raw starches, further studies are necessary to reveal the correlation between the hydrolysis and adsorption rate for this enzyme. Enzymes capable of digesting various raw starch granules are economically attractive as they can increase the range of starch sources for direct hydrolysis. As observed by light microscopy (Fig. 6), after 4 h of hydrolysis the number of raw starch granules was significantly reduced while the structure of visible starch granules was more or less completely damaged. High efficiency of B. licheniformis ATCC 9945a raw starch digesting ␣-amylase was demonstrated by the reduction of numbers of treated raw corn starch granules (Fig. 6, panel 1) and raw horseradish starch granules (Fig. 6, panel 3) as well as by the reduction of number and the existence of holes in the hydrolyzed raw starch potato granules (Fig. 6, panel 2), raw starch triticale granules (Fig. 6, panel 4) and raw starch wheat granules (Fig. 6, panel 5). Since B. licheniformis ATCC 9945a raw starch digesting ␣-amylase showed no complete raw starch adsorbability, raw starch hydrolysis depended on the thrust between the enzyme and specific starch granule. Because of that digested starch showed random style. The potential raw starch digestion ability of B. licheniformis ATCC 9945a ␣-amylase was confirmed by monitoring the hydrolysis of raw starches directly from the dry-milled corn, wheat and triticale flour. After 8 h of incubation, raw starches could not be detected with iodine staining solution (results not shown).
4. Conclusion
Fig. 6. Light microscopies of (A) untreated and (B) treated raw starch granules with ␣-amylase. (1) Raw corn starch, (2) raw potato starch, (3) raw horseradish starch, (4) raw triticale starch, (5) raw wheat starch.
concerning very low units of B. licheniformis ATCC 9945a ␣-amylase applied it can be concluded that this enzyme is highly efficient in raw starch hydrolysis in a short duration of time. Mitsuiki et al. [16] have used 8 U/mL of enzyme for the hydrolysis of 0.6% starch at 40 ◦ C for 5 days to obtain 50% hydrolysis in the best case. Liu and Xu [30] obtained 57.5%, 53% and 45.1% hydrolysis of 1% raw corn, wheat and potato starches, respectively, after 8 h by employing Bacillus sp. YX-1 amylase (1 U/mg of starch). Bacillus subtilis 65
The present study has yielded a highly efficient, thermostable, diverse raw starch digesting ␣-amylase by submerged fermentation from a B. licheniformis ATCC 9945a. The advantages of this amylase compared to previously reported ones are related to a high hydrolytic affinity of this enzyme towards different types of raw starch granules; cereals, tubers and roots. Since the most raw starch digesting alpha amylase reported to date hardly digest potato starch as well as the starch from cereals and tubers with the same high efficiency and having in mind that corn, wheat and potato are the most important sources of starch in EU, B. licheniformis ATCC 9945a raw starch digesting ␣-amylase capable of digesting all these types of raw starches efficiently is economically attractive. Enzyme appears to be a good candidate for the direct hydrolysis of diverse raw starches, using very low doses (0.07 U/mg of starch) and omitting energy intensive and expensive gelatinization step. Due to the importance of these findings, further studies will be carried on in order to commercialize the production process after necessary optimization for enhanced enzyme production.
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