Thermostable a-amylases from hyperthermal springs of Ethiopia bisolation, purification, characterization and application | |
| Author | Haki, Gulelat Desse |
| Call Number | AIT DISS. no. PH-03-06 |
| Subject(s) | Themopiles Amylases |
| Note | A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Technical Sciences |
| Publisher | Asian Institute of Technology |
| Abstract | Thermostable a-amylase producing microorganisms from six Ethiopian hyperthermal springs located at Arbaminch, Awassa, Nazret, Shalla and Abijata, Wenda Genet and Yirgalem were screened during December 2001 to July 2002. The altitude of the hot springs ranged from 1200 to 1690 meters above sea level. The temperature of the water ranged from 36.6°C to 93.4°C and pH was close to neutral, except that of Shalla and Abijata, which are alkaline. Two thousand one hundred and three colonies of water and soil bacteria from the six hyperthermal springs were cultured on starch agar plates. During their metabolism 201 strains (about 10%) formed a clear halo around the colony while flooded with Gram's iodine solution. Nearly 60% of the crude a-amylase extracts obtained from the strains isolated from Shalla and Abijata showed activity at 60°C and only about 7% obtained from Nazret effectively hydrolyzed starch at this temperature. The optimum pH range for the activity of enzymes isolated from 33 most promising microorganisms according to the plate assay method was from 5 to 11. Of the total 1509 fungal isolates, 61 strains (4%) were able to consume starch as a substrate. Two of the best strains selected for further study were identified and named as Bacillus stearothermophilus GRE 1 and Bacillus licheniformis GRE 7. They produced the highest level of enzyme and had an optimum growth temperature of 55°C and 50°C, respectively. The melting temperatures of the genomic DNA isolated from the microorganisms respectively were 86°C and 88°C, which indicated that they are indeed stable at elevated temperatures. The optimum enzyme production medium established for B. stearothermophilus GRE 1 yielded 2.36 U/ml, which was approximately 1.31- fold higher than the yield obtained in the normal standard medium. In an optimized media 1.76 U/ml (1.80-fold higher) of amylase was produced by B. licheniformis GRE 7. Lactose (2.5%) combined with yeast extract (0.5%) enhanced enzyme production by B. stearothermophilus GRE 1 while B. licheniformis GRE 7 preferred 0.5% starch and 1 % peptone. Calcium and magnesium did not bring any change on the enzyme production capacity of both the strains, while copper and zinc were inhibitory. Both strains preferred a neutral pH and a stirrer speed of 200 rpm for enzyme production. Enzyme production by B. stearothermophilus GRE 1 increased more than 3-fold as compared to shake flasks in a well-stirred bioreactor under optimized conditions. Factorial design experiments revealed that the enzyme production medium for B.stearothermophilus GRE 1 should consist 1.02% starch and 3.12% lactose. Complete precipitation of the a-amylase enzyme isolated from B. stearothermophilus GRE 1 in the purification stage was attained at an ammonium sulphate saturation level of 75%. Further purification by dialysis and gel filtration chromatography yielded a purification factor of 33.62-fold and a recovery of 46.52%. The molecular weight of the enzyme was 63,000 and its optimum temperature was determined to be 60-70°C. The optimum pH was in the range of 5.5-6.0. The enzyme maintained 50% of its original activity after 45 m of incubation at 80°C and was stable at pH values of 5.0-9.0. Cu2 +, Zn2 + and Fe2 + strongly inhibited enzyme activity. The enzyme was calcium independent and 94% and 86% relative activity were displayed with low concentrations of Co2 + and Mg2+ respectively. The V max and Km of the enzyme respectively were 42 mg reducing sugar/min and 4.98 mg starch/ml. The enzyme hydrolyzed wheat, com and tapioca starch granules efficiently, but had low activity on potato starch granules at 40 and 50°C. An increase in temperature to 60°C improved hydrolysis of potato starch, and promoted its degradation up to 50% after 36 h. Nearly 50% degradation rate of all the starches was obtained after 12 h of incubation at 70°C. There was no external addition of calcium ion. Overall, thermophilic microorganisms, which seemed to have good potential for thermostable amylase production were screened and the enzyme characterized. They were able to hydrolyze raw starch granules. |
| Year | 2003 |
| Type | Dissertation |
| School | School of Environment, Resources, and Development (SERD) |
| Department | Department of Food, Agriculture and Natural Resources (Former title: Department of Food Agriculture, and BioResources (DFAB)) |
| Academic Program/FoS | Postharvest and Food Process Engineering (PH) |
| Chairperson(s) | Rakshit, Sudip K. |
| Examination Committee(s) | Jindal, Vinod K.;Athapol Noomhorm; Ranamukhaarachchi, S. L. |
| Scholarship Donor(s) | Agricultural Research and Training Project (AUNARTP);Government of Ethiopia |
| Degree | Thesis (Ph.D.) - Asian Institute of Technology, 2003 |