?Purification and characterization of the ?-amylase from locally isolated Xanthomonas campestris bacteria
Abstract:
A total of 18 soil-isolated starch analytic bacteria were isolated from different areas of Mosul University. In order to make an initial differentiation between them in terms of their ability to produce the enzyme ?- amylase, the isolates were subjected to a primary screening process, the isolates then that showed little productivity were excluded. then the secondary screening was performed on five isolates, the result of the final test stage among those isolates led to that the isolate denoted X2 is the most efficient. Consequently, identified it by microscopic diagnostic and biochemical tests that confirmed it is Xanthomonas campestris bacteria. The pure ?-amylase was extracted in several steps. included precipitation with ammonium sulphate at 40-70 % saturation followed by dialysis and gelatin filtration in the Sephadex G-100 column. The number of times of purification was 30.2 and the enzyme yield was 57 %. the result of the characterization of the enzyme showed that the molecular weight was 55 kiloDalton when evaluated by SDS- acrylamide gel electrophoresis (PH 6 ) method, gave the highest efficacy of the enzyme while its maximum efficiency was recorded at 55 °C.

Introduction :
In the last few years, the importance of enzymes had increased due to appear vital role in the existence of life itself, since it acts as a vital catalyst for a wide range of chemical reactions (1). ?- amylase is one of the most important enzymes not only for its ability to catalyze starch and glucose production but also for its ability to decompose starchy substances to simpler sugars such as dextran and maltose(2).

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amylase is an enzyme that hydrolyze starch by breakdown glycosidic bond.it is classification into three main types: alpha-amylase (EC3.2.2.2)(1,4-?-D-glucan glucanohydrolase) which works on the inner of starch chain it is a well-known endoamylase, is able to cleave ?,1-4 glycosidic bond produced dextrin, maltose and glucose. Beta-Amylase (EC 3.2.1.2) 1,4-?-D-glucan maltohydrolase is an exoamylase act on the external glucose residues of starch at ? (4-1) glycosidic bond and produces only maltose. Gluco Amylase (E.C. 3.2.1.3) Glucan 1,4-?-glucosidase(E.C. 3.2.1.3) (Amyloglucosidase and gamma amylase) is different from alpha-amylase and beta-amylase because it cleaves ?(1–6) glycosidic bonds, as well as the last ?(1–4)glycosidic bonds at the nonreducing end of amylose and amylopectin, and the final product is glucose(3, 4).

Alpha-amylase is obtained from various sources including plants, especially wheat, barley and maize which is the primary source of alpha-amylase. It can also be obtained from animals and also found in microorganisms such as fungi, yeasts and bacteria, enzymes from microbial sources are generally used in industrial processes, produced at a large-scale commercial (5, 6). Amylase is utilized in various industrial operation, especially in textiles, paper, bread and alcohol, as well as in the pharmaceutical sector and detergent industries (7). It is also used in the product of dextran, which is used in many pharmaceutical industries (8) and in the product sugar for its ability to improve the filtration of sugar beet juice by breaking down the starch found in the juice(9).

due to absence literature studies on the production of ?-amylase from Xanthomonas campestris bacteria, This study an attempt to obtain local isolation from this bacterium, which has the potentiaesl to produce amylase and purification of the extracted enzyme and study some of its properties.

Materials and methods
Cultivation mediums:
Nutrient agar medium: Prepared the media according to the manufacturer instructions (Oxoid) by melting 28g of powdered nutrient agar in a liter of distilled water. the medium is used to cultivate and preserve bacterial isolates(10(
Preliminary screening medium: this medium is prepared from the following substances g/100 ml: glucose-2, CaCO3-2, yeast extract-1 and agar-1.5. adjust the pH at 7. the medium is used to detect the susceptibility of the bacterial isolates to the production of the amylase(11).

Medium of production of the ?-amylase: Prepare the medium by mixing the following substances g/l: Starch -10, Yeast extract-2, KH2PO4-0.05, MnCL2.4H2O-0.015, MgSO4.7H2O-0.25, CaCL2.2H2O-0.05 and FeSO4.7H2O(12) .

Isolation of bacteria from soil: Decimal dilutions of soil samples were carried out. Spread 0.1 ml of appropriate dilution on the medium surface containing starch in Petri dishes. Incubate of dishes at 28 °C for 48 hours. The well grew colonies were transferred to the same medium and were used to test the susceptibility of bacteria to starch analysis and the production of amylase by adding several drops of iodine solution around the growth zone to observe a transparent halo to indicate starch degradation. The solubility of isolates was calculated by the following equation ( 13) .

Efficiency of enzyme production of ?-amylase = diameter of the decomposition zone diameter of the growth zone
The secondary screening: A swab was inculated with the pollination needle of the isolates selected from the primary screening and at 48 hours in 10 ml of the production medium and incubated at 28 °C for 48 hours. The bacteria were then centrifuged at 10,000 xg for 20 min. The lysate is the crude extract of the enzyme as it is estimated to be effective.

Determination of enzyme efficacy: The method described by(14) was used in estimating the efficacy of the enzyme using the DNSA reagent, adding 0.1 ml of the crude enzymatic extract to a mixture of 0.5 ml of the base material solution with 0.4 mL from the phosphate wells in a hot water bath 55 °C for 10 minutes. Add 1 mL of the DNSA reagent to stop the reaction. Place the tubes in a boiling bath for 5 minutes and refrigerate directly with tap water. Add 10 mL of distilled water to each tube and measure absorbance at a wavelength of 540 nm. The unit of activity was defined as the amount of enzyme that releases 1 micromole of polysaccharides in the form of maltose per minute under experimental conditions.

Diagnosis of selected isolate: cultural diagnostic and physiological , and biochemical tests were used, including tests of indole production, the consumption of jackets, the production of urease enzyme, starch analysis, casein analysis, oxidase production, gelatinization, motion testing, carbohydrate fermentation and hydrogen sulfide gas production, as well as the ability to grow at different temperatures according to (15, 16).

Purification of the enzyme ?- amylase: The process of purification of the enzyme from the bacteria in several steps, including deposition of ammonium sulphate followed by dialysis and then chromatography of gelatin filtration, ammonium sulphate was used to precipitate the enzyme and saturation ranged between 40-70 % followed by centrifugation at 10,000 cycles /minute and for 15 minutes. The precipitate was taken and solved in a quantity of concentrated phosphate solution with a concentration of 0.05 molary and pH 7.0 .The enzymatic extract, protein concentration, and enzymatic efficacy were estimated. This is followed by a process of enzyme deleterization to get rid it of ammonium sulphate salts for 24 hours. The gelatin filtration process was performed using the Sephadex G-100 gel, which was prepared by suspension of 50g of Sephadex powder in 500 ml of distilled water and placed in a water bath at 90-85 °C for 3 hours with continuous stirring. A quantity of it was washed with pH solution(5.0) twice, and the mixture was used to wash the column and then another amount of gel was suspended with the catalyst and the degassing process was carried out. the mixture in the glass column to give the gel dimensions of(30 × 2.5) cm and the balancing of the column with phosphate solution. Transfer the enzymatic solution to the gel filter column Sephadex G-100. The arbitrage and recovery process was carried out by the phosphate precipitate solution for 18 hours with a flow velocity of 30 mL/h by 3 mL per part. The parts that gave enzymatic efficacy within the one end of the column were collected by the collector of the parts, which reached(10) parts. The concentration of protein in the aggregated parts was accompanied by the reading of light absorption at 280 nm and the enzymatic activity of the separated parts was estimated.

Characterization of the enzyme:
The molecular weight of the enzyme was estimated using the SDS-PAGE technique(Sodium-Dodecyl-Sulfate- Polyacrylamide -Gel -Electrophoresis). Using the bi-directional electric migration system using the Bio-RAD method(17).

Optimum pH: The solutions of the basic substrate were prepared by dissolving 50 mg in 10 mm of various reagents and with a range of pH (4.5-9). The efficacy of the enzyme in each solution was estimated and the relationship between PH values ??versus enzyme activity was determined to determine the optimal PH of the enzyme’s efficacy.

Optimal temperature of the estimate enzyme activity of the enzyme: The effectiveness of the enzyme was estimated to a degree of degrees the temperature ranged between 40-70 °C and by a difference of five degrees and then the relationship between enzymatic activity and temperature was determined.

Results and discussion :
Isolation of bacteria: Selection of colonies that were characterized by growth on the nutrient agar from a large number of isolates that appeared on the medium, which was characterized by yellow color of the nature of the gels, which amounted to 18 isolates bacterial. The results of the preliminary tests conducted on the selected isolates are negative for the color of chromium when pigmented, as well as being bacillary in the form.

Preliminary screening: To obtain a clear indication of alpha-amylase- producing isolates, starch has been added to the isolation medium, one of the most common ways to capture the amylase-producing organisms. The isolates were able to secrete the bactin-digesting enzymes by observing the starch breakdown in the regions surrounding the growth of bacterial isolates. By time we discovered the susceptibility of many isolates to its production through a clear halo decomposition diameter, other isolates showed a poor susceptibility to starch degradation which was reflected in their ability to produce amylase as shown in Table (1) .Five of 18 isolates gave a high decomposition rate(2 or more), of which isolate X2 with a higher decomposition rate of 76.3, the isolate of X16, which gave a 3.25 decomposition ratio, and X13, X7 and X8 isolates gave a decomposition rate of 2.50 And 1.66 and 1.53 respectively, so these isolates were selected for testing in the secondary screening. The difference in isolates in their ability to analyze starch may be due to the presence of genetic variation among them. In an initial attempt to screen for a number of bacterial isolates, 18 were able to obtain 9 isolates of Bacillus sp, which showed a clear variation in the production of amylase by calculating the diameter of the halo. It gave the isolate numbered R6, which was later identified as B.subtilis the highest decomposition rate which was 2.8 when developing on starch as the sole source of carbon. (19) mentioned that the isolates of Bacillus sp. isolated from the soil have the ability to decompose starch when shown by an iodine solution. These isolates showed a difference in their ability to produce ?-amylase.

Table(1): Preliminary screening of different bacterial isolates.

Isolates Percentage of the diameter of the decomposition zone/ diameter of the growth zone
Isolates Percentage of the diameter of the decomposition zone/ diameter of the growth zone
X1 1.27 X10 1.42
X2 3.76 X11 1.10
X3 0.88 X12 1.34
X4 1.22 X13 2.50
X5 1.06 X14 1.20
X6 1.50 X15 0.68
X7 1.66 X16 3.25
X8 1.53 X17 1.35
X9 1.51 X18 1.28
Secondary screening: As a result of different reactions with respect to the ability of isolates to produce the enzyme ?-amylase from the primary screening process, the secondary screening stage came on the isolates chosen from the primary screening as isolates producing the enzyme ?-amylase after its development on the production medium, which are five in number and denoted designated by(X2, X7, X8, X13 and X16) to better characterized production. This is a very valuable step to validate the choice of enzyme-producing isolates that have seen the emergence of a star of one of the selected isolates, which has shown a high level of efficiency in the production of ? -amylase as shown in Table(2). The isolate with the symbol(X2) yielded high production of 6.3 mg/ml while the enzyme quantity ranged between 4.8 and 4.3 unit/mL respectively for the X16 and X13 isolates. Both the X7 and X8 isolates were low in the enzyme production, with the amount of enzyme obtained(2.7 and 1.5) unit/ml respectively. Based on the results obtained, the focus of attention has been on highlighting X2 isolate which for its part showed high production of ?-amylase enzyme. Twenty(20) isolates were obtained from B. subtilis, number B5, which produced the amylase enzyme with a production capacity of 166.5 units/ml in the secondary screening process from a large number of isolates. 21 were able isolate 50 local isolates from the soil and showed isolates of B. subtilis, which has a 10-R symbol of higher amylase yield of 228 units/ml in submerged farms.

Table (2): Secondary screening of ?-amylase from selected isolates.

Isolation The amount of enzyme ?-amylase unit / mL
X2 6.3
X7 2.7
X8 1.5
X13 4.3
X16 4.8
Diagnosis of isolates:
The overwhelming success was showed by X2 selected from the primary and secondary screening stages as the best analysing isolate for the starch and the production of ?-amylase, which was an excellent model for the production of the enzyme in urgent need and for the importance of its diagnosis. The taxonomic keys mentioned in(15 , 16), which are the standard means of obtaining bacterial identity, were adopted after a set of morphological diagnostic and agricultural diagnostic tests as shown in Table(3) as well as biochemical tests showing the belonging of the selected isolates to Xanthomonas campestris bacteria. The initial tests, which included the morphological and cultural tests, showed that the colonies are characterized by a creamy yellow color with soft texture, sticky nature and circular shape with regular edges. The bacterial shape under the microscope was found to be bacillus, gram negative when examined with the optical microscope and by the use of the ocular lens. This is in line with what was stated(22) when isolating the Xanthomonas campestris bacteria from different leaves of the plant and also showed the ability of the bacteria to move when they are planted on the medium of the movement test. During the prickling of the pollination pollen, it was also negative for the oxidase test as the color of the colony did not change to violet.While having the ability to produce catalase enzyme through the emergence of gas bubbles when the addition of hydrogen peroxide solution(H2S), and also gave a positive result to test the Indole showed a negative result of the consumption of jackets and was able to analyze starch and galaxies. These tests were sufficient to confirm the diagnosis, which was previously adopted(23,24, 25).

Table (3): The physiological, vegetative and chemical properties of isolate X2.

The cells shape short bacilli
Gram stain –
The nature of growth on the solid medium widespread and convex
Growth on the liquid medium grows on the middle surface
The color of the colony yellow to the creamy
The edges of the colony circular and regular
Motility test +
Oxidase –
Catalysis test
+
Indole test +
H2S test –
Starch hydrolysis test
+
Citrate utilization test –
Gelatin liquefaction test +
Purification of the enzyme ?-amylase: The crude enzyme obtained from the bacterial farm leachate was subjected to a number of purification steps described in Table 4 and starch was used as an enzyme reaction substance. Sequential filtration procedures included precipitation with ammonium sulphate by 70 % saturation, yielding 30.64 unit/mg and 94 % enzymatic yield. The precipitation obtained from centrifugation and disolved in a little amount sterile solution 0.025 and pH 7.4. It was subjected to the permeability process by using a permeable membrane of molecular weight 14000 versus the same buffer solution to get rid of ammonium sulphate. The enzymatic solution, which was collected after the membrane permeability process, was 27 mL. The enzyme’s efficacy and protein concentration was measured. This step achieved enzymatic efficiency of 49.27 units and the quality of 133.16 units/mg. The number of purification times achieved in this step reached 11.8 with an enzyme yield of 79%.The enzymatic solution, which was collected after the membrane permeability process, was 27 mL. The enzyme’s efficacy and protein concentration were measured. This step yielded an enzymatic efficiency of 49.27 units and the quality of 133.16 units/mg. The number of times of purification achieved in this step amounted to 11.8 and the result of enzymatic 79 %. It is noted from the purification table that the concentration of protein has decreased and due to the osmosis of proteins with molecular weights less than 14000 Dalton out of the tubes of dialysis. Also this process lead to the entrance of buffer solution to the bags of dialysis leading to the dilution of enzyme concentration and increasing of purification times. Ammonia sulphate was also used to concentrate the alpha amylase enzyme by(26) product from the Xanthomonas campestris bacteria with a saturation rate of 70%. In the gel filtration step, Fig. 1: The appearance of five protein peaks was one of these peaks, the fourth summit contains an enzyme activity, while the other peaks were completely free of them. The peak of the enzymatic activity was identical to the protein peak. Therefore, the fourth peak of the enzyme was determined which reached 64.35 ml / with Enzymatic result of 57% with purification times 30.2) 27(were able to purify of amylase from Bacillus subtilis BS5 and obtained three protein peaks. The second peak had an enzymatic effect of 127.20 units/ml while the specific efficacy(0.99) during it, the enzymatic result reached 74.62% and the times of purification 30.50.The difference in the high quality and frequency of purification of the purified enzyme from different sources is due to the difference in the source of the enzyme as well as the difference in the techniques used in the purification steps and the type of gel use (28) .

Table(3): Steps to purify the enzyme ?-amylase from the isolation of Xanthomonas campestris.

Purification step
the size
Effectiveness
unit /ml
Protein mg/ml
Specific efficacy
unit / mg Overall effectiveness
Number of times of purification
Enzymatic yield%
The crude enzyme 100 16.82 1.50 11.21 1682.0 1 100
Deposition with ammonium sulphate
52 30.64 0.85 36.04 1593.28 3.2 94
The dialysis
27 49.27 0.37 133.16 1330.29 11.8 79
Gel filtration
15 64.35 0.19 338.68 965.25 30.2 57

Figure(1): Purification of the enzyme ?-amylase extracted from Xanthomonas campestris
Characterization of the enzyme: Determination of molecular weight of the ?-amylase enzyme purified by gelatin filtration method on gel acrylamide with gel electrophoresis and with the presence of SDS. Figure 2 shows that the molecular weight of the enzyme was estimated at about 55 kilo Dalton. This value approximates the findings of 26 when estimating the molecular weight of alpha-amylase from Xanthomonas campestris, which was 52 kilo Dalton at electrophoresis on the poly acrylamide gel and with SDS. (29) showed that the molecular weight of the alpha-amylase purified from the Xanthomonas campestris strain was 42 kilo Dalton and the presence of SDS.

Figure(2): Molecular weight of the ?-amylase enzyme.

Optimal pH for enzyme efficacy: Figure(3) shows the optimal PH curve for the efficacy of alpha-amylase, which had an upward trend up to pH 6, after which enzymatic activity was reduced. The decrease in enzyme effectiveness is due to the effect of pH of the reaction medium in specific groups that were ionizable within the enzyme structure and in the base material on which the enzyme works. Results of comparing the results revealed that the optimal pH for the efficacy of alpha-amylase from the isolation of Xanthomonas campestris is 6.2 (26). Within the same field, 30 indicated that the optimal pH for the efficacy of alpha amylase from Bacillus subtilis ATCC6633 is 7.0.

Figure (3) Optimal pH curve for ?-amylase enzyme activity.

The optimum temperature of the enzyme efficacy : Results in Fig.4 showed increased efficacy of the refined ?-amylase with temperature increase, which reached a maximum of 55 °C, after which the effect was gradually reduced with increasing temperature. The increase in the speed of enzymatic reactions with a temperature increase to a certain extent is due to increased collisions between the enzyme molecules and the base material. High temperatures cause a decrease in the enzyme’s effectiveness due to an enzyme inhibition, which changes the structure and body of the active site. Similar studies have shown an a proximal result (29) mentioned that the optimal temperature of the alpha-amylase enzyme from Xanthomonas campestris is 50 °C. The optimal temperature of the enzyme’s efficacy varies with the source of the enzyme. The optimal temperature of the alpha-amylase enzyme from B. subtilis is 60 °C.

Figure(4): The optimal temperature curve for the efficacy of ?-amylase enzyme.

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