Enzymes are macromolecular biocatalysts that initiate and increase the rate of thousands biochemical and metabolic reactions in living cells . Amylase is a general term for enzymes that hydrolyze starch and glycogen. There are many kinds of amylases, and amylases can be divided into α-amylase and β-amylase according to the isomeric types of hydrolysates. Alpha-amylase (1,4-a-D- glucanohydrolase; EC 188.8.131.52) is one of the enzymes that have the ability to hydrolyze and cleave the α-1,4-glucosidic linkage and catalyze the breakdown of starch and starch derivatives; amylopectin and amylose; oligosaccharides and dextrin, liberating reducing groups   . Alpha-amylase represents about 30% of the whole enzyme market in the world. Proteases are the first  .
Alpha-amylase is widely used in industrial production   . α-amylase is used in starch processing industry , such as the manufacture of glucose, maltose, various kinds of dextrin, etc. α-amylase is used in food industry, such as bread baking, monosodium glutamate, spices, fruit juice, etc. α-amylase is used in brewing and fermentation industries, such as beer, liquor, soy sauce, vinegar, etc. In textile industry, α-amylase is used in chemical fiber, high-quality silk and man-made cotton desizing process. In pharmaceutical, medical, α-amylase can be made into different kinds of industrial enzymes, medical enzymes, diagnostic enzymes, digestive drugs, etc. In the detergent industry , α-amylase acts synergistically with alkaline protease, lipase, etc, to give it a better wash. It is also widely used in paper industry and feed industry.
Plants, animals and microorganisms have the ability to produce amylases  . Amylases derived from microbial sources are preferred because of its plasticity, vast availability and they have a great potential in fulfilling industrial demands. Bacillus species is considered the best producers of α-amylase that play essential roles in starch processing industry . Amylases isolated from different Bacillus species have significantly different properties, such as the optimum pH and temperature as well as metal ion requirements .
In this study, taking Bacillus subtilis QM3 as the research object, production of α-amylase by B. subtilis QM3 was identification by iodine test. The amylase activity of B. subtilis QM3 was determined by 3,5-dinitrosalicylic acid (DNS), and the enzymatic properties were studied. It provides a theoretical basis for the application of B. subtilis QM3.
2. Materials and Methods
2.1.1. Bacteria Materials
Bacillus subtilis QM3 used in the present study come from the microbiological lab, College of Life Science, Shanxi Normal University, China .
Basal culture medium (g/100mL): peptone 1.0, beef extract 0.3, sodium chloride 0.5, agar 2.0, pH 7.4-7.6, 121˚C for 20 min.
Screening medium (g/100mL): peptone 0.5, NaCl 0.3, soluble starch 0.2, agar 2.0, pH 7, 121˚C for 20 min.
Seed medium (g/100mL): yeast extract 0.5, peptone 1.0, NaCl 0.5, soluble starch 0.2, monopotassium phosphate 0.1, water, pH 7.0, 121˚C for 20 min.
Fermentation medium (g/100mL): bran 3.0, dipotassium phosphate 0.1, sodium nitrate 0.1, water, pH 7.0, 121˚C for 20 min.
2.2.1. Identification of Alpha-Amylase Produced by B. subtilis QM3
B. Subtilis QM3 strain was activated and inoculated into culture dish of screening medium and cultured for 48 h. After growing colony, 0.04 g iodine was lightly sprinkled on the cover of the culture dish, Inverted Petri dish, and the iodine was fumigated for 5 minutes, and the appearance of transparent ring was observed .
2.2.2. Determination of α-Amylase Activity
Preparation of crude enzyme solution: The B. Subtilis QM3 were inoculated into seed medium and cultured at 37˚C for 24 h. The fermentation medium was inoculated with 5% inoculum and incubated at 150 r/min and 37˚C for 48 h. The bacterial liquid was centrifuged at 5000 r/min for 10 min. The supernatant is the crude enzyme fluid to be measured.
Alpha-amylase activity was measured using 3,5-dinitrosalicylic acid (DNS) . The unit of enzyme activity was defined as follows: at 70˚C, pH = 6, amylase catalyzed starch hydrolysis for 5 min to produce maltose μmol number, representing one unit of enzyme activity.
2.2.3. Effect of pH on the Enzyme Activity and Stability
The activity of α-amylase was measured at pH 3, 4, 5, 6, 7, 8, 9 and 10, respectively. The enzyme solution was placed at pH 3, 4, 5, 6, 7, 8, 9 and 10 for 60 min, and then the activity of α-amylase was measured at the corresponding pH. The relative enzyme activity was calculated by comparing the enzyme activity with that of untreated enzyme solution.
2.2.4. 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˚C - 90˚C. The optimum temperature for the alpha-amylase stability was also considered by incubating the enzyme at the respective temperatures for 1 h. The relative enzyme activity was calculated by comparing the enzyme activity with that of untreated enzyme solution.
2.2.5. Effect of Metal Ions and EDTA on the Enzyme Activity
Various metal ions (Ca2+, Na+, K+, Mg2+, Cu2+, Fe2+, Mn2+, Zn2+) and EDTA were added into the enzyme solution to be tested, respectively, and treated for 60 min. The enzyme activity was measured under the action of different metal ions and EDTA. Enzyme activity in the absence of the chemical agents or the metal ions was considered as 100%.
2.3. Statistics Analysis
All data are the average of three repetitions. The obtained results were analyzed statistically by SPSS (Statistical Product and Service Solutions) statistical software. Data were analyzed by the analysis of variance and treatment mean comparison by using least significance difference (LSD; p < 0.05).
3. Results and Discussion
3.1. Bacillus subtilis QM3 Produces α-Amylase
B. Subtilis QM3 grew on the flat plate of amylase medium, and transparent ring appeared on the flat plate by iodine fumigation (Figure 1). The results indicated that B. subtilis QM3 could produce α-amylase.
3.2. Effect of Temperature on Activity of α-Amylase
The influence of temperatures on the activity of α-amylase produced by B. subtilis QM3 is shown in Figure 2. It can be seen that low temperature can inhibit the activity of the enzyme. At 30˚C - 70˚C, with the increase of temperature, the activity of the enzyme also continues to increase, and the activity of the enzyme reaches the highest at 70˚C. After 70˚C, the enzyme activity decreased continuously, and when 90˚C, the enzyme activity was very low. The results showed that the optimum reaction temperature of amylase was 70˚C.
Figure 1. The starch hydrolysate circle produced by B. subtilis QM3.
Figure 2. Effect of temperature on α-amylase activity.
3.3. Temperature Stability of α-Amylase
As can be seen from Figure 3, when the enzyme solution was left at 30˚C - 90˚C for 60 minutes, the activity of alpha-amylase decreased. The enzyme activity was above 60% at 30˚C - 70˚C. When the temperature exceeded 70˚C, the enzyme activity decreased greatly, and the enzyme activity basically lost at 90˚C. It shows that the highest temperature of amylase tolerance is 70˚C, and then the amylase is inactivated because of high temperature.
3.4. Effect of pH on Activity of α-Amylase
The influence of different pH on the activity of α-amylase produced by B. subtilis QM3 is shown in Figure 4. The α-amylase activity increased first and then decreased with the increase of pH value. When PH was 6, the activity of alpha-amylase was the highest, which indicated that the optimum pH of the enzyme was 6.0. Enzymatic reactions have an optimal pH, and the enzyme activity decreases when the pH is above or below the optimal pH. Because the pH of the solution affects the binding ability of the active center of the enzyme and the substrate, and the binding ability and activity is the highest at the optimal pH .
Figure 3. Stability of α-amylase to temperature.
Figure 4. Effect of pH on α-amylase activity.
3.5. pH Stability of α-Amylase
As can be seen from Figure 5, when the enzyme solution was placed at a pH of 3 - 9 for 60 min, the activity of α-amylase decreased. The activity of α-amylase was high at pH 6 - 7, but was low or even partially inactivated when pH was below 5 or above 8.
3.6. Effect of EDTA and Metal ions on Activity of α-Amylase
The activity of the α-amylase was determined under the action of different metal ions and EDTA as shown in Figure 6. Ca2+ could promote the activity of α-amylase significantly, the relative enzyme activity was 130.1%. Na+, K+ and Mg2+ also promoted the activity of alpha-amylase, and relative enzyme activities were 118.1%, 115.9% and 110.4% respectively. Fe2+, Zn2+, Cu2+ and Mn2+ inhibited the activity of α-amylase, the relative enzyme activities were 86.1%, 77.8%, 67.4%, and 64.2% respectively. EDTA inhibited the activity of α-amylase significantly, and its relative enzyme activity was less than 50%.
Figure 5. Stability of α-amylase to pH.
Figure 6. Effect of EDTA and metal ions on activity of α-amylase.
Most α-amylase is metalloenzyme. The influence of metal ions on the activity of amylase is mainly due to the metal ions can combine with the active center of α-amylase, thus affect the combination of α-amylase and substrate or the concept of α-amylase, thus affect the activity of α-amylase .
The results of our study revealed that B. subtilis QM3 has ability to produce α-amylase. Activity of α-amylase was 50.58 IU/mL. The optimum temperature is 70˚C, and its stability is high at 30˚C - 70˚C. The optimum pH is 6.0, and its stability is high at pH = 6.0 - 7.0. Ca2+, Na+, K+ and Mg2+ promoted the activity of alpha-amylase, Fe2+, Zn2+, Cu2+, Mn2+ and EDTA inhibited the activity of α-amylase.
This work was financially supported by the Natural Science Foundation Project of Shanxi Province (201801D121206) and the Natural Science Foundation of Modern College of Humanities and Sciences of Shanxi Normal University (2018JCYJC01).
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