2.2 Outline who may be involved in advanced care planning.
Advance car planning centres on discussions with a person who has capacity to make decisions about their care and treatment. If the individual wishes, their family, friends and health and social care professionals may be involved, it is recommended that with the individual’s agreement that discussions, regularly reviewed and communicated to key people involved in their care.
2.3 Describe the type of information an individual may need to enable them to make informed decisions.
Statements of wishes and preferences can include personal preferences, such as where one would wish to live, have a shower rather than a Bath, or just wanting to sleep with the light on and the bedroom door open. Sometimes people may wish to express their values, e.g. The welfare of their husband and children are taken in to consideration when decisions are made about the place of care. Sometimes people may have views about the treatment they do not wish to receive, but do not want to formalise their views as a specific advance decision to refuse treatment. These views should be considered when acting in a person’s best interest but will not be legally binding. A statement of wishes and preferences cannot be made in relation to any act which is illegal. E.g. assisted suicide.

?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).

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

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.

References:
Abel-Nabey,H.M. and Farag, A.M.(2016). Production, optimization and characterization of tracellular amylase from halophile Bacillus lichineformis AH214. Biotechnol., 15(17): 670-683.

Abe,JI., Shibata,Y; Fujisue,M. and Hizukuri,S.( 1996). Expression of periplasmic amylase of Xanthomonas campestris K=II 151 in Escherichia coli and its action on maltose. Microbiol., 142, 1505-1 512.

Garg,D. and Kaur,DR.M.(2013). Extraction, Purification and characterization of enzyme amylase from Bacillus amyloliq- uefaciens. Inter JAdvEng.Sci.,3(3): 158-159.

Vijayan, M.; Jothinathan,M.K.D.; Murugesan,S.K.; Rangasamy,N.; Sathasivam,V. and Shanthini,T.(2015). Microbial production of amylase from cassava waste. Res in Pharmacy., 5: 20-28.

Ashwini,K.; Karthik,L.; Kumar,G.and Rao,K.V.B.(2013). Purific- ation and activity of amylases of marine Halobacillus sp amylase., 429(6): 781-785.

Aygan,A.; Sariturk,S.; Kostekci,S. and Tanis,H.(2014). Production and characterization of alkaliphilic alpha-amylase from Bacillus subtilis A10 isolated from soils of Kahramanmaras, Turkey., 210 (8): 2168-2173.

Asad,W.; Asif,M. and Rasool,S.A.(2011). Extracellular enzyme production by indigenous thermophilic bacteria partial purification and characterization of ?-amylase by Bacillus sp. WA21., Pak. J. Bot., 43(2): 1045-1052.
Kumar,R. and Mehta, A.(2013). Isolation, Optimization and chara -cterization of ?-Amylase from Bacillus alcalophilus. IJSR., 29(70): 2319-7064.

Abu,T.F.; Enujiugha,V.N.; Sanni,D.M. and Bamidele,O.S.(2014) .Purification and characterization of ?-Amylase from Bacillus subtilis isolated from ermented African locust bean (PARKIA BIGLOBOSA) seeds. LifeSc. Bt ; Pharm. Res.,39(4): 2250-3137.

Sudharhsan S, Senthilkumar S, Ranjith K.(2007). Physical and nutritional factors affecting the production of amylase from species of Bacillus isolated from spoiled food waste. Afr.J. Biotechnol., 6(4): 430-435.

Saha, B.C.( 2002) . Production, purification and properties of xylanase from a newly isolated Fusarium proliferatum. Process Biochemistry., 37: 1279-1284.

Hamilton,L.M.; Kelly,C.T. and Fogarty,W.M.(1999) Production and properties of the raw starch-digesting ?- amylase of Bacillus sp. IMD 435. Process Biochem 35: 27-31.

Amoozegar,M.A.; maletzadeh,F. and malik,K.A.(2003). production of amylase by newly isolated moderate halophile, Halobacillus sp. Strain MA-2. Microbiol.meth., 52: 353-359.

Miller, G. L.(1959) . Use of dinitrosalicylic acid reagent for the determination of reducing sugar. Anal. Chem. ,31: 426-428.

Holt,J.G.N.(1979). The Shorter bergeys manual of Determ- inative bacteriology . Williams and wilkins . U.S.A.

Roohie,R.K. and Umesha,S.(2012). Development of multi- plex PCR for the specific selection of Xanthomonas campestris pv.campestris in cabbage and correlation with disease incidence. Pathol. Microbiol.,3: 1-9.
Samiallah,T. R.; Bakhah, A.; Rao, A.Q. and Naz,M.(2009). Isolation, purification and characterization of extracellular B-glucosidase from Bacillus sp. Advan in Environ. Biol. ,3(3): 269-277.

Verma,V.; Avasthi,M.S.; Gupta,A.R. and Kushwaha,A. (2011). Amylase production and purification from bacteria isolated from a waste potato dumpsite in district Farvukhabad U.P. state India. Euro.J.Bio.,1(30): 107-113.

Kim,D.H.; Morimoto,N.; Saburi,W.; takehana,T.; Koike,S. and Matsui,H.( 2017). Biosci. Biotechnol. Biochem., 76( 7): 1378-1383.

Abdel-fahah,Y.R.; Solima,N.S.; El-Toukhy,N. and Ahmed, R.S.( 2013). Production , purification and characterization of thermostable ?-amylase produced by Bacillus licheniformis isolate AI20. Chem.,2013, Artide ID. 673173,11.

Bukhari,D.A. and Rehman ,A.(2015). Purification and characterization of ?-amylase produced from Bacillus subtilis isolated from local environment. Pak.J.Zool., 4794): 905-911.

Roohie, R.K. and Umesha , S. ( 2013).DNA polymorphism analysis of Xanthomonas campestris pv. Campestris using single strand conformation polymorphism (SSCP) and random amplified polymorphic DNA. Biotechnol., 12: 6913-6921.
Gracelin,D.H.S..; Britto,A.J. and Kumar.,J.R.(2011). Dete- ction and identification of Xanthomonas campestris PV. Centellae on leaves of centella asiatica collected in tamilnadu. Asian J. Pharm Clin.Res., 5(10): 111-113.
Popovic,T.;Josic,D.;Starovic,M.;Postic,D.and Stanlcovic,S. (2013). Phenotypic and genotypic characterization of Xanthomonas campestris strains isolated from cabbage, kale and broccoli. Arch.Biol.Sci., 65(20): 585-593.
Bila,J.; Mortensen,C.N.; Andresen,M.; Vicente,J.G. and Wulff, E.G.(2014). Xanthomonas campestris pv. campestris race 1 is the main causal agent of black rot of Brassicas in Southern Mozambique .Biotechnol. 12(6): 602-610.
Abe,J.I.; Onitsuka,N.; Nakano,T.; Shiba,Y.; Hizukuri,S. and Entani,E.(1994). Purificationand characterization of periplasmic a-Amylase from Xanthomonas campestris K-1 1 151.Biotechnol. 176( 12) : 3584-3588.

Femi-Ola,T.O. and Olowe,B.W.( 2011). Characterization of ? amylase from Bacillus subtilis BS5 isolated from Amitermes evuncifer Silvestri.Microbiol.,6: 140-146.
Baltas,n.; Dhcer,B.; Ekinci,A. and Adiguzel,A.(2016). Purif- ication and characterization of extracellular ? amylase from thermophilic Anoxybacillus thermarum A4 strain. Braz. Arch. Biol. technol.,59: e1616346.
Hongge,C.; Jian,C. and Xu,W.(2005). Study on properties of ? amylase from Xanthomonas campestris -PV campestris 8004. Chines academy of agricultural sciences. ,33(92): 143-146.

Maity, S.; Mallik,S.; Basuthakur, R.and Gupta, S.(2015). Optimization of solid state fermentation conditions and character- ization of thermostable alpha amylase from Bacillus subtilis (ATCC 6633). Bioprocess Biotech., 5(4): 2-7.
Sani,I.; Abdulhamid,A.; Bello,F.; Yahaya,M. and Bagudo, A.I.(2014). Isolation, partial purification and characterization of ?-amylase from Bacillus subtilis. Microbiol. Biotech. Res.,4 (1):49-54.

2.0 LITERATURE REVIEW
2.1 Conceptual Definitions
2.2 Motivation
The research presented is about the theories of motivation, which prove there is a need for motivation in all workplaces and effective ways of motivating the workforce. Motivation has become the main factor in the most researched part of management in today’s working environment. Many theories were designed which greatly influenced and still influence many organizational performance. According to Bulkus ; Green (2009), motivation come from the word “motivate”, which means a move, push or influence to continue for satisfying a want. In the view of Bedian (2003), motivation is an internal motivation to satisfy an unsatisfied need and the will to achieve it. In addition, motivation is an advancement of moving and supporting goal-directed behavior (chowdhury, M.S, 2006).

In the mid twentieth century were several motivational theories created by scientists and psychologists, namely Maslow’s hierarchy of needs (1943), Herzberg’s two-factor theory (1959) and Vroom’s expectancy theory (1964). Those researchers positioned main on employee motivation and in general particularly. In the previous year’s different definitions of motivation were defined, eg. Herzberg (1959) defined employee motivation as performing a work related action because you want to.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

According to Mullin (1996), motivation is the force within an individual that influence his or her path, the passion and perseverance to perform well or not. On a further note, he also that motivated employees are willing to make an effort, toward incentive or reward value of the goal. The management of business organizations set this kind of strategy to fulfill their employees’ common tasks, objectives and goals. According to Warren (2010), the workforce motivation is an important concept in an organization. On the other hand, Wisconsin put forward that organizational goals help to focus our learning on the system’s highest priorities. In this way, professional development and organizational improvement are integrally related. The growth of professionals can contribute to the organization as a whole, but individual growth without the organizational context is not enough and inefficient in helping the organization to achieve its strategic goals. In addition, motivation is an evolution of supporting the goal-directed behavior (Chowdhury, M.S, 2006).
The first question that can arise is: “why management need to motivate employees?” (Herzberg, 1959). According to Smith (1994), it is because of the survival and success of the business. Amabile (1993) said that managers must know how to motivate and deal effectively with their employee’s motivation as a motivated workforce influence the success of an organization by achieving the organizational goals and objectives. Motivation is a decision making process, through which someone chooses the preferred outcomes and sets in motion the behavior appropriate to them. Motivation can therefore be thought of as the degree to which an individual WANTS and CHOOSES to engage in certain behavior (Matoka, 2011).

According to some researchers, there are many different theories motivation and have made remarkable development for explaining motivation that can be interpreted into the working environment. The following theories are still being used today. These include Maslow’s hierarchy of needs, Herzberg’s two-factor theory and different types of motivation, such as intrinsic and extrinsic.
2.3 Types of Motivation
There are two types of motivation at work are the Extrinsic and Intrinsic motivation:
Extrinsic outcomes however are what action is done to motivate employees. It is related with rewards such as pay, working conditions and fringe benefits, job security, promotion, the working environment, conditions, punishments such as disciplinary action, and these are done to motivate the employees (Armstrong, 2006). Extrinsic motivation are objects or actions that have immediate influence on employees but may not necessary last long.
In general sense, Intrinsic Motivation is actually originates from the content of the job the direct relationship between the employee and the task, own efforts from the employees. All the jobs have such potential opportunities where such outcomes engage the feeling of motivation of achievement, accomplishment, recognition, challenges and goals will be achieved.
2.4 Techniques of Motivation
According to Gupta, (2005), there are two main method of motivation as explained below:
2.4.1 Carrot and stick Approach to Motivation
The Carrot and the Stick is a motivation approach to motivation, which is used in order to encourage desired behavior such as rewards and punishment. Under the approach employee who perform well are given rewards in terms of monetary rewards, rewards capable of measurement in terms of money and non-monetary rewards. On the other hand, punishments are to be imposed on employees, for not acting in the desired way or avoiding work are given punishment in the form of non-monetary punishment, non-monetary rewards and monetary punishment. Carrot and stick approach is, therefore a reward and punishment system for motivating employees.
The following steps may be taken to make the carrot and stick, approach more effective:

Reward (carrot) is more effective motivator when it is connected with the performance. Accurate and unbiased appraisal of performance. Performance appraisal is an ongoing process of evaluating employee performance.
Punishment (stick) is applied at the time when the underperforming actually occurs such as non-monetary punishment, non-monetary rewards and monetary punishment.
2.4.2 Motivation through Job Enrichment
Job enrichment is a non-financial method of motivation from Herzberg’s’ two factor theory of motivation. Job enrichment is based on the assumption factors, which is closed to the work and are not effective motivators of performance. In order to motivate the staff the work itself must provide opportunities for achievement, recognition, responsibility, advancement and personal growth. Job enrichment is the way the job is design in order to build in the opportunities for professional development such as achievement, recognition, responsibility and personal growth. It provides greater opportunities in terms of decentralization of decision-making authority and responsibility by carrying out a given task and with timely feedback on the performance (Gupta, 2005). It is said that the Job enrichment is the key of higher motivation and productivity.
Employee Motivation
There are many factors such as working environment, finance and human resources influences the performance of an organization. However, employees are the most important resources for nearly all organization. The workforce have a direct influence on the organization’s continuous success and ultimately its stability (Milliman et al., 2008). Coulter (2006) defines employee performance as the entire productivity of an employee activities and actions in an organization. She has also been said that the level of employee performance may be described as low, moderate and high performance levels as employee performance is usually measured by using effectiveness, efficiency, quality, innovation, creativity, commitments, satisfaction, cohesiveness, flexibility, customer relations, communication patterns and employee efforts towardsthe organizational goals (Robbins, 2003)
2.4.3 Job Performance
According to Daniel et, al. (2002), job performance can be defined as employees performance contribute to organizational goals. The Performance can be viewed as an individual or organizational task performance. Firms needs to know how to improve employee’s performance, and therefore they have to find out why employees fail to perform (Muchinsky, 1993). Employee performance can be defined as an employee ability to accomplish tasks assigned to them in an organizational context (Arverty& Murphy, 1998). There are several causes that can affect the level of performance of employees and according to Korman (1971), there are some internal factors, which are very important factors that affect job performance. The internal factors can be divided into two main aspects that is the skills and abilities for a given job task that employee has and which can influence the work performance. In addition to the internal factors that affect employee, performance can be communication that is very important among employees and managers. Motivation is essential for performance and this push employee to perform well and also reduces the rate of absenteeism and encourage stability and loyalty with the firm.
2.3 Theoretical Literature Review
This part focused on various theories of motivation
2.3.1 The Hierarchy of Need Theory
Different researchers have shown different description on how motivation can have great influence within a firm. The famous amongst them is Abraham Maslow with the theory of “Maslow’s Hierarchy of needs”. In Maslow’s Hierarchy, there are five different levels of needs and it is shown in a pyramid shape (Maslow, 1954) with the most essential needs at the bottom and at the top with the need for self-actualization. According to Maslow, people are motivated to satisfy the lower level needs before moving to the next level to satisfy the higher need, which start with the ascending order with the lower needs; the needs are psychological, safety needs, social, esteem needs and self-actualization needs. Firstly, individuals are motivated by Psychological needs: are basic life needs require for human survival such as the air, food and water. These needs are satisfied through an income from employment high enough to meet essential needs and their needs tend to move to the second level needs. It also include good working conditions, which is important for every job in the banking sector. The second level is the security needs: which is the most essential need for people as it expressed with some job security in banks and by providing some generous benefits include health insurance and company sponsored retirement plans for the employee. The third level of needs by Maslow was the social needs. After having a job security, the social needs may be satisfied by having a friendly working environment, build up good working relationship and provide a good workplace to collaborate and communicate with others. The fourth level of Esteem needs which is the recognition where the person must have feelings of self-respect and the need for respect from others, status and recognition of achievement. The last level of the hierarchy of needs is self-actualization. In this particular stage, the person’s always strive to be better and use their talents in new ways. Maslow believes that no one is ever completely self-actualized and this is important to motivate someone in order to fulfill their needs and strive for the next level until they reach self-actualization and this will give a sense of achievement for opportunities to develop and apply new skills will increase their potential. The Figure 1 illustrates Maslow’s five hierarchy of needs.

Figure 1 Maslow´s five hierarchy of needs
2.3.2 Herzberg Two Factor Theory
Frederick Herzberg (1959) a behavioral scientist who introduced the two-factor theory, two-factor theory is based on need achievement because of their interest in how to satisfy employees. According to the study of Herzberg, two-factor theory are the motivators and hygiene factors which influence employees and he suggested that the job itself could be the source of job satisfaction. This theory is very important to current understanding of employer and employee relationship, mutual understanding and alignment within the psychological contract. On the researcher theoretical framework, as shown in Figure 3, there are six motivational factors that may motivate staff in the banking sector in achieving organizational goals. The six motivational goals are recognition, employee empowerment, career progression, personal growth, sense of achievement and, interesting and challenging work. The researcher’s defined that recognition means appreciating the efforts of employee through giving rewards, promotion or show appreciation. Employee empowerment means authorizing employee by means of allowing them to make their own decision in a specific work related situation. Career progression is one of the motivational factors, which mean to grow in terms of career development in. Then next motivational factor is personal growth, which can be defined as improvement in personal development such as talents potential and improvement on employee’s personality. Lastly, interesting and challenging work, encouraging employees to use their full potential when assigning a work to them. On the other hand, the hygiene factors are extrinsic variables, remuneration and benefits is receiving compensation in exchange of showing efforts, sacrifices, leadership and hard work. Working conditions refer to the working environment. Job security is the assurance that an employee are on a permanent basis and there will be continuity of employment for their work life. Relationships with immediate the supervisor or Branch manager have a direct impact the engagement level of the staff. A strong connection existed between an employee’s level of satisfaction and dissatisfaction. Relationship with colleagues at work make the environment more productive and more motivated team. Organization values and policies is about the organizational missions and directions, which help in keeping the employee loyalty. The researcher’s states that the theory for people who are working in a bank specifically in rank-and-file employees and they are the essence of organizational success and rank-and-file employees represent the bank to the customers. However, even by eliminating the poor hygiene factors does not prove that will solve the motivation problem. Similarly, when employees have job satisfaction, motivators are present, but by eliminating the motivators does not automatically lead to dissatisfaction. He advised, there are three ways in which this could be done which are through job enrichment, job rotation and job enlargement.

Figure 2. Frederick Herzberg Motivational Theory

2.3 SOFTWARE REVIEW
2.3.1 SOLIDWORKS
27 Solid Work is a software which is used for solid modeling computer aided design (CAD) and computer aided engineering (CAE). Through this software we can easily sketch 2D structure and by extruding feature we can get it 3D model very easily. From this software we can design separate parts according to our own dimensions and assemble those parts together easily. And also from this software we can designed mechanical system as well as we can simulate through this software. But in this research we have used different kind of software to simulate the solid work design.

2.3.2 BASIC CONCEPT OF SOLID WORK
28 From sketch option we can create different kind of shapes like rectangle, circle, lines, curves and etc. And also from this we can insert smart dimensions, so that we could able to make a design according to our own dimensions. Mirror option also could be used through this sketch option.
29 Through the feature option we can convert 2D model to 3D model easily by using extrude option. And if we want to make a hole or cut in that 3D object we could use extrude cut option in this feature panel. If we want some smooth edges, some other features like fillet, shell and draft could be used. To create airfoil, curve feature has used in the designing stage.
30 We can flow simulate through the solid work Flow Simulation option but it is not much accurate as Open Foam and other simulation software. So throughout this experiment we didn’t use that option in solid work.
31 We can assembly parts through this software. We can sketch different parts of model in different pages and after completing the parts, it can be assembled together and complete with one solid 3D model.
32 We can use different kind of constraints while drawing the sketch such as horizontal, perpendicular, vertical, coincident and etc.
2.3.2 ANALYSIS OF OPEN FOAM
33 OpenFOAM is a structure for creating application executables that utilization bundled usefulness contained inside an accumulation of roughly 100 C+ libraries. OpenFOAM is dispatched with around 250 pre-incorporated applications that fall with two classifications: solvers, that are each intended to take care of a speci?c issue in ?uid (or continuum) mechanics; and utilities, that are intended to perform assignments that include information control. The solvers in OpenFOAM cover an extensive variety of issues in fluid dynamics. Some of them are compressible, multiphase, incompressible, heat transfer etc. The users in OpenFOAM can expand the accumulation of solvers, utilities and libraries in OpenFOAM, utilizing some pre-essential learning of the hidden strategy, physics and programming procedures included. The pre-processing and post-processing conditions are made associated with OpenFOAM. The interface to the pre-and post-preparing are themselves OpenFOAM utilities, subsequently guaranteeing steady information dealing with over all conditions. The post handling is went with ParaView programming.
34 There are some limited numbers of CFD simulations done so far using dynamic mesh in openFOAM. These simulation projects are done with pimpleDyMFoam solver. For example: the simulation of the wind turbines and propellers. Therefore we have proceeded with our CFD project on Bell 412 main rotor with the very close studies of the simulations of wind turbine and propeller. Most of the techniques and ideas are drawn from these existing simulations and modified appropriately for our CFD project.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

2.4 APPLYING COMPUTATIONAL FLUID DYNAMIC
35 Computational Fluid Dynamic (CFD) is one of the main tool to perform in Researches and the industrial applications. From this CFD analysis we can predict, how the system component are working, how the fluid flow behavior and it provides a qualitative and quantitative prediction of fluid flows by means of following methods,
Numerical Method
Software tools
Mathematical Modeling
So that we can implement our design and make necessary development in design. And it has been using in industry for many years. Some of basic applications are given bellow;
Flow and heat transfer in industrial processes
Aerodynamics of ground vehicles, aircraft, missiles.
Film coating, thermoforming in material processing applications.
Flow and heat transfer in propulsion and power generation systems.
Ventilation, heating, and cooling flows in buildings.
Heat transfer for electronics packaging applications.
36 CFD is the latest branch of engineering In CFD it used numerical method and the algorithm method to solve and analyze the problem in fluid flows. This analysis have done through the basic governing equation in CFD which are in partial differential form. This equation will convert in to computer programs by using high level computer languages. Existing commercial CFD codes are capable of simulating a very wide variety of physical processes besides fluid flow. This CFD describe the pressure, temperature, density and the velocity of the moving fluid, which given in the Naiver-stoke equations. In Naiver-Stock equation it contain energy equation, momentum equation and the continuity equation which are given bellow.

Continuity Equation:
??/?t+ ?( ?u/?x+ ?v/?y+ ?w/?z )=0 (1)
Momentum Equations;
For X direction;
??u/?t+ (?(?uu))/?x+ (?(?uv))/?y+ (?(?uw))/?z= -?p/?x+ ?((?^2 u)/??x?^2 + (?^2 u)/??y?^2 + (?^2 u)/??z?^2 ) (2)

For Y direction;
??u/?t+ (?(?uu))/?x+ (?(?uv))/?y+ (?(?uw))/?z= -?p/?y+ ?((?^2 v)/??x?^2 + (?^2 v)/??y?^2 + (?^2 v)/??z?^2 ) (3)
For z direction;
??u/?t+ (?(?uu))/?x+ (?(?uv))/?y+ (?(?uw))/?z= -?p/?z+ ?((?^2 w)/??x?^2 + (?^2 w)/??y?^2 + (?^2 w)/??z?^2 ) (4)

Energy Equation;
??E/?t+ (?(?uE))/?x+ (?(?vE))/?y+ (?(?wE))/?z= -?pu/?x-?pv/?y- ?pw/?z+S (5)
Where,
x, y and z – three different directions component
? – Density of air
u, v and w – Velocity component in different direction.
37 From this CFD analysis, it can have great control over the physical process and provides the ability to isolate specific phenomena for study. And from experiment we could only have data in limited number of locations in the system but through the CFD simulation it can analysis data in large number of locations and give comprehensive set of flow parameters for examination. Experimental process may get much expensive compare to the CFD process and the cost of CFD process may get reduce when the computers get more powerful. The simulation could be executed in short period of time as well as we could simulate in real conditions. This are the main advantage of computational fluid dynamic.
38 When we discuss about the limitation of CFD, the CFD solutions relay in physical model of real world processes such as compressibility, chemistry, turbulence and many more. Through the CFD it can get much accurate data as the physical model on which they are based on. When the computer solve the equation it invariably introduce numerical errors which include round-off errors and due to the approximation in numerical mode it will give truncation errors. The accuracy of the solution mainly depend on the initial boundary conditions given in to the numerical mode.
39 In CFD it divided in to three main processing which are pre-processing, solving and post-processing. In pre-processing, it need to be created Mesh for the solid work model. For that software like Open Foam and Gambit could be used according to our own boundary conditions.

2.5 GENERAL TURBULANCE MODELS
40 To solve CFD problems it consist of three main components which are geometry and grid generation, setting up a physical model and post processing the compute data. In the turbulence it results in increasing energy dissipation, mixing, heat transfer and the drag. The way geometry and the grid are generated and the set problem is computed are very well known. Precise theories are available. But it is not true for setting up a physical model for turbulence flow. There for it need to create the ideal model with the minimum amount of complexity. The complexity of the model will increase with the amount of information required about the flow field. The key elements of turbulence are time dependent and the three dimensional. 17
41 Turbulence models can be categorized in to several different approaches which are by solving the Reynolds-averaged Navier-Stokes equations with suitable models for turbulent quantities or by computing them directly.
Reynolds-Averaged Navier-Stokes (RANS) Models
Eddy Viscosity Model (EVM)
Non-linear Eddy Viscosity Model (NLEVM)
Differential Stress Model (DSM)
Detached eddy simulation (DES)
Large-eddy simulation (LES)
Direct numerical simulation (DNS)
Reynolds stress transport models
Direct numerical simulations

2.6 REYNOLDS-AVERAGED NAVIER-STOKES MODELS (RANS)
42 This method is the mainly use method in Engineering industry. This can be categorized according to the wall function, number of variables and their types. So we mainly focus on following models in RANS.
Spalart-Allmaras
K-Epsilon(?) Model
K-Omega(?) Model
43 Here this k-Epsilon model further divided in to two types of models, which are standard K-Epsilon model (SK-?) and the Realizable K-Epsilon model (RNGK-?). And also this K-omega model also divided in to two models which are standard K-omega model (SK-?) and the shear stress transport K-Omega model (SSTK-?).
2.6.1 SPALART-ALLMARAS
44 This equation solves a modelled transport equation for kinematic eddy turbulent viscosity. It easy to resolve near the wall. From this model it shows good results for boundary layer subjected to adverse pressure gradient in especially wall bounded flows involve in aerospace applications. This could be used for the supersonic and transonic applications. This model is not calibrated for the general industrial flows. This model is very effective in low Reynolds numbers. Minimum boundary layer resolution of 10-15 cells should be there to resolve the equation. The formulation provide wall shear stress and heat transfer coefficient. This model cannot rely on the turbulence isotropic calculations. 18
2.6.2 K-EPSILON(?) MODEL
45 This model mainly focus on the affect the turbulent kinetic energy. In this model it take the kinetic viscosity is isotropic as an assumption, or the ratio between rater of deformation and the Reynolds’ number is same in all directions. This model used commonly in industrial applications rather than the other two models. This model gives reasonably accurate results. Under different pressure gradients it gives the equilibrium boundary layers and free shear flows. This usually use for free shear layer flow with small pressure gradient. This model poorly perform in strong separations, large pressure gradients, unconfined flows, curved boundary layers, rotating flows and flows in non-circular ducts. Among the two type of this model (RNG) K-? model perform better than the SK-? model.
For k and ? it use two transport equations for turbulent length and the viscosity.
Equation for turbulent length;
l=k^(3/2)/? (6)
Equation for turbulent viscosity;
v_t=c_? k^(1/2) l=c_? k^2/? (7)

Turbulent kinetic energy;
?/?t (?k)+ ?y/(?x_i ) (?ku_i )= ?y/(?x_i )(?+?_t/?_k ) ?k/(?x_i )+P_k+P_d+??+Y_M+S_k (8)

Dissipation ?;
?y/?x (??)+ ?y/(?x_i ) (??u_i )= ?/(?x_j )(?+?_t/?_? ) ??/(?x_j )+C_1? ?/k (P_k+?C_3? P?_b )-C_2? ? ?^2/k+S_k (9)

Where,
C1? = 1.44, C2? = 1.92, C3? = 0.09, ?k = 1.0, ?? = 1.3

2.6.3 K-OMEGA (?) MODEL
46 It is two equation model which means it use two transport equations to represent the turbulent properties of the flow. This also a common equation model. This can be integrated to the wall without using the wall functions. From this equations, it accounts history effects such as diffusion and convection of turbulence energy. Here kinetic energy (k) is one of variable. It determines the energy in turbulence. The other variable is dissipation (?), it determine the scale of turbulence.
For kinematic eddy viscosity;
v_t=(a_1 k)/(max?(a,?,?SF?_2)) (10)
Turbulence kinetic energy;
?y/?x+U_j ?k/(?x_j )=P_k-?^* k?+?/(?x_j )(v+?_k+v_T ) ?k/(?x_j ) (11)

Specific dissipation rate;
??/?t+U_j ??/(?x_j )=?S^2-??^2+?/(?x_j ) (v+?_k v_T ) ??/(?x_j )+2(1-F_1)?_(?^2 ) 1/? ?k/(?x_j ) ??/(?x_j ) (12)
?

2.6 GENOME PLASTICITY AND EVOLUTION OF ESCHERICHIA COLI
Like all life forms, new strains of E. coli evolve through the natural biological processes of mutation, gene duplication, and horizontal gene transfer; in particular, 18% of the genome of the laboratory strain MG1655 was horizontally acquired since the divergence from Salmonella. E. coli K-12 and E. coli B strains are the most frequently used varieties for laboratory purposes. Some strains develop traits that can be harmful to a host animal. These virulent strains typically cause a bout of diarrhea that is often self-limiting in healthy adults but is frequently lethal to children in the developing world. (Futadar et al., 2005). More virulent strains, such as O157:H7, cause serious illness or death in the elderly, the very young, or the immunocompromised.
The genera Escherichia and Salmonella diverged around 102 million years ago (credibility interval: 57–176 mya), which coincides with the divergence of their hosts: the former being found in mammals and the latter in birds and reptiles. (Wang et al., 2009). This was followed by a split of an Escherichia ancestor into five species (E. albertii, E. coli, E. fergusonii, E. hermannii, and E. vulneris). The last E. coli ancestor split between 20 and 30 million years ago.
The long-term evolution experiments using E. coli, begun by Richard Lenski in 1988, have allowed direct observation of genome evolution over more than 65,000 generations in the laboratory. For instance, E. coli typically do not have the ability to grow aerobically with citrate as a carbon source, which is used as a diagnostic criterion with which to differentiate E. coli from other, closely, related bacteria such as Salmonella. In this experiment, one population of E. coli unexpectedly evolved the ability to aerobically metabolize citrate, a major evolutionary shift with some hallmarks of microbial speciation.
2.7 INCUBATION PERIOD
The time between ingesting the STEC bacteria and feeling sick is called the “incubation period”. The incubation period is usually 3–4 days after the exposure, but may be as short as 1 day or as long as 10 days. The symptoms often begin slowly with mild belly pain or non-bloody diarrhea that worsens over several days. HUS, if it occurs, develops an average of 7 days after the first symptoms, when the diarrhea is improving.

2.7.1 DISCOVERY OF ANTIBIOTICS
• History of antibiotics – 1
19th century:Louis Pasteur & Robert Koch
• History of antibiotics – 2
Plant extracts
– Quinine (against malaria)
– Ipecacuanha root (emetic, e.g. in dysentery)
Toxic metals
– Mercury (against syphilis)
– Arsenic (Atoxyl, against Trypanosoma)
• Dyes
– Trypan Blue (Ehrlich)
– Prontosil (azo-dye, Domagk, 1936)
• History of antibiotics – 3
Paul Ehrlich
• started science of chemotherapy
• Systematic chemical modifications
(“Magic Bullet”) no. 606 compound = Salvarsan (1910)
• Selective toxicity.
• Developed the Chemotherapeutic Index
• History of antibiotics – 4
Penicillin- the first antibiotic – 1928• Alexander Fleming observed the
killing of staphylococci by a fungus (Penicillium notatum)
• observed by others – never exploited
• Florey & Chain purified it by freeze-drying (1940) – Nobel prize 1945
• First used in a patient: 1942
• World War II: penicillin saved 12-15% of lives
• History of antibiotics – 5
Selman Waksman – Streptomycin (1943), was the first scientist who discovered antibiotic active against all Gram-negatives for examples; Mycobacterium tuberculosis
– Most severe infections were caused by Gram-negatives and Mycobacterium
tuberculosis, extracted from Streptomyces – extracted from Streptomyces
– 20 other antibiotics include. neomycin, actinomycin
2.8 CHARACTERISTICS OF ANTIBIOTICS
According to the Oxford Dictionary, the term Antibiotics encompasses medicines (such as penicillin or its derivatives) that inhibit the growth of or destroys microorganisms. Antibiotics are naturally occurring substances that exhibit inhibitory properties towards microbial growth at high concentrations. (Zaffiri, et al., 2012).
-Antibiotics are selective in their effect on different microorganisms, being specific in their action not only against genera and species but even against strains and individual cells. Some of these agents act mainly on gram-positive bacteria, while others inhibit only gram-negative ones.
-Some antibiotics are produced by some organism, from different strains of penicillin.
-Bacteria are sensitive to the antibiotic which enable them to developed resistance after contact, for several periods.

We Will Write a Custom Essay Specifically
For You For Only $13.90/page!


order now

2.9 ROLE OF ANTIBIOTICS
Based on the clinical use of antibiotics, it may appear that these compounds play a similar role as microbial weapons in nature, yet this seems unlikely due to the fact that the concentrations used in the clinical setting are significantly higher than that produced in nature (Fajardo et al., 2008). Due to experimental evidence, it makes more sense to see antibiotics as small, secreted molecules involved in cell-to-cell communication within microbial communities.
(Martinez, 2008). Diverse Studies have been conducted in which different antibiotics and antibiotic-like structures were administered to different bacterial species at levels below the compounds minimum inhibitory concentrations (MIC). (Fajardo et al., 2008). that was

x

Hi!
I'm Belinda!

Would you like to get a custom essay? How about receiving a customized one?

Check it out