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Characterization of a thermostable, CaCl2-activated and raw-starch hydrolyzing alpha-amylase from Bacillus licheniformis AT70: Production under solid state fermentation by utilizing agricultural wastes
Accepted Manuscript Title: Characterization of a thermostable, CaCl2 -activated and raw-starch hydrolyzing alpha-amylase from Bacillus licheniformis AT70: Production under solid state fermentation by utilizing agricultural wastes Author: Saideh Afrisham Arastoo Badoei-Dalfard Abdolhamid Namaki-Shoushtari Zahra Karami PII: DOI: Reference:
S1381-1177(16)30117-5 http://dx.doi.org/doi:10.1016/j.molcatb.2016.07.002 MOLCAB 3393
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
28-2-2016 2-7-2016 3-7-2016
Please cite this article as: Saideh Afrisham, Arastoo Badoei-Dalfard, Abdolhamid Namaki-Shoushtari, Zahra Karami, Characterization of a thermostable, CaCl2activated and raw-starch hydrolyzing alpha-amylase from Bacillus licheniformis AT70: Production under solid state fermentation by utilizing agricultural wastes, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of a thermostable, CaCl2-activated and raw-starch hydrolyzing alpha-amylase from Bacillus licheniformis AT70: Production under solid state fermentation by utilizing agricultural wastes
Saideh Afrisham, Arastoo Badoei-Dalfard*, Abdolhamid Namaki-Shoushtari, Zahra Karami
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran.
*Corresponding
author: Dr. Arastoo Badoei-Dalfard, Department of Biology, Faculty of
Sciences, Shahid Bahonar University of Kerman, Kerman, Iran; Tel:+9834 31322044; Fax: +9834 33222032; Email: [email protected]
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Graphical abstract
Screening
Medium optimization
Solid state fermentation
partial purification
Ca2+-activated thermal stability
Raw-Corn starch digestion
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Highlights
The production of AT70 alpha-amylase was optimized about 10 folds using starch (0.1 %) and ammonium chloride (2.0 %).
This is the first report for the production of bacterial alpha-amylase by using date waste in SSF.
The very salient improvement (2.5 folds) of the enzyme thermo-stability was observed after addition of Ca2+ into reaction mixture.
AT70 alpha-amylase showed about 34 % activity toward commercial solid detergents Shooma.
AT70 alpha-amylase exhibited remarkable hydrolytic activity in 14-20% (w/v) of raw corn starch at 55 ℃.
AT70 alpha-amylase is a thermostable, CaCl2-activated and has potent raw-starch hydrolyzing alpha-amylase
Abstract B. licheniformis AT70 which produced a thermophilic, raw-starch degrading alpha-amylase was isolated from Gorooh hot springs in Kerman province. Maximum production of AT70 alphaamylase was obtained in the presence of starch (as a carbon source) and ammonium chloride (as a nitrogen source) with 388 and 329 U/ml enzyme yield, respectively. SSF was also carried out using various agricultural and kitchen wastes and results showed that the maximum yield of AT70 alpha-amylase production was obtained by date waste and wheat bran, respectively (10 %, w/v). The thermal stability of the AT70 alpha-amylase was increased about 2.5 folds at 60 ℃. AT70 alpha-amylase showed the maximum activity at 1.5 M NaCl by 42% and local detergent Shooma enhanced the alpha-amylase activity about 34%, compared to control. Furthermore, AT70 alpha-amylase exhibited remarkable hydrolytic activity in a range of 14-20% (w/v) of raw corn starch at 55 ℃. These results indicated that AT70 alpha-amylase has great potential applications for the raw-starch degrading.
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Keywords: Alpha-amylase, Agricultural wastes, Bacillus, Raw-corn starch, Solid state fermentation, Thermophilic
1. Introduction Starch is a major constituent of agriculture and domestic wastes. These cheap agricultural wastes could become appreciated resources to be converted into high value compounds. In recent years, a global interest has been focused on the raw-starch hydrolyzing amylases to simplify the procedure of starch digestion [1, 2]. Alpha-amylase is a key enzyme in the industrial processes such as starch saccharification, textile, paper, brewing, food, distilling industries and pharmaceuticals [3]. Alpha-amylase (E.C 3.2.1.1 1,4-α-glucan-glucanohydrolase) catalysis the hydrolysis of the internal α-D-(1,4) glycosidic linkages in starch at random locations. This enzyme constitutes a class of industrial enzymes that account for ~25% of the enzyme market [4]. Alpha-amylase can be derived from several sources, such as animals, plants and microorganisms. However, enzymes from microbial sources generally meet industrial demands [4]. Whereas many of the commercial alpha-amylases do not accordance industrial process conditions, isolation and characterization of alpha-amylases with favorite features such as thermostability, alkaline stability and halophilicity is of special importance [5]. Activity of alphaamylases at higher temperatures is desirable for gelatinization and liquefaction of starch to economize the processes; as the need to continually search for more thermophile and thermostable alpha-amylases is increasing [4]. Using thermostable alpha-amylase in the industrial processes has advantages, including the decreased risk of contamination, cost of external cooling, increased diffusion rate, a better solubility of substrates, a lesser viscosity permitting accelerated mixing and pumping. Moreover, they showed a resistance in the presence of denaturing agents, solvents and proteolytic enzymes [3]. Among the genus Bacillus, B. licheniformis and B. amyloliquifaciens are identified as good producers of thermostable alphaamylase. Due to significant thermal resistance, the alpha-amylase produced by B. licheniformis widely used in the starch liquefaction process [6]. Bacterial organisms are now being progressively considered for the production of enzymes by solid-state fermentation (SSF) [7]. SSF has several advantages over submerged fermentation (SmF) because of the low capital 4
investment, the simple technique, low energy requirement, end-product inhibition, low waste water output and better product recovery [8]. SSF has been engaged in the production of thermophilic alpha-amylase by Bacillus subtilis and B. cereus MTCC 1305 [9]. In the present research, we report the biochemical characterization of a Ca2+-activated, thermophilc and detergent-stable alpha-amylase from B. licheniformis AT70 and its potential to degrade raw starch granules was also investigated. The production of the alpha-amylase was also performed by using different kinds of agro-residues and the kitchen wastes, which some of them were not reported yet i.e. Hajibadam sweets, Sehen Komath sweets in SSF manner. The properties of this enzyme, including its pH and temperature profiles, kinetic parameters, irreversible thermoinactivation and raw starch digestibility revealed that it has significant potential for the starch industry.
2. Materials and methods 2.1. Identification of the microbial strain and culture conditions Bacillus licheniformis AT70 strain which used in this research was isolated from Gorooh hot spring, located in Jiroft city, Iran. For identification of the isolated strain, a number of morphological and biochemical tests described in Bergey and especially 16S rRNA gene sequence analysis were performed [10, 11]. The isolate was detected to be an alpha-amylase producer on the starch-agar medium composed of (%) soluble starch 1.0, yeast extract 0.2, peptone 0.5, NaCl 0.1, MgSO4.7H2O 0.1, CaCl2.2H2O 0.02, after incubating at 55 ℃ for 72h. Alpha-amylase production was indicated by flooding the plates with 1% (w/v) I2 and 2% (w/v) KI solution.
2. 2. Production of alpha-amylase Alpha-amylase production was performed in the culture medium containing (g/l): KH2PO4 1.0, Na2HPO4·2H2O 3.13, tryptone 2.0, (NH4)2SO4 2.0, MgSO4·7H2O 0.05, CaCl2·2H2O 0.05, soluble starch 1.0 (pH 7.0) [12]. The medium seeded with 10% (v/v) of the pre-culture composed of nutrient broth medium and incubation was carried out at 55 ℃ with 150 rpm for 72 h. The cells were harvested after centrifugation at 10000 g for 10 min at 4 ℃ and cell-free supernatants were collected as crude enzymes. 5
2.3. Alpha amylase assay
Alpha amylase activity was measured using 3,5-dinitrosalicylic acid (DNS) reagent according to Bernfeld [13]. A mixture of 0.5 ml of enzyme solution and 0.5 ml of soluble starch (1.0%) in phosphate buffer (50 mM, pH 7.0), as substrate, was incubated at 55 ℃ for 20 min. The reaction was stopped by adding 1 ml of DNS reagent. The mixture was boiled for 5 min and after cooling in room temperature, the mixture was diluted with distilled water. The amount of reducing sugars released during the starch hydrolyzing was measured by recording absorbance at 540 nm and using the glucose standard curve. A unit of enzyme activity was defined as the amount of enzyme required to release of 1µmol of sugar reducing per 1 min under enzyme assay conditions.
2.4. Optimization of enzyme production conditions
The alpha-amylase production was optimized in the presence of the different carbon sources by supplementing the basal medium (without carbon source) with 0.1% (w/v) of starch, glucose, galactose, maltose, fructose [14]. For this order, the mediums were inoculated with 10% of an overnight culture of the AT70 isolate. After 48 h of incubation at 55 ℃, the culture mediums were harvested by centrifugation with 11000 g and 4 ℃ for 10 min and the obtained supernatants were used for the investigation of the alpha-amylase production. The organic nitrogen sources, including yeast extract, gelatin, peptone and inorganic nitrogen sources, including ammonium chloride and sodium nitrate replaced by ammonium sulphate in the basal medium at a concentration of 2.0 % [14]. In each case, the culture medium was inoculated with 10% of the overnight grown bacterial culture. After 48 h, the culture medium was centrifuged at 11000 g and 4 ℃ for 10 min and the alpha- amylase activity was assayed for the resulting supernatants. The effect of different metal ions on the enzyme production was also investigated by adding a concentration of 1.0 % of KCl, MgSO4.7H2O, NaCl and CaCl2 in the basal medium (without metal ion) [14]. Alpha-amylase activity was measured after 48 h of incubation. The effect of pH on the alpha-amylase production was studied by growing the bacterium in the culture media with
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various pH 5.0, 6.0, 7.0, 8.0 and 9.0. After 48 h of incubation, the alpha-amylase activity of the resulting supernatants was measured under the standard conditions.
2.5. Solid state fermentation
The production of alpha-amylase was investigated under solid state fermentation method on the eight different types of local agricultural wastes as substrate including banana peel, potato peel, orange peel, Hajibadam sweets, Sehen Komath sweets; a kind of local cake, wheat bran [9], rice bran and a kind of date waste. First of all, the AT70 isolate was inoculated into 100 ml of the pre-culture medium containing (%) glucose 1.0, peptone 0.25, yeast extract 0.2, NaCl 0.15, KH2PO4 0.05, MgSO4 0.05, CaCl2 0.01 and was incubated at 55 ℃ for 16h. After this period, 3 ml of pre-culture was added within the culture medium composed of 10 gr of agricultural waste and 25 ml of optimized salt solution containing ammonium chloride 2.0 gr/l, CaCl2 1.0 gr/l and KH2PO4 1.0. After 7 days, 30 ml of sodium phosphate buffer (50 mM, pH 7.0) was added to the culture mediums and the obtained mixture was passed from a textile. Then, centrifugation was performed in 11000 g for 10 min at 4 ℃. The gathered extract was used as enzyme and its activity was assayed under the standard conditions by using the DNS method.
2.6. Partial purification of the alpha-amylase
A Q-Sepharose column pre-equilibrated with 50 mM phosphate buffer pH 7.6 was used for the partial purification of the alpha-amylase produced by B. licheniformis AT70. The supernatant was passed through the pre-equilibrated column. The attached proteins were removed from the column using a linear gradient of NaCl (0.0-5.0 M) in the same buffer at a flow rate of 1.0 ml/min [15, 16]. The fractions were used to determine the alpha-amylase activity and protein content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was also performed according to the Laemmli method to determine the purity and the molecular mass of alpha-amylase [17]. Proteins were observed by silver staining method [18]. Zymography of 7
alpha-amylase was also performed in non-denatured gel containing 10% polyacrylamide and 1% soluble starch for the confirmation of the amylolytic activity [12]. Enzyme activity was detected using staining with KI/I2 solution as a white zone in dark background.
2.7. Biochemical characterization 2.7.1. Effect of pH on the enzyme activity and stability The effect of pH on the enzyme activity was studied under standard assay conditions in the following buffer systems at a concentration of 50 mM: glycine (pH 2.0 and 3.0), sodium acetate (pH 4.0 and 5.0), sodium phosphate (pH 6.0 and 7.0), Tris (pH 8.0-10.0), glycine (pH 11.0 and 12.0). pH stability was also measured by pre-incubation of the enzyme solution at room temperature for 1 h in the same buffer systems and then the residual activity was measured under standard assay conditions [5].
2.7.2. Effect of temperature on the enzyme activity and stability To investigate the effect of temperature on the alpha-amylase activity, the enzyme assay was carried out over the range of 30-80 ℃. The optimum temperature for the alpha-amylase stability was also considered by incubating the enzyme at the respective temperatures for 1 h [19]. Then, 1% soluble starch was added and the remaining enzyme activity was measured under standard assay conditions. Irreversible thermal inactivation was also investigated by pre-incubating enzyme at temperature ranges of 60-80 ℃ for 7 h, with and without 20 mM CaCl2.6H2O [20]. Remaining alpha-amylase activity was assayed in the regular intervals of 30 min under standard conditions. The activity of the enzyme at time zero was considered as 100%.
2.7.3. Effect of metal ions, chelators and surfactants on the enzyme activity Enzyme activity was determined under standard assay conditions by using different compounds in sodium phosphate buffer (pH 7.0, 50 mM). To assay in the presence of metal ions, different metal ions in the form of FeSO4, CuSO4, CoCl2, ZnSO4, MnSO4, MgSO4, CaCl2, HgCl2 and KCl were used [12]. EDTA and 2-mercaptoetanol were used as chelating agents, H2O2 as oxidizing agent, SDS and Triton X-100 as surfactant [5, 21]. Enzyme activity in the absence of the chemical agents or the metal ions was considered as 100%. 8
2.7.4. Effect of detergents on the enzyme activity The enzyme activity in the presence of local detergents was studied under standard conditions using 1.0% solution of Kaf, Shooma, Dioxigeneh, Tage, Darya, Barf and Banoo in the sodium phosphate buffer (50 mM, pH 7.0) [21]. The activity of the sample with no detergent was considered as 100%.
2.7.5. Effect of salt concentration on the enzyme activity To study the effect of salt concentration, enzyme activity was assayed under standard condition in the presence of 0.0-4.0 M of NaCl [5]. Activity in the absence of NaCl was considered as 100%.
2.7.6. Effect of organic solvents on the enzyme activity Different organic solvents (40 % v/v) including isopropanol, 1-botanol, dimetylformamide, dimethyl sulfoxide (miscible solvents), diethyl ether, cyclohexane, chloroform and toluene (immiscible solvents) were used to investigate the enzyme activity in the presence of organic solvents [22, 23]. Each organic solvent was prepared in the sodium phosphate buffer (pH 7.0, 50 mM). Activity was done under standard assay condition and the sample without organic solvent was considered as 100 %.
2.8. Determination of kinetic parameters To determine Km and Vmax values, the enzyme was incubated with different concentrations of soluble starch ranging (0.0-2.0 %) in phosphate buffer (50 mM, pH 7.0) under standard conditions. Then, the Michaelis-Menten and Lineweaver-Burk plots were drawn.
2.9. The investigation of the raw starch hydrolysis The AT70 alpha-amylase activity on the raw-starch was considered via adding the different concentrations of raw corn starch (0.0-20%) as solid to the reaction mixture under standard assay condition [2, 24]. The mixture was incubated in a shaking incubator at 150 rpm and 45 ℃ for 1 h
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and then centrifuged at 12000 g for 10 min at 4 ℃. Then, the reaction was stopped by adding 1ml of DNS and the alpha-amylase activity was determined by DNS method.
3. Result 3.1. Identification of bacterial strain AT70 isolate showed the highest clear halo on the plate containing starch when flooding with KI/I2 solution after 48 h of incubation at 55 ℃. Various morphological and biochemical tests indicated that this isolate was gram positive, rod-shaped, aerobic, catalase positive, gelatin and casein hydrolyzing, citrate utilizing and acid producing from glucose, galactose and sucrose. The accession number from Genbank data base and nucleotides sequence length of the isolated strain were KT948060 and 1190 nucleotide, respectively. On the basis of the morphological and biochemical properties and following 16S rRNA sequencing, this strain was identified as B. licheniformis with 100 % sequence similarity.
3.2. Partial optimization of the alpha-amylase production In this research, the alpha-amylase production from B. licheniformis AT70 was optimized in the presence of several factors, including carbon and nitrogen sources, metal ions and pHs after 48 hours of incubation at 55 ℃. The data obtained are summarized in Table 1. Results showed that, starch has induced the highest enzyme production, while fructose was seen as a repressor of alpha-amylase production. Among organic and inorganic nitrogen sources, yeast extract showed decrease in the alpha-amylase production, but the other nitrogen sources enhanced the alphaamylase production and the maximum enzyme production was obtained when ammonium chloride was added to the basal medium. The AT70 alpha-amylase production was investigated in the presence of different metal ions by incorporating sodium chloride, calcium chloride, potassium chloride and magnesium sulphate in the basal medium individually at a concentration of 0.1%. The results in Table 1 showed that a slight increase in the alpha-amylase production was occurring in calcium chloride, in which the other sources were observed to repress the enzyme production extremely. In other research, calcium chloride was also found to be an inducer of the alpha-amylase production. The initial pH of the production medium is very effective on the alpha-amylase yield. In the current research, the highest amount of alpha-
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amylase production was seen to be at pH 8.0 and the lowest alpha-amylase production was obtained at pH 5.0 and 6.0. To confirm the additive effect of all factors, the AT70 isolate was inoculated into the basal medium containing 1% soluble starch and the optimum medium including (g/l) soluble starch 5.0, ammonium chloride 2.0, CaCl2 1.0, KH2PO4 1.0, which its pH was adjusted in 8.0 and then the mediums were incubated at 55 ℃ for 72 h. After 24, 48 and 72 h of incubation, the enzyme production was investigated in the both mediums and it was found that the yield of enzyme production was excellently raised in the optimal medium (about 10 folds compare to the basal medium) and also 48 h was observed to be the favored time for the alpha-amylase production (Fig. 1).
3.5. Solid state fermentation Despite SmF is a customary way to produce alpha-amylase, but in the recent years the SSF system has become a remarkable alternative method over SmF. In this study, among the mentioned kitchen and agricultural wastes, the maximum yield of AT70 alpha-amylase was exploited by date waste (209 U/g) and wheat bran (184 U/g) (Fig. 2).
3.3. Partial purification and zymoghraphy The cell-free extract was applied onto Q-Sepharose column chromatography and then fractionated by a stepwise elution using the stated buffer with increasing concentration of NaCl (0.0–0.5 M) and the alpha-amylase was recovered in the fraction obtained with the concentration of 70 % NaCl gradient. SDS–PAGE was performed to evaluate protein purify and the molecular mass of alpha-amylase. The purified alpha-amylase was detected as a single band with a molecular mass of approximately 85 kDa by gel silver staining. Zymoghraphy of AT70 alphaamylase revealed white region in the dark background of non-denaturing gel contained 1% starch, which it was exhibited the alpha-amylase activity (Fig. 3).
3.4. Characterization of alpha-amylase 3.4.1. Effect of pH on the activity and stability of enzyme
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The results of activity and stability of the AT70 alpha-amylase in the different pH indicated that the optimum enzyme activity and stability was at pH 8.0 and 9.0, respectively, and the enzyme retained the most of its activity in the alkaline conditions (Fig. 4a). 3.4.2. Effect of temperature on the enzyme activity and stability The stability and activity of the AT70 alpha-amylase were examined at different temperatures. Results showed that, the optimum temperature of this enzyme was at 60 ℃, while the activity reduced slightly at temperatures beyond and less than 60 ℃ (Fig. 4b). The maximum stability of the AT70 alpha-amylase was detected in 55 ℃, but the half-life obtained at 70 ℃ (Fig. 4c). The irreversible thermoinactivation of AT70 alpha-amylase was studied at a temperature range from 60 to 80 ℃ in the presence and absence of 20 mM CaCl2 for 7 h. As shown in Fig. 4d, thermal stability of the AT70 alpha-amylase was decreased with rising temperature from 60 to 80 ℃ in the presence or in the absence of Ca2+. Thermal stability was improved by the addition of Ca2+, as the half-life was enhanced to more than 6 h at 70 ℃, but the Ca2+ wasn't affective to keep the activity at 80 ℃.
3.4.3. Effect of metal ions, cheaters and surfactants on the enzyme activity The effect of metal ions (Ca2+, Fe2+, Cu2+, Co2+, Zn2+, Mg2+, Mn2+, K+, Hg2+) and some of the chemical compounds such as EDTA, 2-mercaptoetanol, SDS, Triton X-100 and H2O2 on the alpha-amylase activity was studied at a concentration of 4 mM. Among the metal ions studied, Mg2+ and Ag+ decreased the enzyme activity (13% and 15%, respectively), while the activity was increased about twofold by Mn2+ (Table 2). 3.4.4. Effect of solid commercial detergents on the enzyme activity Hydrolytic enzymes, in particular proteases and amylases, are the main components of automatic dishwashing and laundry detergents that is a duo to the reduction of alkalinity and dosage as lower wash temperature. Therefore, in the present study, the effect of seven solid commercial local detergents including Kaf, Shooma, Di-oxigeneh, Taghe, Darya, Barf and Banoo (at a concentration of 1.0 %) on the alpha-amylase activity was investigated. As the results showed (Table 3), among the detergents tested, Shooma and Taghe improved the alpha-amylase activity in comparison to the control (without detergents) by 34% and 13%, respectively. In contrast,
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Kaf, Darya, Di-oxygeneh and Barf had been reduced the enzyme activity by 15, 12, 12 and 5%, respectively, and was seen no significant change in the enzyme activity with Darya.
3.4.5. Effect of organic solvents on the enzyme activity The effect of organic solvents on the AT70 alpha-amylase activity was tried by using water miscible solvents (isopropanol, 1-botanol, dimethylformamide and dimethylsulfoxide) and water immiscible solvents (toluene, cyclohexane, chloroform and diethyl ether) (40%, v/v). On the basis of these results (Table 4), the enzyme demonstrated the excellent stability against all of the mentioned organic solvents exceptions of dimethylformamide. Furthermore, the activity of the enzyme was increased in the presence of dimethylsulfoxide, diethyl ether and chloroform by 60, 30 and 26 %, respectively. Nonetheless, the enzyme was destabilized in the presence of some water miscible solvents; as the enzyme was inhibited by the addition of DMF to more than 50% and incubation of enzyme solution with isopropanol resulted in reduction of the activity by 20%. However, water miscible solvents identify as the stoppers of the enzyme activity toward water immiscible solvents.
3.4.6. Effect of salt concentration on the enzyme activity The alpha-amylase activity was also determined in the presence of 0.0–4.0 M NaCl. The enzyme activity demonstrated an increase in the presence of different concentrations of NaCl and the maximum activity was observed at 1.5 M NaCl by 42% (Table 5). Furthermore, the enzyme also showed increased activity in the range of 1.0-2.0 M NaCl by 30-40 %, this finding clearly explains halophilicity of the enzyme.
3.7. Determination of kinetic parameters Kinetic parameters were also determined by the incubation of the purified enzyme solution in the presence of different concentrations of soluble starch, 0.0-7.0 mg/ml. The Michaelis-Menten constant (Km) and Vmax values estimated to be 1.203 mg/ml and 0.01 mg/ml/min from the Michaelis-Menten and Lineweaver-Burk plots (Fig. 5).
3.6. Raw starch hydrolysis property 13
The results of raw corn starch digestion by the AT70 alpha-amylase were shown in Fig. 6, which explained that the enzyme was very effective on the raw corn granules and the optimal activity was obtained at 14-20 % (w/v) of the raw corn granules.
4. Discussion In this research, a strain namely Bacillus licheniformis AT70 was isolated from Gorooh hot spring in Kerman and found to produce a thermostable alpha-amylase with desirable properties for biotechnology. In the previous reports, great attention has been paid on the determination of the optimal conditions for the alpha-amylase production from Bacillus. sp, as well as in the present study [25, 26]. Maximum production of the AT70 alpha-amylase was obtained in the presence of starch (as a carbon source) and ammonium chloride (as a nitrogen source) with 388 and 329 U/ml enzyme yield, respectively. Babu and Satyanarayana reported that soluble starch was the best carbon source and fructose was a repressor of the alpha-amylase production by B. stearothermophilus [27]. Unlikely, the stimulating effect of the organic nitrogen sources such as yeast extract and peptone was reported by Hamilton and Fogarty [28]. But, it is agreed to the report published by Das et al [29] that B. subtilis DM-03 produced the maximum alpha-amylase in the medium with ammonium chloride as the nitrogen source. Application of agro-industrial wastes as available substrates for the production of enzyme under solid state fermentation (SSF) lead to reduce the provided costs and economize the production process [30]. Therefore, we nominated a number of kitchen and agricultural wastes specially date waste and wheat bran as SSF suitable substrates for the production of AT70 alpha-amylase. Results indicated that AT70 alpha-amylase produced about 209 U/g and 184 U/g, respectively. However, this is the first report for the production of bacterial alpha-amylase by using date waste as substrate containing starch in solid state fermentation. The molecular weight estimated for the AT70 alpha-amylase was 85 kDa, obtaining on the basis of SDS-PAGE results, which it is higher than the range measured for the majority of alphaamylases [21, 24, 31, 32]. The AT70 alpha-amylase exposed substantial properties against various factors, which can be aided to its selection for the particular processes in industry. The pH stability profile of the AT70 alpha-amylase showed that the optimal stability was in pH 9.0 and the enzyme retained 60-73 % of its activity in pH 6.0 to 10.0. The enzyme also be active well in the range of pH 7.0–10.0, with 14
an optimum activity in pH 8.0. According to these results, AT70 alpha-amylase was more active and stable than majority of alpha-amylases from Bacillus. sp and hence the enzyme was recognized as an alkalophilic alpha-amylase which can be widely used in the food and pharmaceutical fields. Kim et al reported that, alkaline amylases adopt a unique structure and catalytic mechanism to adequate function on the starch carbohydrate in these alkaline conditions. They also suggested that, existence of an un-ionized carboxylic acid group in the active site of GM8901 alpha-amylase at high pH values [33].
Since the most industrial processes are performed at high temperatures, finding enzymes, which are able to maintain their structure and activate in the high-temperatures is very important. The alpha-amylase activity of B. licheniformis AT70 was kept in the highest amount at 60 ℃ and was decreased by 47 % at 70 ℃. Optimum activity of the thermostable alpha-amylases in the temperature range of 65-90 ℃ provide their usability in industrial liquefaction of starch [34, 35]. Oziengbe and Onilude suggested that, increased enzyme activity observed for thermostable alpha-amylases toward increase in temperature can be the result of enhanced smash of the enzyme and substrate [35]. In this research, we also found the dramatic thermal stability for AT70 alpha-amylase at 60 ℃ as not only it’s original activity was entirely protected up to 7 h but also was slightly increased. The very salient improvement of the enzyme thermostability was observed after addition of Ca2+ into reaction mixture. Residual activity in the presence of CaCl2 at 60 ℃ increased more than 2 folds of sample without CaCl2 at the same temperature. Although, the improvement of thermal stability at the same condition was observed for some thermostable alpha-amylases including Thermus filiformis, Bacillus licheniformis NH1, Bacillus licheniformis M27 and Bacillus amyloliquefaciens [20, 6, 36, 37], but, this fantastic improvement in the alpha-amylases thermal stability didn’t report up to now. Machius and co-workers reported that improvement of thermal stability with calcium ion can be explained by the creation of a calcium–sodium–calcium in the main Ca2+ binding site, bridging the domains A and B of the enzyme [38]. Furthermore, the stabilizing effect of Ca2+ on the thermostability of the alpha-amylase can be clarified due to the salting out of hydrophobic residues by Ca2+ in the protein, hence triggering the adoption of a compact structure [39] and has also been stated in other organisms [40-42].
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The alpha-amylase activity was considerably impressible from metal ions and chemical compounds. Exceptions of Mg2+ and Hg2+, all of metal ions increased the enzymatic activity of AT70 alpha-amylase. Activation of the AT70 alpha-amylase in Cu2+ disagree with the behavior observed for the most alpha-amylases [5, 43, 44] and it is assumed that Cu2+ interact with some of amino acid residues in the active site of the enzyme. Yet, some of the alpha-amylases i.e. alpha-amylase from Natronococcus sp. strain Ah-36 were activated by Cu2+ [45]. Nies reported that, the reduction of alpha-amylase activity by heavy ion Hg2+ is the evidence of thiol groups in the alpha-amylase structure [46]. Although, Kolcuoğlu and co-workers reported that, Mn2+ increased the activity of Geobacillus caldoxylosilyticus TK4 alpha-amylase about 51% [47] and the maximum activity of A. veronii NS07 alpha-amylase was also observed in the presence of Mn2+ [12], but the enzymatic activity of the AT70 alpha-amylase improved about twofold in the presence of Mn2+. These results indicated that the semi-rigid and flexible structure of the enzyme, which improve enzyme linkage to substrate [48].
Despite of the reduction of enzyme activity of the most alpha-amylases in the presence of EDTA [49-51], the activity of the AT70 alpha-amylase increased about 145% in the same condition. This result is agree with the improvement of the activity of Bacillus. sp L1711 alpha-amylase in the presence of 5 mM EDTA [52]. Arikan B., reported that alkaline amylases vary in their response to the chelator EDTA with some being unaffected [4]. In addition, Hagihara et al., reported that Amy-K38 retained full activity in the presence of 1mM EDTA as high as 100 mM while Egas et al., reported 88% activity with 10 mM EDTA [53, 54]. It is probable that metal ions can be activated AT70 alpha-amylase but they don’t essential for the starch hydrolysis process. However, the molecular investigations of the AT70 alpha-amylase are in progress to find the better understanding of these results. AT70 alpha-amylase showed not only good resistant toward commercial solid detergents, but also its activity was increased around 34% and 13% by Shooma and Taghe detergents. In contrast, the alpha-amylase reported by Chakraborty et al, retained 35–70% of its original activity when pre-incubated in the presence of commercial detergents [55]. Due to remarkable stability and increase the enzyme activity in the presence of several commercial detergents, the
16
AT70 alpha-amylase could be considered as a talented candidate in the formulation of industrial detergents in order to clean starch-containing stains. AT70 alpha-amylase had completely retained its original activity at 0.0-4.0 M NaCl and even it,s activity was increased about 42% at 1.5 M. It is more than the halophilicity results of the alpha amylases from the other microbial sources such as Streptomyces sp. D1 [55] and Bacillus dipsosauri [41]. Activity and stability of the halophilic amylases in the various concentrations of different salts can led to adaptation of the microorganisms to the harsh environments, mainly salterns [56].
Kinetic parameters of the AT70 alpha-amylase were approved under standard assay conditions using several concentrations of soluble starch as substrate. As obtained from the Lineweaver– Burk plot (Fig. 5), the Km and Vmax values were 1.203 mg/ml and 0.01 mg/ml/min, respectively. The Vmax and Km values of the alpha-amylase from Bacillus amyloliquefaciens for soluble starch was found to be 4.11 mg/min and 3.076 mg, respectively [57]. Goyal and co-workers reported that Km and Vmax values was 3.44 mg/ml and 0.45 mg hydrolyzed starch/ml/min at 55°C, respectively, for Lactobacillus manihotivorans [58]. However, Km value of AT70 alpha-amylase is lower than Km values of the other thermostable alpha-amylases from Bacillus lichentformis and various Bacillus sp. [59, 60], that it demonstrate the high tendency of the AT70 alphaamylase for bond to substrate.
Upshot, one of the other attractive properties of the AT70 alpha-amylase was strong hydrolysis power on the high concentrations (14-20%) of raw corn granules. To the best of our knowledge, this is the first report of Bacillus licheniformis alpha-amylase with potency of raw-starch digesting. In general, there are a small number of bacterial amylases capable of strong hydrolysis of raw corn starch at a high concentration [61]. In view of raw corn starch digesting ability of the AT70 alpha-amylase, it could have potential applications in the food and fermentation industries that can be due to the global availability of raw corn starch. Existence of a separate starchbinding domain (SBD) in carboxyl-terminal region has been proven for the most of starchdigesting enzymes and has been found to be very important in binding to raw starch [62]. The molecular mechanism of the AT70 amylase can be appeared after determination of three dimensional structure. 17
5. Conclusion Nowadays, thermostable raw-starch degrading alpha-amylase has been attending a great interest for the starch hydrolyzing. Results of AT70 alpha-amylase characterization showed that this enzyme was stable at the wide ranges of temperatures from 40-70 ℃ and showed good activity at alkaline pH and over a broad range of NaCl concentration (0.5-3.0 M). It, s thermal stability was improved interestingly about 2.5 folds by adding Ca2+. SSF results indicated that the maximum alpha-amylase production was achieved by date wastes and wheat bran, as cheap agricultural wastes. Moreover, raw starch digesting effect by AT70 alpha-amylase was considerably observed on the high concentrations of raw corn starch. Due to these excellent properties, the AT70 alpha-amylase can be used as a good candidate in the starch industry especially in the rawstarch digestion.
Acknowledgment The authors express their gratitude to the Research Council of the Shahid Bahonar University of Kerman (Kerman, Iran) for financial support during the course of this project.
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Legends: Fig. 1. The comparison of the alpha-amylase production in the basal medium (containing % 0.1 of soluble starch) and the optimal medium (encompassing all of the effective factors) after 72 h of incubation at 55 ℃.
Fig. 2. Investigation of the alpha-amylase production from Bacillus licheniformis AT70 using agro residues under SSF procedure. After 7 days of incubation, the supernatants exploited from centrifugation of the culture mediums and applied to assay enzyme activity.
Fig. 3. (A) SDS-page of AT70 alpha-amylase. Lane 1, active fraction from ion exchange chromatography. Lane 2, Protein markers. (B) Activity staining of the alpha-amylase enzyme in native PAGE by iodine solution.
Fig. 4. A) Influence of different pH values on the alpha-amylase activity and stability. pH activity was measured in the buffer systems presented in section 2 under standard assay conditions. For determination of pH stability, pre-incubation of the alpha-amylase was performed in the same buffers for 60 min. B) Activity profile of the AT70 alpha-amylase at the different temperatures from 30-80 ℃. Activity was measured in standard assay conditions using 1% soluble starch. C) The thermal stability was determined by pre-incubating the enzyme at 3080 ℃ for 60 min and then assaying the residual activity under standard conditions. D) The thermal stability in the presence of CaCl2 was determined by pre-incubating the enzyme at 30-80 ℃ for 60 min and then assaying the residual activity under standard conditions.
Fig. 5. Alpha-amylase activity was determined in the presence of various concentrations of soluble starch as substrate. Km and Vmax values was determined from Michaelis-Menten and Lineweaver-Burk plots.
Fig. 6. Determination of the alpha-amylase activity rate on the different concentrations of raw corn starch. The activity was assayed after 5 h of incubation at 55 ℃.
24
Fig. 1.
25
Fig. 2.
26
A
1
B
2
Fig. 3.
27
Fig. 4.
28
B)
Fig. 5.
29
Fig. 6.
30
Table 1. Influence of various factors on the alpha amylase production. Bacillus licheniformis AT70 was incubated at different condition such as, carbon and nitrogen sources, different ions and pHs for 48 h and then the alpha amylase production was considered at standard condition.
Factors Carbon sources: Glucose Galactose Maltose Fructose Soluble starch Control Nitrogen sources: Peptone Yeast extract Gelatin NaNO3 NH4Cl (NH4)2 SO4 Control Metal ions: CaCl2 MgSO4 KCl NaCl Control Different pHs: pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0
Enzyme activity (U/ml) 256±5 264±6 228±6 144±7 388±5 213±5 260±5 52±6 255±7 278±7 329±5 294±4 105±5 451±4 20±5 29±6 60±6 424±5 72±5 74±7 251±8 288±6 146±6
31
Table 2. Effect of some metal ions and chemical additive on the alpha-amylase activity. Metal ions and chemical additives
Activity (%)
Fe2+
143±3
Cu2+
114±3
Co2+
130±5
Zn2+
131±6
Mn2+
257±5
Ca2+
108±5
Hg2+
85±7
K+
117±4
Mg2+
87±3
T.X.100
88±4
2-Mercaptoethanol
107±4
EDTA
145±3
SDS
75±4
H2O2
117±5
Control
100±4
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Table 3. Effect of commercial detergents on the alpha-amylase activity. Commercial detergents
Activity (%)
Barf
94±5
Shooma
134±5
Tage
113±4
Banoo
101±4
Dioxigeneh
88±3
Darya
88±3
Kaf
85±4
Control
100±2
The enzyme activity in any detergent calculated as a percent of the activity in the control sample (contained no detergent).
33
Table 4. Effect of various water miscible and water immiscible organic solvents on the alpha-amylase activity. Organic solvents
Activity (%)
No ion
100±2
Isopropanol
80±3
1-botanol
110±3
Diethyl ether
160±4
DMF
42±3
Cyclohexane
115±4
Chloroform
126±4
Toluene
111±3
DMSO
130±2
Methanol
90±1
34
Table 5. Alpha-amylase activity in the presence of NaCl.
NaCl (M)
Activity (%)
0
100±1
0.5
115±1
1.0
133±3
1.5
142±2
2.0
139±3
2.5
120±3
3.0
112±2
3.5
105±2
4.0
101±1
For determination of the salt activity, AT70 alpha-amylase was incubated In the presence of different concentrations of NaCl (0.0-4.0 M).
35
S1381-1177(16)30117-5 http://dx.doi.org/doi:10.1016/j.molcatb.2016.07.002 MOLCAB 3393
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date: Revised date: Accepted date:
28-2-2016 2-7-2016 3-7-2016
Please cite this article as: Saideh Afrisham, Arastoo Badoei-Dalfard, Abdolhamid Namaki-Shoushtari, Zahra Karami, Characterization of a thermostable, CaCl2activated and raw-starch hydrolyzing alpha-amylase from Bacillus licheniformis AT70: Production under solid state fermentation by utilizing agricultural wastes, Journal of Molecular Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2016.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Characterization of a thermostable, CaCl2-activated and raw-starch hydrolyzing alpha-amylase from Bacillus licheniformis AT70: Production under solid state fermentation by utilizing agricultural wastes
Saideh Afrisham, Arastoo Badoei-Dalfard*, Abdolhamid Namaki-Shoushtari, Zahra Karami
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran.
*Corresponding
author: Dr. Arastoo Badoei-Dalfard, Department of Biology, Faculty of
Sciences, Shahid Bahonar University of Kerman, Kerman, Iran; Tel:+9834 31322044; Fax: +9834 33222032; Email: [email protected]
1
Graphical abstract
Screening
Medium optimization
Solid state fermentation
partial purification
Ca2+-activated thermal stability
Raw-Corn starch digestion
2
Highlights
The production of AT70 alpha-amylase was optimized about 10 folds using starch (0.1 %) and ammonium chloride (2.0 %).
This is the first report for the production of bacterial alpha-amylase by using date waste in SSF.
The very salient improvement (2.5 folds) of the enzyme thermo-stability was observed after addition of Ca2+ into reaction mixture.
AT70 alpha-amylase showed about 34 % activity toward commercial solid detergents Shooma.
AT70 alpha-amylase exhibited remarkable hydrolytic activity in 14-20% (w/v) of raw corn starch at 55 ℃.
AT70 alpha-amylase is a thermostable, CaCl2-activated and has potent raw-starch hydrolyzing alpha-amylase
Abstract B. licheniformis AT70 which produced a thermophilic, raw-starch degrading alpha-amylase was isolated from Gorooh hot springs in Kerman province. Maximum production of AT70 alphaamylase was obtained in the presence of starch (as a carbon source) and ammonium chloride (as a nitrogen source) with 388 and 329 U/ml enzyme yield, respectively. SSF was also carried out using various agricultural and kitchen wastes and results showed that the maximum yield of AT70 alpha-amylase production was obtained by date waste and wheat bran, respectively (10 %, w/v). The thermal stability of the AT70 alpha-amylase was increased about 2.5 folds at 60 ℃. AT70 alpha-amylase showed the maximum activity at 1.5 M NaCl by 42% and local detergent Shooma enhanced the alpha-amylase activity about 34%, compared to control. Furthermore, AT70 alpha-amylase exhibited remarkable hydrolytic activity in a range of 14-20% (w/v) of raw corn starch at 55 ℃. These results indicated that AT70 alpha-amylase has great potential applications for the raw-starch degrading.
3
Keywords: Alpha-amylase, Agricultural wastes, Bacillus, Raw-corn starch, Solid state fermentation, Thermophilic
1. Introduction Starch is a major constituent of agriculture and domestic wastes. These cheap agricultural wastes could become appreciated resources to be converted into high value compounds. In recent years, a global interest has been focused on the raw-starch hydrolyzing amylases to simplify the procedure of starch digestion [1, 2]. Alpha-amylase is a key enzyme in the industrial processes such as starch saccharification, textile, paper, brewing, food, distilling industries and pharmaceuticals [3]. Alpha-amylase (E.C 3.2.1.1 1,4-α-glucan-glucanohydrolase) catalysis the hydrolysis of the internal α-D-(1,4) glycosidic linkages in starch at random locations. This enzyme constitutes a class of industrial enzymes that account for ~25% of the enzyme market [4]. Alpha-amylase can be derived from several sources, such as animals, plants and microorganisms. However, enzymes from microbial sources generally meet industrial demands [4]. Whereas many of the commercial alpha-amylases do not accordance industrial process conditions, isolation and characterization of alpha-amylases with favorite features such as thermostability, alkaline stability and halophilicity is of special importance [5]. Activity of alphaamylases at higher temperatures is desirable for gelatinization and liquefaction of starch to economize the processes; as the need to continually search for more thermophile and thermostable alpha-amylases is increasing [4]. Using thermostable alpha-amylase in the industrial processes has advantages, including the decreased risk of contamination, cost of external cooling, increased diffusion rate, a better solubility of substrates, a lesser viscosity permitting accelerated mixing and pumping. Moreover, they showed a resistance in the presence of denaturing agents, solvents and proteolytic enzymes [3]. Among the genus Bacillus, B. licheniformis and B. amyloliquifaciens are identified as good producers of thermostable alphaamylase. Due to significant thermal resistance, the alpha-amylase produced by B. licheniformis widely used in the starch liquefaction process [6]. Bacterial organisms are now being progressively considered for the production of enzymes by solid-state fermentation (SSF) [7]. SSF has several advantages over submerged fermentation (SmF) because of the low capital 4
investment, the simple technique, low energy requirement, end-product inhibition, low waste water output and better product recovery [8]. SSF has been engaged in the production of thermophilic alpha-amylase by Bacillus subtilis and B. cereus MTCC 1305 [9]. In the present research, we report the biochemical characterization of a Ca2+-activated, thermophilc and detergent-stable alpha-amylase from B. licheniformis AT70 and its potential to degrade raw starch granules was also investigated. The production of the alpha-amylase was also performed by using different kinds of agro-residues and the kitchen wastes, which some of them were not reported yet i.e. Hajibadam sweets, Sehen Komath sweets in SSF manner. The properties of this enzyme, including its pH and temperature profiles, kinetic parameters, irreversible thermoinactivation and raw starch digestibility revealed that it has significant potential for the starch industry.
2. Materials and methods 2.1. Identification of the microbial strain and culture conditions Bacillus licheniformis AT70 strain which used in this research was isolated from Gorooh hot spring, located in Jiroft city, Iran. For identification of the isolated strain, a number of morphological and biochemical tests described in Bergey and especially 16S rRNA gene sequence analysis were performed [10, 11]. The isolate was detected to be an alpha-amylase producer on the starch-agar medium composed of (%) soluble starch 1.0, yeast extract 0.2, peptone 0.5, NaCl 0.1, MgSO4.7H2O 0.1, CaCl2.2H2O 0.02, after incubating at 55 ℃ for 72h. Alpha-amylase production was indicated by flooding the plates with 1% (w/v) I2 and 2% (w/v) KI solution.
2. 2. Production of alpha-amylase Alpha-amylase production was performed in the culture medium containing (g/l): KH2PO4 1.0, Na2HPO4·2H2O 3.13, tryptone 2.0, (NH4)2SO4 2.0, MgSO4·7H2O 0.05, CaCl2·2H2O 0.05, soluble starch 1.0 (pH 7.0) [12]. The medium seeded with 10% (v/v) of the pre-culture composed of nutrient broth medium and incubation was carried out at 55 ℃ with 150 rpm for 72 h. The cells were harvested after centrifugation at 10000 g for 10 min at 4 ℃ and cell-free supernatants were collected as crude enzymes. 5
2.3. Alpha amylase assay
Alpha amylase activity was measured using 3,5-dinitrosalicylic acid (DNS) reagent according to Bernfeld [13]. A mixture of 0.5 ml of enzyme solution and 0.5 ml of soluble starch (1.0%) in phosphate buffer (50 mM, pH 7.0), as substrate, was incubated at 55 ℃ for 20 min. The reaction was stopped by adding 1 ml of DNS reagent. The mixture was boiled for 5 min and after cooling in room temperature, the mixture was diluted with distilled water. The amount of reducing sugars released during the starch hydrolyzing was measured by recording absorbance at 540 nm and using the glucose standard curve. A unit of enzyme activity was defined as the amount of enzyme required to release of 1µmol of sugar reducing per 1 min under enzyme assay conditions.
2.4. Optimization of enzyme production conditions
The alpha-amylase production was optimized in the presence of the different carbon sources by supplementing the basal medium (without carbon source) with 0.1% (w/v) of starch, glucose, galactose, maltose, fructose [14]. For this order, the mediums were inoculated with 10% of an overnight culture of the AT70 isolate. After 48 h of incubation at 55 ℃, the culture mediums were harvested by centrifugation with 11000 g and 4 ℃ for 10 min and the obtained supernatants were used for the investigation of the alpha-amylase production. The organic nitrogen sources, including yeast extract, gelatin, peptone and inorganic nitrogen sources, including ammonium chloride and sodium nitrate replaced by ammonium sulphate in the basal medium at a concentration of 2.0 % [14]. In each case, the culture medium was inoculated with 10% of the overnight grown bacterial culture. After 48 h, the culture medium was centrifuged at 11000 g and 4 ℃ for 10 min and the alpha- amylase activity was assayed for the resulting supernatants. The effect of different metal ions on the enzyme production was also investigated by adding a concentration of 1.0 % of KCl, MgSO4.7H2O, NaCl and CaCl2 in the basal medium (without metal ion) [14]. Alpha-amylase activity was measured after 48 h of incubation. The effect of pH on the alpha-amylase production was studied by growing the bacterium in the culture media with
6
various pH 5.0, 6.0, 7.0, 8.0 and 9.0. After 48 h of incubation, the alpha-amylase activity of the resulting supernatants was measured under the standard conditions.
2.5. Solid state fermentation
The production of alpha-amylase was investigated under solid state fermentation method on the eight different types of local agricultural wastes as substrate including banana peel, potato peel, orange peel, Hajibadam sweets, Sehen Komath sweets; a kind of local cake, wheat bran [9], rice bran and a kind of date waste. First of all, the AT70 isolate was inoculated into 100 ml of the pre-culture medium containing (%) glucose 1.0, peptone 0.25, yeast extract 0.2, NaCl 0.15, KH2PO4 0.05, MgSO4 0.05, CaCl2 0.01 and was incubated at 55 ℃ for 16h. After this period, 3 ml of pre-culture was added within the culture medium composed of 10 gr of agricultural waste and 25 ml of optimized salt solution containing ammonium chloride 2.0 gr/l, CaCl2 1.0 gr/l and KH2PO4 1.0. After 7 days, 30 ml of sodium phosphate buffer (50 mM, pH 7.0) was added to the culture mediums and the obtained mixture was passed from a textile. Then, centrifugation was performed in 11000 g for 10 min at 4 ℃. The gathered extract was used as enzyme and its activity was assayed under the standard conditions by using the DNS method.
2.6. Partial purification of the alpha-amylase
A Q-Sepharose column pre-equilibrated with 50 mM phosphate buffer pH 7.6 was used for the partial purification of the alpha-amylase produced by B. licheniformis AT70. The supernatant was passed through the pre-equilibrated column. The attached proteins were removed from the column using a linear gradient of NaCl (0.0-5.0 M) in the same buffer at a flow rate of 1.0 ml/min [15, 16]. The fractions were used to determine the alpha-amylase activity and protein content. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was also performed according to the Laemmli method to determine the purity and the molecular mass of alpha-amylase [17]. Proteins were observed by silver staining method [18]. Zymography of 7
alpha-amylase was also performed in non-denatured gel containing 10% polyacrylamide and 1% soluble starch for the confirmation of the amylolytic activity [12]. Enzyme activity was detected using staining with KI/I2 solution as a white zone in dark background.
2.7. Biochemical characterization 2.7.1. Effect of pH on the enzyme activity and stability The effect of pH on the enzyme activity was studied under standard assay conditions in the following buffer systems at a concentration of 50 mM: glycine (pH 2.0 and 3.0), sodium acetate (pH 4.0 and 5.0), sodium phosphate (pH 6.0 and 7.0), Tris (pH 8.0-10.0), glycine (pH 11.0 and 12.0). pH stability was also measured by pre-incubation of the enzyme solution at room temperature for 1 h in the same buffer systems and then the residual activity was measured under standard assay conditions [5].
2.7.2. Effect of temperature on the enzyme activity and stability To investigate the effect of temperature on the alpha-amylase activity, the enzyme assay was carried out over the range of 30-80 ℃. The optimum temperature for the alpha-amylase stability was also considered by incubating the enzyme at the respective temperatures for 1 h [19]. Then, 1% soluble starch was added and the remaining enzyme activity was measured under standard assay conditions. Irreversible thermal inactivation was also investigated by pre-incubating enzyme at temperature ranges of 60-80 ℃ for 7 h, with and without 20 mM CaCl2.6H2O [20]. Remaining alpha-amylase activity was assayed in the regular intervals of 30 min under standard conditions. The activity of the enzyme at time zero was considered as 100%.
2.7.3. Effect of metal ions, chelators and surfactants on the enzyme activity Enzyme activity was determined under standard assay conditions by using different compounds in sodium phosphate buffer (pH 7.0, 50 mM). To assay in the presence of metal ions, different metal ions in the form of FeSO4, CuSO4, CoCl2, ZnSO4, MnSO4, MgSO4, CaCl2, HgCl2 and KCl were used [12]. EDTA and 2-mercaptoetanol were used as chelating agents, H2O2 as oxidizing agent, SDS and Triton X-100 as surfactant [5, 21]. Enzyme activity in the absence of the chemical agents or the metal ions was considered as 100%. 8
2.7.4. Effect of detergents on the enzyme activity The enzyme activity in the presence of local detergents was studied under standard conditions using 1.0% solution of Kaf, Shooma, Dioxigeneh, Tage, Darya, Barf and Banoo in the sodium phosphate buffer (50 mM, pH 7.0) [21]. The activity of the sample with no detergent was considered as 100%.
2.7.5. Effect of salt concentration on the enzyme activity To study the effect of salt concentration, enzyme activity was assayed under standard condition in the presence of 0.0-4.0 M of NaCl [5]. Activity in the absence of NaCl was considered as 100%.
2.7.6. Effect of organic solvents on the enzyme activity Different organic solvents (40 % v/v) including isopropanol, 1-botanol, dimetylformamide, dimethyl sulfoxide (miscible solvents), diethyl ether, cyclohexane, chloroform and toluene (immiscible solvents) were used to investigate the enzyme activity in the presence of organic solvents [22, 23]. Each organic solvent was prepared in the sodium phosphate buffer (pH 7.0, 50 mM). Activity was done under standard assay condition and the sample without organic solvent was considered as 100 %.
2.8. Determination of kinetic parameters To determine Km and Vmax values, the enzyme was incubated with different concentrations of soluble starch ranging (0.0-2.0 %) in phosphate buffer (50 mM, pH 7.0) under standard conditions. Then, the Michaelis-Menten and Lineweaver-Burk plots were drawn.
2.9. The investigation of the raw starch hydrolysis The AT70 alpha-amylase activity on the raw-starch was considered via adding the different concentrations of raw corn starch (0.0-20%) as solid to the reaction mixture under standard assay condition [2, 24]. The mixture was incubated in a shaking incubator at 150 rpm and 45 ℃ for 1 h
9
and then centrifuged at 12000 g for 10 min at 4 ℃. Then, the reaction was stopped by adding 1ml of DNS and the alpha-amylase activity was determined by DNS method.
3. Result 3.1. Identification of bacterial strain AT70 isolate showed the highest clear halo on the plate containing starch when flooding with KI/I2 solution after 48 h of incubation at 55 ℃. Various morphological and biochemical tests indicated that this isolate was gram positive, rod-shaped, aerobic, catalase positive, gelatin and casein hydrolyzing, citrate utilizing and acid producing from glucose, galactose and sucrose. The accession number from Genbank data base and nucleotides sequence length of the isolated strain were KT948060 and 1190 nucleotide, respectively. On the basis of the morphological and biochemical properties and following 16S rRNA sequencing, this strain was identified as B. licheniformis with 100 % sequence similarity.
3.2. Partial optimization of the alpha-amylase production In this research, the alpha-amylase production from B. licheniformis AT70 was optimized in the presence of several factors, including carbon and nitrogen sources, metal ions and pHs after 48 hours of incubation at 55 ℃. The data obtained are summarized in Table 1. Results showed that, starch has induced the highest enzyme production, while fructose was seen as a repressor of alpha-amylase production. Among organic and inorganic nitrogen sources, yeast extract showed decrease in the alpha-amylase production, but the other nitrogen sources enhanced the alphaamylase production and the maximum enzyme production was obtained when ammonium chloride was added to the basal medium. The AT70 alpha-amylase production was investigated in the presence of different metal ions by incorporating sodium chloride, calcium chloride, potassium chloride and magnesium sulphate in the basal medium individually at a concentration of 0.1%. The results in Table 1 showed that a slight increase in the alpha-amylase production was occurring in calcium chloride, in which the other sources were observed to repress the enzyme production extremely. In other research, calcium chloride was also found to be an inducer of the alpha-amylase production. The initial pH of the production medium is very effective on the alpha-amylase yield. In the current research, the highest amount of alpha-
10
amylase production was seen to be at pH 8.0 and the lowest alpha-amylase production was obtained at pH 5.0 and 6.0. To confirm the additive effect of all factors, the AT70 isolate was inoculated into the basal medium containing 1% soluble starch and the optimum medium including (g/l) soluble starch 5.0, ammonium chloride 2.0, CaCl2 1.0, KH2PO4 1.0, which its pH was adjusted in 8.0 and then the mediums were incubated at 55 ℃ for 72 h. After 24, 48 and 72 h of incubation, the enzyme production was investigated in the both mediums and it was found that the yield of enzyme production was excellently raised in the optimal medium (about 10 folds compare to the basal medium) and also 48 h was observed to be the favored time for the alpha-amylase production (Fig. 1).
3.5. Solid state fermentation Despite SmF is a customary way to produce alpha-amylase, but in the recent years the SSF system has become a remarkable alternative method over SmF. In this study, among the mentioned kitchen and agricultural wastes, the maximum yield of AT70 alpha-amylase was exploited by date waste (209 U/g) and wheat bran (184 U/g) (Fig. 2).
3.3. Partial purification and zymoghraphy The cell-free extract was applied onto Q-Sepharose column chromatography and then fractionated by a stepwise elution using the stated buffer with increasing concentration of NaCl (0.0–0.5 M) and the alpha-amylase was recovered in the fraction obtained with the concentration of 70 % NaCl gradient. SDS–PAGE was performed to evaluate protein purify and the molecular mass of alpha-amylase. The purified alpha-amylase was detected as a single band with a molecular mass of approximately 85 kDa by gel silver staining. Zymoghraphy of AT70 alphaamylase revealed white region in the dark background of non-denaturing gel contained 1% starch, which it was exhibited the alpha-amylase activity (Fig. 3).
3.4. Characterization of alpha-amylase 3.4.1. Effect of pH on the activity and stability of enzyme
11
The results of activity and stability of the AT70 alpha-amylase in the different pH indicated that the optimum enzyme activity and stability was at pH 8.0 and 9.0, respectively, and the enzyme retained the most of its activity in the alkaline conditions (Fig. 4a). 3.4.2. Effect of temperature on the enzyme activity and stability The stability and activity of the AT70 alpha-amylase were examined at different temperatures. Results showed that, the optimum temperature of this enzyme was at 60 ℃, while the activity reduced slightly at temperatures beyond and less than 60 ℃ (Fig. 4b). The maximum stability of the AT70 alpha-amylase was detected in 55 ℃, but the half-life obtained at 70 ℃ (Fig. 4c). The irreversible thermoinactivation of AT70 alpha-amylase was studied at a temperature range from 60 to 80 ℃ in the presence and absence of 20 mM CaCl2 for 7 h. As shown in Fig. 4d, thermal stability of the AT70 alpha-amylase was decreased with rising temperature from 60 to 80 ℃ in the presence or in the absence of Ca2+. Thermal stability was improved by the addition of Ca2+, as the half-life was enhanced to more than 6 h at 70 ℃, but the Ca2+ wasn't affective to keep the activity at 80 ℃.
3.4.3. Effect of metal ions, cheaters and surfactants on the enzyme activity The effect of metal ions (Ca2+, Fe2+, Cu2+, Co2+, Zn2+, Mg2+, Mn2+, K+, Hg2+) and some of the chemical compounds such as EDTA, 2-mercaptoetanol, SDS, Triton X-100 and H2O2 on the alpha-amylase activity was studied at a concentration of 4 mM. Among the metal ions studied, Mg2+ and Ag+ decreased the enzyme activity (13% and 15%, respectively), while the activity was increased about twofold by Mn2+ (Table 2). 3.4.4. Effect of solid commercial detergents on the enzyme activity Hydrolytic enzymes, in particular proteases and amylases, are the main components of automatic dishwashing and laundry detergents that is a duo to the reduction of alkalinity and dosage as lower wash temperature. Therefore, in the present study, the effect of seven solid commercial local detergents including Kaf, Shooma, Di-oxigeneh, Taghe, Darya, Barf and Banoo (at a concentration of 1.0 %) on the alpha-amylase activity was investigated. As the results showed (Table 3), among the detergents tested, Shooma and Taghe improved the alpha-amylase activity in comparison to the control (without detergents) by 34% and 13%, respectively. In contrast,
12
Kaf, Darya, Di-oxygeneh and Barf had been reduced the enzyme activity by 15, 12, 12 and 5%, respectively, and was seen no significant change in the enzyme activity with Darya.
3.4.5. Effect of organic solvents on the enzyme activity The effect of organic solvents on the AT70 alpha-amylase activity was tried by using water miscible solvents (isopropanol, 1-botanol, dimethylformamide and dimethylsulfoxide) and water immiscible solvents (toluene, cyclohexane, chloroform and diethyl ether) (40%, v/v). On the basis of these results (Table 4), the enzyme demonstrated the excellent stability against all of the mentioned organic solvents exceptions of dimethylformamide. Furthermore, the activity of the enzyme was increased in the presence of dimethylsulfoxide, diethyl ether and chloroform by 60, 30 and 26 %, respectively. Nonetheless, the enzyme was destabilized in the presence of some water miscible solvents; as the enzyme was inhibited by the addition of DMF to more than 50% and incubation of enzyme solution with isopropanol resulted in reduction of the activity by 20%. However, water miscible solvents identify as the stoppers of the enzyme activity toward water immiscible solvents.
3.4.6. Effect of salt concentration on the enzyme activity The alpha-amylase activity was also determined in the presence of 0.0–4.0 M NaCl. The enzyme activity demonstrated an increase in the presence of different concentrations of NaCl and the maximum activity was observed at 1.5 M NaCl by 42% (Table 5). Furthermore, the enzyme also showed increased activity in the range of 1.0-2.0 M NaCl by 30-40 %, this finding clearly explains halophilicity of the enzyme.
3.7. Determination of kinetic parameters Kinetic parameters were also determined by the incubation of the purified enzyme solution in the presence of different concentrations of soluble starch, 0.0-7.0 mg/ml. The Michaelis-Menten constant (Km) and Vmax values estimated to be 1.203 mg/ml and 0.01 mg/ml/min from the Michaelis-Menten and Lineweaver-Burk plots (Fig. 5).
3.6. Raw starch hydrolysis property 13
The results of raw corn starch digestion by the AT70 alpha-amylase were shown in Fig. 6, which explained that the enzyme was very effective on the raw corn granules and the optimal activity was obtained at 14-20 % (w/v) of the raw corn granules.
4. Discussion In this research, a strain namely Bacillus licheniformis AT70 was isolated from Gorooh hot spring in Kerman and found to produce a thermostable alpha-amylase with desirable properties for biotechnology. In the previous reports, great attention has been paid on the determination of the optimal conditions for the alpha-amylase production from Bacillus. sp, as well as in the present study [25, 26]. Maximum production of the AT70 alpha-amylase was obtained in the presence of starch (as a carbon source) and ammonium chloride (as a nitrogen source) with 388 and 329 U/ml enzyme yield, respectively. Babu and Satyanarayana reported that soluble starch was the best carbon source and fructose was a repressor of the alpha-amylase production by B. stearothermophilus [27]. Unlikely, the stimulating effect of the organic nitrogen sources such as yeast extract and peptone was reported by Hamilton and Fogarty [28]. But, it is agreed to the report published by Das et al [29] that B. subtilis DM-03 produced the maximum alpha-amylase in the medium with ammonium chloride as the nitrogen source. Application of agro-industrial wastes as available substrates for the production of enzyme under solid state fermentation (SSF) lead to reduce the provided costs and economize the production process [30]. Therefore, we nominated a number of kitchen and agricultural wastes specially date waste and wheat bran as SSF suitable substrates for the production of AT70 alpha-amylase. Results indicated that AT70 alpha-amylase produced about 209 U/g and 184 U/g, respectively. However, this is the first report for the production of bacterial alpha-amylase by using date waste as substrate containing starch in solid state fermentation. The molecular weight estimated for the AT70 alpha-amylase was 85 kDa, obtaining on the basis of SDS-PAGE results, which it is higher than the range measured for the majority of alphaamylases [21, 24, 31, 32]. The AT70 alpha-amylase exposed substantial properties against various factors, which can be aided to its selection for the particular processes in industry. The pH stability profile of the AT70 alpha-amylase showed that the optimal stability was in pH 9.0 and the enzyme retained 60-73 % of its activity in pH 6.0 to 10.0. The enzyme also be active well in the range of pH 7.0–10.0, with 14
an optimum activity in pH 8.0. According to these results, AT70 alpha-amylase was more active and stable than majority of alpha-amylases from Bacillus. sp and hence the enzyme was recognized as an alkalophilic alpha-amylase which can be widely used in the food and pharmaceutical fields. Kim et al reported that, alkaline amylases adopt a unique structure and catalytic mechanism to adequate function on the starch carbohydrate in these alkaline conditions. They also suggested that, existence of an un-ionized carboxylic acid group in the active site of GM8901 alpha-amylase at high pH values [33].
Since the most industrial processes are performed at high temperatures, finding enzymes, which are able to maintain their structure and activate in the high-temperatures is very important. The alpha-amylase activity of B. licheniformis AT70 was kept in the highest amount at 60 ℃ and was decreased by 47 % at 70 ℃. Optimum activity of the thermostable alpha-amylases in the temperature range of 65-90 ℃ provide their usability in industrial liquefaction of starch [34, 35]. Oziengbe and Onilude suggested that, increased enzyme activity observed for thermostable alpha-amylases toward increase in temperature can be the result of enhanced smash of the enzyme and substrate [35]. In this research, we also found the dramatic thermal stability for AT70 alpha-amylase at 60 ℃ as not only it’s original activity was entirely protected up to 7 h but also was slightly increased. The very salient improvement of the enzyme thermostability was observed after addition of Ca2+ into reaction mixture. Residual activity in the presence of CaCl2 at 60 ℃ increased more than 2 folds of sample without CaCl2 at the same temperature. Although, the improvement of thermal stability at the same condition was observed for some thermostable alpha-amylases including Thermus filiformis, Bacillus licheniformis NH1, Bacillus licheniformis M27 and Bacillus amyloliquefaciens [20, 6, 36, 37], but, this fantastic improvement in the alpha-amylases thermal stability didn’t report up to now. Machius and co-workers reported that improvement of thermal stability with calcium ion can be explained by the creation of a calcium–sodium–calcium in the main Ca2+ binding site, bridging the domains A and B of the enzyme [38]. Furthermore, the stabilizing effect of Ca2+ on the thermostability of the alpha-amylase can be clarified due to the salting out of hydrophobic residues by Ca2+ in the protein, hence triggering the adoption of a compact structure [39] and has also been stated in other organisms [40-42].
15
The alpha-amylase activity was considerably impressible from metal ions and chemical compounds. Exceptions of Mg2+ and Hg2+, all of metal ions increased the enzymatic activity of AT70 alpha-amylase. Activation of the AT70 alpha-amylase in Cu2+ disagree with the behavior observed for the most alpha-amylases [5, 43, 44] and it is assumed that Cu2+ interact with some of amino acid residues in the active site of the enzyme. Yet, some of the alpha-amylases i.e. alpha-amylase from Natronococcus sp. strain Ah-36 were activated by Cu2+ [45]. Nies reported that, the reduction of alpha-amylase activity by heavy ion Hg2+ is the evidence of thiol groups in the alpha-amylase structure [46]. Although, Kolcuoğlu and co-workers reported that, Mn2+ increased the activity of Geobacillus caldoxylosilyticus TK4 alpha-amylase about 51% [47] and the maximum activity of A. veronii NS07 alpha-amylase was also observed in the presence of Mn2+ [12], but the enzymatic activity of the AT70 alpha-amylase improved about twofold in the presence of Mn2+. These results indicated that the semi-rigid and flexible structure of the enzyme, which improve enzyme linkage to substrate [48].
Despite of the reduction of enzyme activity of the most alpha-amylases in the presence of EDTA [49-51], the activity of the AT70 alpha-amylase increased about 145% in the same condition. This result is agree with the improvement of the activity of Bacillus. sp L1711 alpha-amylase in the presence of 5 mM EDTA [52]. Arikan B., reported that alkaline amylases vary in their response to the chelator EDTA with some being unaffected [4]. In addition, Hagihara et al., reported that Amy-K38 retained full activity in the presence of 1mM EDTA as high as 100 mM while Egas et al., reported 88% activity with 10 mM EDTA [53, 54]. It is probable that metal ions can be activated AT70 alpha-amylase but they don’t essential for the starch hydrolysis process. However, the molecular investigations of the AT70 alpha-amylase are in progress to find the better understanding of these results. AT70 alpha-amylase showed not only good resistant toward commercial solid detergents, but also its activity was increased around 34% and 13% by Shooma and Taghe detergents. In contrast, the alpha-amylase reported by Chakraborty et al, retained 35–70% of its original activity when pre-incubated in the presence of commercial detergents [55]. Due to remarkable stability and increase the enzyme activity in the presence of several commercial detergents, the
16
AT70 alpha-amylase could be considered as a talented candidate in the formulation of industrial detergents in order to clean starch-containing stains. AT70 alpha-amylase had completely retained its original activity at 0.0-4.0 M NaCl and even it,s activity was increased about 42% at 1.5 M. It is more than the halophilicity results of the alpha amylases from the other microbial sources such as Streptomyces sp. D1 [55] and Bacillus dipsosauri [41]. Activity and stability of the halophilic amylases in the various concentrations of different salts can led to adaptation of the microorganisms to the harsh environments, mainly salterns [56].
Kinetic parameters of the AT70 alpha-amylase were approved under standard assay conditions using several concentrations of soluble starch as substrate. As obtained from the Lineweaver– Burk plot (Fig. 5), the Km and Vmax values were 1.203 mg/ml and 0.01 mg/ml/min, respectively. The Vmax and Km values of the alpha-amylase from Bacillus amyloliquefaciens for soluble starch was found to be 4.11 mg/min and 3.076 mg, respectively [57]. Goyal and co-workers reported that Km and Vmax values was 3.44 mg/ml and 0.45 mg hydrolyzed starch/ml/min at 55°C, respectively, for Lactobacillus manihotivorans [58]. However, Km value of AT70 alpha-amylase is lower than Km values of the other thermostable alpha-amylases from Bacillus lichentformis and various Bacillus sp. [59, 60], that it demonstrate the high tendency of the AT70 alphaamylase for bond to substrate.
Upshot, one of the other attractive properties of the AT70 alpha-amylase was strong hydrolysis power on the high concentrations (14-20%) of raw corn granules. To the best of our knowledge, this is the first report of Bacillus licheniformis alpha-amylase with potency of raw-starch digesting. In general, there are a small number of bacterial amylases capable of strong hydrolysis of raw corn starch at a high concentration [61]. In view of raw corn starch digesting ability of the AT70 alpha-amylase, it could have potential applications in the food and fermentation industries that can be due to the global availability of raw corn starch. Existence of a separate starchbinding domain (SBD) in carboxyl-terminal region has been proven for the most of starchdigesting enzymes and has been found to be very important in binding to raw starch [62]. The molecular mechanism of the AT70 amylase can be appeared after determination of three dimensional structure. 17
5. Conclusion Nowadays, thermostable raw-starch degrading alpha-amylase has been attending a great interest for the starch hydrolyzing. Results of AT70 alpha-amylase characterization showed that this enzyme was stable at the wide ranges of temperatures from 40-70 ℃ and showed good activity at alkaline pH and over a broad range of NaCl concentration (0.5-3.0 M). It, s thermal stability was improved interestingly about 2.5 folds by adding Ca2+. SSF results indicated that the maximum alpha-amylase production was achieved by date wastes and wheat bran, as cheap agricultural wastes. Moreover, raw starch digesting effect by AT70 alpha-amylase was considerably observed on the high concentrations of raw corn starch. Due to these excellent properties, the AT70 alpha-amylase can be used as a good candidate in the starch industry especially in the rawstarch digestion.
Acknowledgment The authors express their gratitude to the Research Council of the Shahid Bahonar University of Kerman (Kerman, Iran) for financial support during the course of this project.
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Legends: Fig. 1. The comparison of the alpha-amylase production in the basal medium (containing % 0.1 of soluble starch) and the optimal medium (encompassing all of the effective factors) after 72 h of incubation at 55 ℃.
Fig. 2. Investigation of the alpha-amylase production from Bacillus licheniformis AT70 using agro residues under SSF procedure. After 7 days of incubation, the supernatants exploited from centrifugation of the culture mediums and applied to assay enzyme activity.
Fig. 3. (A) SDS-page of AT70 alpha-amylase. Lane 1, active fraction from ion exchange chromatography. Lane 2, Protein markers. (B) Activity staining of the alpha-amylase enzyme in native PAGE by iodine solution.
Fig. 4. A) Influence of different pH values on the alpha-amylase activity and stability. pH activity was measured in the buffer systems presented in section 2 under standard assay conditions. For determination of pH stability, pre-incubation of the alpha-amylase was performed in the same buffers for 60 min. B) Activity profile of the AT70 alpha-amylase at the different temperatures from 30-80 ℃. Activity was measured in standard assay conditions using 1% soluble starch. C) The thermal stability was determined by pre-incubating the enzyme at 3080 ℃ for 60 min and then assaying the residual activity under standard conditions. D) The thermal stability in the presence of CaCl2 was determined by pre-incubating the enzyme at 30-80 ℃ for 60 min and then assaying the residual activity under standard conditions.
Fig. 5. Alpha-amylase activity was determined in the presence of various concentrations of soluble starch as substrate. Km and Vmax values was determined from Michaelis-Menten and Lineweaver-Burk plots.
Fig. 6. Determination of the alpha-amylase activity rate on the different concentrations of raw corn starch. The activity was assayed after 5 h of incubation at 55 ℃.
24
Fig. 1.
25
Fig. 2.
26
A
1
B
2
Fig. 3.
27
Fig. 4.
28
B)
Fig. 5.
29
Fig. 6.
30
Table 1. Influence of various factors on the alpha amylase production. Bacillus licheniformis AT70 was incubated at different condition such as, carbon and nitrogen sources, different ions and pHs for 48 h and then the alpha amylase production was considered at standard condition.
Factors Carbon sources: Glucose Galactose Maltose Fructose Soluble starch Control Nitrogen sources: Peptone Yeast extract Gelatin NaNO3 NH4Cl (NH4)2 SO4 Control Metal ions: CaCl2 MgSO4 KCl NaCl Control Different pHs: pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0
Enzyme activity (U/ml) 256±5 264±6 228±6 144±7 388±5 213±5 260±5 52±6 255±7 278±7 329±5 294±4 105±5 451±4 20±5 29±6 60±6 424±5 72±5 74±7 251±8 288±6 146±6
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Table 2. Effect of some metal ions and chemical additive on the alpha-amylase activity. Metal ions and chemical additives
Activity (%)
Fe2+
143±3
Cu2+
114±3
Co2+
130±5
Zn2+
131±6
Mn2+
257±5
Ca2+
108±5
Hg2+
85±7
K+
117±4
Mg2+
87±3
T.X.100
88±4
2-Mercaptoethanol
107±4
EDTA
145±3
SDS
75±4
H2O2
117±5
Control
100±4
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Table 3. Effect of commercial detergents on the alpha-amylase activity. Commercial detergents
Activity (%)
Barf
94±5
Shooma
134±5
Tage
113±4
Banoo
101±4
Dioxigeneh
88±3
Darya
88±3
Kaf
85±4
Control
100±2
The enzyme activity in any detergent calculated as a percent of the activity in the control sample (contained no detergent).
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Table 4. Effect of various water miscible and water immiscible organic solvents on the alpha-amylase activity. Organic solvents
Activity (%)
No ion
100±2
Isopropanol
80±3
1-botanol
110±3
Diethyl ether
160±4
DMF
42±3
Cyclohexane
115±4
Chloroform
126±4
Toluene
111±3
DMSO
130±2
Methanol
90±1
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Table 5. Alpha-amylase activity in the presence of NaCl.
NaCl (M)
Activity (%)
0
100±1
0.5
115±1
1.0
133±3
1.5
142±2
2.0
139±3
2.5
120±3
3.0
112±2
3.5
105±2
4.0
101±1
For determination of the salt activity, AT70 alpha-amylase was incubated In the presence of different concentrations of NaCl (0.0-4.0 M).
35