Strain of Rhodococcus rhodochrous as a producer of nitrile hydra

Details Strain of Rhodococcus rhodochrous as a producer of nitrile hydra Strain of Rhodococcus rhodochrous as a producer of nitrile hydra

1995-08-15

1998-10-27

Naff, David M.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing nitrogen-containing organic compound

435128, 4352521, 435822, 435183, 435170, C12P 1300, C12P 1302, C12N 120

Patent

active

058276998

DESCRIPTION:

BRIEF SUMMARY
BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to biotechnology and to preparing a new strain of bacteria having a high nitrile hydratase activity, which strain is intended for use in processes for producing amides from nitriles.
2. Description of Related Art
An enzyme of a nitrile hydratase capable to catalyze the conversion of nitriles into amides has been detected in a number of genera of bacteria. In order to produce nitrile hydratase in the process of growing bacteria, it is necessary to add an inducer to the nutrient medium. Nitriles and amides of organic acids (Ref. U.S. Pat. No. 4,555,487; European Patent No. 0 109 083; European Patent No. 0 204 555), urea or its derivatives (European Patent Application No. 0 362 829) may be used as inducers. Known in the prior art are strains of Corynebacterium N 774 (U.S. Pat. No. 4,248,968) and Rhodococcus sp. S-6 (U.S. Pat. No. 5,179,014) for which no inducer is required. Disadvantages of the strain N774 reside in its low nitrile hydratase activity (its specific activity is 50-60 units/mg, here and further on measured in mcM of acrylamide/min/mg of cells, based on dry matter weight), narrow range of substrate nitriles (only aliphatic nitriles), a low thermal stability of the enzymes (the optimum effect temperature for N774 is 35.degree. C.). A disadvantage of the strain S-6 resides in its capacity to hydrate the produced amides to acids, whereby the quality of the amides is sharply affected. Furthermore, the strain S-6 has a low thermal stability of nitrile hydratase (not higher than 30 C.) and a low productivity (it is capable of accumulating not more than 20% of acrylamide in the solution). Expensive components of nutrient media (such as peptone, yeast extract, meat extract) are used to grow both strains, N774 and S-6.
In terms of its technical essence and the result achieved, the strain Rhodoccus rhodochrous J1 (European Patent Application No. 0 362 829) possessing a nitrile hydratase activity with respect to aliphatic and aromatic nitriles comes nearest to the present invention. However, the above-cited strain is disadvantageous by the fact that it is necessary to use a nutrient medium containing vitamins, yeast extract and peptone to cultivate it. Another disadvantage lies in the fact that the strain J1 necessitates the use of urea as inducer in high concentrations (7.5-12 g/l). Thus, when the strain is cultivated in a medium without urea, the specific nitrile hydratase activity is as low as 3.35 units/mg (total activity is 17.7 units/ml, here and further when measured in micro-moles of acrylamide/min/ml of a cultural liquid). With the presence of 7.5 g/l of urea in the medium, the specific activity reaches 497 units/mg, the total activity being 2480 units/ml. It should be noted that the urea plays a dual role, namely: it is used as a source of nitrogen, and as an inducer for the nitrile hydratase. An amount of urea which does not exceed 2 g/l is sufficient for growth of the strain (whereby the specific nitrile hydratase activity of the strain J1 reaches 36.5 units/mg, the total activity being 189 units/ml), while high urea concentrations are necessary only for induction. For this reason, a substantial amount of the urea remains in the medium after cultivating therein the strain J1.


SUMMARY DESCRIPTION

It is the object of the present invention to produce a strain featuring a high nitrile hydratase activity, while being grown in simple synthetic media which does not contain any vitamins, amino acids, or other compounds serving as an inducer for synthesis of nitrile hydratase (nitriles, amides or urea).
The above-formulated object is accomplished by developing a strain Rhodocosccus rhodochrous M33 which would be capable in the absence of inducers to constitutively produce nitrile hydratase for catalyzing the hydrolysis of aliphatic nitriles, such as, e.g. acrylonitrile, and aromatic nitriles, such as, e.g. 3-cyano pyridine, into corresponding amides, such as, e.g. acrylamide and nicotinamide. The strain of the present

REFERENCES:
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