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Isolation and primary characterization of an amidase from Rhodococcus rhodochrous.
E K Kotlova1, G G Chestukhina, O B Astaurova
1Institute of Genetics and Selection of Industrial Microorganisms, Moscow, 113545, Russia. lab11@vnigen.msk.su.
Biochemistry. Biokhimiia
|May 8, 1999
Summary
Researchers purified amidase (EC 3.5.1.4) from Rhodococcus rhodochrous M8. This enzyme shows optimal activity at high temperatures and a specific pH range, preferring aliphatic amides as substrates.
Area of Science:
- Biochemistry
- Enzymology
- Microbial Biotechnology
Background:
- Amidase (EC 3.5.1.4) plays a crucial role in nitrogen metabolism and industrial biocatalysis.
- Understanding amidase properties is key for optimizing biotechnological applications.
Purpose of the Study:
- To purify and characterize amidase from Rhodococcus rhodochrous M8.
- To determine the enzyme's optimal conditions, substrate specificity, and sensitivity to inhibitors.
- To compare its properties with other known amidases.
Main Methods:
- Homogeneous purification using isopropanol fractionation and Mono Q ion-exchange chromatography.
- Determination of molecular weight, optimal temperature, and pH range.
- Enzyme activity assays with various substrates and inhibitors.
Main Results:
- Purified amidase comprises four identical subunits (42+/-2 kD).
- Optimal activity observed between 55-60°C and pH 5-8.
- Enzyme is sensitive to Hg2+ and Cu2+ but not EDTA, o-phenanthroline, PMSF, or DIFP.
- Exhibits hydrolytic and acyl transferase activity, with aliphatic amides as preferred substrates.
Conclusions:
- The characterized amidase from R. rhodochrous M8 possesses distinct biochemical properties.
- Its substrate preference and stability suggest potential for specific industrial applications.
- Enzyme characteristics are comparable to amidases from Arthrobacter sp. J-1 and Brevibacterium sp. R312.