Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Preserving or maintaining micro-organism
Reexamination Certificate
2001-05-14
2004-01-13
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Preserving or maintaining micro-organism
C435S182000, C435S183000, C435S244000, C435S195000, C435S188000
Reexamination Certificate
active
06677149
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of microbiology and molecular biology. More specifically, a method has been developed to stabilize the nitrilase activity of unimmobilized or immobilized microbial cells and to preserve the integrity of the microbial cells, where the unimmobilized or immobilized microbial cells are stored in an aqueous solution containing from about 0.100 M to the saturation concentration of an inorganic salt of bicarbonate, carbonate, and carbamate, including ammonium, sodium, and potassium salts of bicarbonate, carbonate, and carbamate.
BACKGROUND OF THE INVENTION
The occurrence of nitrile hydrolyzing enzymes has been widely described. Within this family of enzymes, two broad classes are generally recognized. The first includes the nitrile hydratases, which catalyze the addition of one molecule of water to the nitrile, resulting in formation of the corresponding amide:
The second group includes the nitrilases which catalyze adding two molecules of water to the nitrile resulting in the direct formation of the corresponding carboxylic acid plus ammonia, without the intermediate formation of the corresponding amide:
Cells containing aromatic or aliphatic nitrilases have been used to directly convert a nitrile to the corresponding carboxylic acid ammonium salt in aqueous solution without the intermediate formation of an amide (Kobayashi et al.,
FEMS Microbiol Lett.
120:217-234 (1994)). Cells having nitrilase activity include
Rhodococcus rhodochrous
K22 (Kobayashi et al.,
Tetrahedron
46:5587-5590 (1990); Kobayashi et al.,
J. Bacteriology
172:4807-4815 (1990)),
Acidovorax facilis
72W (Gavagan et al.,
Appl. Microbiol. Biotechnol.
52:654-659 (1999)),
Comamonas testosteroni
(U.S. Pat. No. 5,629,190 and U.S. Pat. No. 5,635,391; Lévy-Schil et al.,
Gene
161:15-20 (1995)),
Rhodococcus rhodochrous
NCIMB 11216 (Gradley et al.,
Biotechnology Lett.
16:41-46 (1994); Bengis-Garber et al.,
Appl. Microbiol. Biotechnol.
32:11-16 (1989)), and
Rhodococcus rhodochrous
J1 (Kobayashi et al.,
Eur. J. Biochem.
182:349-356 (1989)). Of those nitrilases that have been sequenced, a unique conserved active-site cysteine has been identified as the functional group responsible for enzymatic nitrile hydration (Lévy-Schil et al.,
Gene
161:15-20 (1995); Cowan et al.,
Extremophiles
2:207-216 (1998)). The typical method for stabilizing the cysteine-catalyzed nitrilase activity has been to include mM concentrations of dithiothreitol (DTT) and/or ethylenediamine-tetraacetic acid (EDTA) in the aqueous buffer containing the isolated enzyme or whole cell.
A method for preserving suspensions of cells or immobilized cells has been described in EP 0707061 A1, where the storage solution contains at least one inorganic salt selected from the group consisting of phosphates, borates, sulfates, sulfites, and hydrochlorides. Types of salts included sodium, potassium, and ammonium salts. The concentration of these salts ranged from 100 mM to the saturation concentration of the inorganic salt. The use of these inorganic salts to preserve the nitrilase activity of
Gordona terrae
MA-1 was demonstrated. Japanese patent application JP 10042885 A2 describes the production of amides and/or organic acids by treating nitrites with culture media, cells, cell preparations, enzymes, or enzyme preparations from microorganisms having nitrile-hydrolyzing activities in aqueous reaction mixture containing H
2
CO
3
or carbonate salts, where the inclusion of H
2
CO
3
or carbonate salts is reported to increase the observed activity of the nitrile-hydrolyzing enzyme towards the nitrile; no disclosure of an improvement in the stability of the nitrile-hydrolyzing activity of the cells over time when stored was made.
Methods for the preservation of nitrile hydration activity of cells containing a nitrile hydratase (where an aliphatic nitrile is enzymatically converted to an amide without subsequent conversion of the amide to the corresponding carboxylic acid ammonium salt) are described in U.S. Pat. No. 4,931,391 and U.S. Pat. No. 4,900,672. These methods for the stable preservation of nitrile hydratase activity of unimmobilized or immobilized microorganisms add at least one compound selected from the group consisting of nitrites, amides, or organic acids to a suspension of the unimmobilized or immobilized cells.
U.S. Pat. No. 4,343,900 describes a process to produce acrylamide from acrylonitrile using a microorganism having a nitrilasic activity (that is, a nitrile hydratase) which includes in the reaction mixture an alkali metal carbonate or bicarbonate. The alkali metal carbonate or bicarbonate is added to prevent a loss of enzyme activity that occurs due to a swelling of fixed cells during the reaction, and this loss of activity is not normally observed when physiological saline or phosphate buffer is added to the reaction mixture. In this same method, the addition of alkali metal carbonate or bicarbonate to reaction mixtures for the production of acrylamide from acrylonitrile is preferably done in combination with the addition of an organic carboxylic acid, thereby maintaining the enzymatic activity for a long period of time while acrylonitrile is converted to acrylamide.
The problems to be solved are to stabilize nitrilase activity and to reduce microbial contamination or putrefaction in suspensions of unimmobilized or immobilized cells. Different methods have been used to stabilize cells having either nitrile hydratase or nitrilase activity, where these two enzyme activities are produced by different types of enzymes which have different mechanisms for catalyzing nitrile hydrolysis to amides or acids, respectively. An examination of the effect of various carboxylic acid salts on the storage stability of nitrilase activity did not prevent the loss of nitrilase activity with time as was seen with nitrile hydratase. Similarly, the use of various inorganic salts previously disclosed as preserving the nitrilase or nitrile hydratase of microorganisms also did not stabilize the nitrilase activity of the microbial cells of the present invention upon storage as aqueous suspensions.
SUMMARY OF THE INVENTION
The invention solves the stated problem. It is a method for preserving microbial cells having nitrilase activity and for stabilizing the nitrilase activity thereof, the method comprising adding to an aqueous suspension of immobilized or unimmobilized microbial cells having nitrilase activity at least one compound selected from the group consisting of inorganic carbonate salts, inorganic bicarbonate salts, and inorganic carbamate salts, wherein the resulting total concentration of the inorganic salts in the aqueous suspension ranges from 100 mM to the saturation concentration of the inorganic salts. The microbial cells having nitrilase activity are selected from the group consisting of
Acidovorax facilis
72W (ATCC 55746),
Acidovorax facilis
72-PF-15 (ATCC 55747) and
Acidovorax facilis
72-PF-17 (ATCC 55745). Additionally, transformed microbial cells containing nitrilase activity are useful in this invention. Examples of such a transformed microbial cell are
E. coli
SS1001 (ATCC PTA-1177) and
E. coli
SW91 (ATCC PTA-1175) which contain
A. facilis
72W nitrilase activity. The inorganic salt(s) is preferably ammonium carbamate. The conditions for the method include where the pH of the aqueous suspension of microbial cells having nitrilase activity is from about pH 6 to about pH 10 and the temperature of the aqueous suspension of microbial cells ranges from about 0° C. to about 45° C. The microbial cells are preferably immobilized in polyacrylamide gel, alginate, or carrageenan. In a preferred embodiment, the microbial cells are immobilized in carrageenan, the inorganic bicarbonate salt is selected from the group consisting of ammonium bicarbonate and potassium bicarbonate, and the resulting total concentration of the inorganic bicarbonate salt(s) in the aqueous suspension ranges from about 100 mM to about 1000 mM. In another preferred embodiment of the method, the
Ben-Bassat Arie
Dicosimo Robert
Fallon Robert D.
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