الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
Amylases are responsible for the hydrolysis of starch producing smaller units, including mono and/or di - sugars to finally produce glucose (the basic unit for the construction of complex starch). These enzymes account for 65% of enzyme market in world, which illustrates the importance of globally production, and application many industries, including food, paper, textile, pharmaceuticals and detergents.
The plan of this study included isolation and identification of the most efficient starch degrading bacteria, using some cheap available agro-industrial wastes as nutritional sources (carbon or nitrogen) for bacterial growth and amylases production. Also study the interaction between nutritional and environmental factors to improve the production based on Response Surface Methodology. Studies were focused on enzymes extraction, partial purification, kinetics behaviour of enzymes and physicochemical properties, immobilization and amylases application.
Results could be summarized in the following points:
1- One hundred and thirty three starch degrading bacterial isolates were isolated from different plants rhizosphere on starch agar medium (82 mesophilic isolates and 51 thermophilic isolates at 30 °C and 50 °C, respectively).
2- Three out of the 133 starch degrading bacterial isolates were selected based on starch hydrolysis ratio (SHR) on solid medium and enzyme activity in liquid medium, which gave the highest values of SHR ranged from 2.50 to 3.06 and α-amylase activity ranged from 72.5 to 77.0 Uml-1 after 48 h at 50 °C they were recorded by B85, B87 and E109. These isolates were identified based on phenotypic characteristics and confirmed by 16S rRNA gene analysis (genotypic characterizes), the isolates B85, B87 and E109 were identified as Bacillus megaterium, B. licheniformis and B. amyloliquefaciens, respectively.
3- Growth curve, specific growth rate (µ), doubling time (td), multiplication rate (MR) and number of generation (N) were calculated for the most efficient starch degrading bacteria using a suitable medium (starch broth medium)
a. The specific growth rate (µ) was 0.20, 0.13 and 0.10 h-1 for B. megaterium, B. licheniformis and B. amyloliquefaciens, respectively.
b. Doubling time (td) was 5.3, 4.0, and 9.0 h for B. megaterium, B. licheniformis and B. amyloliquefaciens, respectively.
c. Multiplication rate (MR) was 0.26, 0.19 and 0.11 for B. megaterium, B. licheniformis and B. amyloliquefaciens, respectively.
d. Number of generations was 3.6, 5.0 and 2.6 for B. megaterium, B. licheniformis and B. amyloliquefaciens, respectively.
The results also showed that B. megaterium and B. licheniformis achieved the highest growth during 10 - 24 h of incubation periods while B. amyloliquefaciens grew exponentially during the period between 12 - 36 h.
4- The optimum incubation period was found to be during 18 – 24, which coincide with the highest values of amylases activity by the most efficient tested bacteria.
5- The original carbon source of the basal medium was replaced by each of 9 different sources to study their effect on amylases activity. Corn starch, broken rice and potato starchy waste proved to be the best carbon source for B. megaterium, B. licheniformis and B. amyloliquefaciens, respectively. The suitable concentration of the best carbon sources for the production of the highest enzymes was 2% by the tested bacteria.
6- Thirteen different nitrogen sources were also investigated for amylases production. Ammonium sulphate was the best nitrogen source for B. megaterium at concentration of 0.44 gl-1. Corn steep liquor was favorable nitrogen source for B. licheniformis at 3.65 gl-1 concentration. Soybean meal was the suitable nitrogen source with 1.21 gl-1 concentration for B. amyloliquefaciens.
7- Evaluation of 7 factors (broken rice concentration, corn steep liquor concentration, pH, temperature, agitation, inoculum size, incubation period) affecting amylases activity by B. licheniformis in submerged batch culture was investigated using the Placket-Burman statistical experimental design. The most positive significant variables affecting amylases production (broken rice, corn steep liquor and incubation temperature) were further optimized by using Response Surface Methodology (RSM) based central composite design (CCD). By using the surface plots and response optimizer of statistical software package Design-Expert software 9.0.0 (Stat-Ease, Inc., Minneapolis, MN 55413, USA 2014), the maximum α-amylase activity of 444.7 Uml-1 was predicted when broken rice was 2.25%, corn steep liquor 4.02 gl-1 and incubation temperature 60 °C. Maximum β-amylase activity was predicted being 8.74 Uml-1, at the most proper concentrations (2.25%) broken rice, (3.65 gl-1) corn steep liquor and temperature at 55 °C. Whereas the highest γ-amylase activity (9.18 Uml-1) was attained at 2.25% broken rice, 4.38 gl-1 corn steep liquor and incubation temperature at 55 °C.
8- Partial purification of amylases produced by B. licheniformis using ammonium sulphate fractionation (0 - 80%) and organic solvents (ethanol, acetone and acidified acetone / methanol), revealed that ammonium sulphate fraction 40 - 60 % followed by dialysis proved to be the best performance on precipitation of the produced amylases. Alpha, β & γ-amylases were partially purified by 2.40, 1.80 & 2.25 fold increase with yield being 66.8, 68.6 and 69.5%, respectively.
9- Characterization of the partially purified amylases produced by B. licheniformis
• The optimal temperature of amylases activity was 70, 50 or 60 °C for 15 min of α, β and γ-amylase respectively.
• The maximum α-amylase activity was attained at pH ranged from 7.0 - 9.0, with stability at pH 8.0 for 15 min, while β and γ-amylases were found to be stable at pH ranged from 4.0 - 5.0 for 15 min.
• Studying the effect of different concentrations of starch (0.25 - 5.0) as substrate showed that 1% starch was the best concentration for enzymes activity.
• The Km and Vmax values were calculated using Michaelis-Menten equation and Lineweaver-Burk plot equation, it was found that Km value being 0.50, 0.69 and 0.43 mg ml-1, while Vmax, was 333.3, 6.25 and 7.14 Umin-1 for α, β and γ- amylase respectively.
• Effect of 13 different elements (activators and inhibitors) on amylases activity was tested. Results showed that use of some elements like Ca++, Mg++ & Na+ enhanced α & γ -amylase and Ca++, Co++ & Mg++ enhanced β-amylase. While Cu++, Mn++ , Zn++, Pb++, Fe++ & Ni++, Cd++, Na+, EDTA and SDS showed inhibitory effect.
10- Immobilization of partially purified amylases produced by B. licheniformis
• Sodium alginate (2.0%) was used for immobilization of amylases and the activity of immobilized enzymes was estimated. It was found that immobilized α, β and γ- amylase activity yield being 84.5, 93.2 and 93.8%, respectively.
• The results on reusability indicated that the highest operational stability was 10 reuses with retained activity of 88.9, 87.3 and 91.7% for α, β and γ- amylase, respectively.
11- Some applications of the partially purified enzymes produced by B. licheniformis
• Saccharification efficiency of some different starch wastes
The highest percentage of starch saccharification and glucose formation were obtained after 12 h incubation time with combination of partially purified amylases (α + β + γ), whereas crude enzymes achieved starch saccharification after 18 h.
• As detergent additive for stain removal
Wash performance analysis of chocolate, bread jam and ketchup stains removal after enzyme treatment. However, enzyme in conjugation with detergent at 70 °C proved to be the best.
• Desizing operation
A 95.5% desizing of grey fabric (starch removal) was obtained with 254.0 Uml-1 of α-amylase at 70 °C and pH 7.0 for 1 h.