Biochemical study of Alcoholdehydrogenase produced from hydrocarbon utilizing pseudomonas Sp.

number: 
716
English
Degree: 
Imprint: 
Biotechnology
Author: 
Noora Adil Al-Khilfa
Supervisor: 
Dr. Majid H. Al-Gelawi
Dr.Ghazi Munim Aziz
year: 
2003

Number of efficient hydrocarbon utilizing bacteria of Pseudomonas spp. were tested for their ability to produce Alcoholdehydrogenase (ADH) enzyme. Pseudomonas sp. RB30 isolate was selected according to it's the highest ADH enzyme production, and this bacterium was identified as Pseudomonas fluorescens RB30. The enzyme was yielded the maximal volume activity of 0.34 U/ml when extracted by using lysozyme treatment as compared with low volume activity 0.19 U/ml resulted from sonication of the cells. Optimum carbon source and concentration for the ADH production using salts medium were investigated and it was found that optimum carbon source and , concentration for ADH production was glycerol at 1 % that gave the highest specific activity 11.2 U/mg protein . The enzyme purification was carried out in three steps including concentration by ammonium sulfate 25-65% saturation, ion-exchange chromatography by DEAE-cellulose column and finally using gel filtration on sephadex G-200 column. The overall purification fold was 4.1 with 49.2% yield. The characterization of the partial purified enzyme showed that the molecular weight of partial purified ADH enzyme was 166 KD. Results indicate that the optimal pH for ADH activity was 8-9 and the enzyme was stable at pH values ranging from 8-9.5 The partial purified enzyme retained its activity at 30-50°C after 15 min. of incubation while the activity was lost after 15 min. incubation at 60°C i The enzyme also retained its original activity for up to 20 min. when incubated at 40°C The effect of some salts such as ZnSO4, HgCl2, FeCl2, ZnCl2 and CuCl2 on the enzyme activity was examined and the result showed that the partial purified ADH enzyme was activated by incubation with ZnSO4 and ZnCl2 The catalytic activity of the partial purified ADH enzyme was inhibited to about .63 and 90.3 % after incubation for 15 min. with 2 mM and 5 mM EDTA respectively, while treatment with 2 mM and 5 mM sodium azide for 15 min showed that '53.4% and 78% ADH activity was lost, indicating that this enzyme was metallo-enzyme. It was found also that the presence of 2-mercaptoethanol in the enzyme storage buffer increase enzyme stability. Some of the enzyme kinetics such as mickales constant (Km) and maximum velocity (Vmax) were determined by using Linweaver-Bruk method, and it was 5.6 mM and 6.6 mM.min"1 respectively. The role of Pseudomonas fluorescens RB30 plasmid in the enzyme production was studied. It was found that the treatment of this, bacterium with SDS resulted in numbers of cured colonies and some of these colonies were checked for their ability to produce ADH enzyme. It was found that ADH specific activity for cured colonies was decreased, it was concluded that the plasmid of Pseudomonas fluorescens RB30 might have a regulatory role in ADH production. In an attempt to get highly ADH producer mutants from Pseudomonas fluorescens RB30, molecular mutagenesis by Mini-Mu transposon was performed. Several mutants were isolated and some of them (two mutants) showed an increase in enzyme production. While the other mutants showed low level of enzyme production.