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Method for stabilizing nitrilase activity and preserving microbial cells

A technology of microbial cells and nitrilase, applied in the direction of microorganism-based methods, preserved microorganisms, biochemical equipment and methods, etc.

Inactive Publication Date: 2002-07-31
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0013] The present invention solves the problems stated above

Method used

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  • Method for stabilizing nitrilase activity and preserving microbial cells
  • Method for stabilizing nitrilase activity and preserving microbial cells
  • Method for stabilizing nitrilase activity and preserving microbial cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Storage Stability of Nitrilase Activity of Unimmobilized Acidovorax facilis Cells

[0069] Acidovorax facilis 72W cells were separated from the culture medium by centrifugation, and then suspended in potassium phosphate solution (0.10 or 1.00M), sodium acetate solution (0.10 or 1.00M) or sodium carbonate solution (0.10 or 0.30M) at pH 7.3 ), the concentration is 1-5% of the dry weight of the cells, and the final suspension is stored in a capped glass bottle at 5° C. After a long time, samples are taken from the cell suspension to analyze the nitrilase activity.

[0070] Table 1 illustrates the relative nitrilase activity of the stored cell suspensions, the nitrilase activity of the suspension stored for 0 days was defined as 100%. Cells stored in 0.10M or 0.30M sodium carbonate solution retained a substantially higher percentage of initial nitrilase activity at 92 days compared to cells stored in 0.10M or 1.0M sodium acetate or potassium phosphate solution.

[007...

Embodiment 2

[0073] Immobilization of Acidovorax facilis 72W in carrageenan particles

[0074] Acidovorax facilis 72W wet cell pellet (45 g) was mixed with 45 ml of 0.88% NaCl and the final suspension was heated at 50°C for 1 hour to inactivate the undesired nitrile hydratase activity. Then the cell suspension at 50° C. was added to 135.0 g of 5% by weight Pronova ISAGE L300 carrageenan solution at 55° C. with mixing. The final suspension was immediately cooled to 5°C ice / water bath for 1 hour to gel. The final gel was cut into particles with a diameter of about 2 mm, and the immobilized cell particles were kept in 450 ml of 0.30 M potassium chloride, 20 mM potassium dihydrogen phosphate (adjusted to pH 7.0 with potassium hydroxide) solution at 5 ° C for 18 hours And hardened. The sclerosing solution was removed by washing the immobilized cell pellet three times with 5 mM potassium chloride, 20 mM potassium dihydrogen phosphate (pH 7.0) at 5°C.

Embodiment 3

[0076] Immobilization of Acidovorax facilis 72W in Polyacrylamide Gel Particles

[0077] A solution of 13.8 g of acrylamide and 1.20 g of methylenebisacrylamide dissolved in 15 ml of water at 25 ° C was added with stirring to 10 ° C 30 g (wet weight of cells) Acidovorax facilis 72W was added with stirring 82 ml of 0.10 M potassium dihydrogen phosphate (pH7 .0) in suspension. The final mixture was added to diaminobutane (0.45ml) and 7.5ml of a 5% (w / v) potassium persulfate solution at 25°C. The final polymer gel was kept in an ice bath at 10° C. for 1 hour, and then cut into particles with a diameter of about 2 mm. The immobilized cell pellet was washed twice with 50 mM potassium dihydrogen phosphate (pH 7.0) solution at 5°C.

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Abstract

A method for preserving immobilized or unimmobilized microbial cells having nitrilase activity and for stabilizing the nitrilase activity of unimmobilized or immobilized microbial cells has been developed. The unimmobilized or immobilized microbial cells are stored in an aqueous solution containing from about 0.10 M to the saturation concentration of an inorganic salt of bicarbonate or carbonate, including ammonium, sodium and potassium salts of bicarbonate or carbonate. Aqueous suspensions containing at least 100 mM bicarbonate or carbonate limit microbial contamination of the stored enzyme catalyst, as well as stabilize the desired nitrilase activity of the unimmobilized or immobilized cells. Microorganisms which are characterized by a nitrilase activity and are stabilized and preserved by this method include Acidovorax facilis 72-PF-15 (ATCC55747), Acidovorax facilis 72-PF-17 (ATCC55745), Acidovorax facilis 72W (ATCC55746), and transformed microbial cells having nitrilase activity, preferably Escherichia coli SS1001 (ATTCCPTA-1177) which is transformed with Acidovorax facilis 72W nitrilase activity.

Description

field of invention [0001] The present invention relates to the fields of microbiology and molecular biology. More specifically, a method was developed for stabilizing the nitrilase activity and preserving the integrity of unimmobilized or immobilized microbial cells stored in a Aqueous solutions of inorganic bicarbonates or carbonates, including ammonium, sodium, potassium bicarbonates or carbonates, from a concentration of about 0.100M to saturation. Background of the invention [0002] The presence of nitrilases has been extensively described. Within this family of enzymes, two main types are generally recognized. The first class includes nitrile hydratases, which catalyze the addition of a molecule of water to a nitrile, resulting in the corresponding amide: [0003] Reaction 1: [0004] <chemistry num="001"> <chem file="00810328_cml001.xml" / > < / chemistry> [0005] The second group includes nitrila...

Claims

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Application Information

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IPC IPC(8): C12N1/04C12N9/78C12N9/96C12N11/04C12N11/08C12N11/10C12P13/00C12R1/01
CPCC12P13/00C12N11/10C12N9/78C12N11/04C12N11/08C12N1/04C12N9/96C12N11/087
Inventor R·迪科斯莫A·本-巴萨特R·D·法伦
Owner EI DU PONT DE NEMOURS & CO
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