METHOD OF CULTIVATING MICROORGANISMS HAVING NITRILE HYDRATASE ACTIVITY

Abstract

The present invention relates to methods for cultivating a nitrile hydratase producing microorganism, compositions for cultivating a nitrile hydratase producing microorganism, and use of compositions comprising a saccharide and an organic acid for cultivating a nitrile hydratase producing microorganism. The composition provided in and to be employed in context with the present invention is particularly suitable for inducing both, growth and nitrile hydratase production of corresponding microorganisms.

Claims

1. A method for cultivating a nitrile hydratase producing microorganism, comprising: contacting a nitrile hydratase producing microorganism with an aqueous composition comprising a saccharide and an organic acid; and cultivating the microorganism in the aqueous composition.

2. A composition, comprising: a saccharide; an organic acid; and a nitrile hydratase producing microorganism.

3. (canceled)

4. The method of claim 1, wherein the saccharide is a monosaccharide.

5. The method of claim 1, wherein the number of carbon atoms contained in the saccharide is at least 5.

6. The method of claim 1, wherein the saccharide is at least one selected from the group consisting of glucose, fructose, ribose, and mannose.

7. The method of claim 1, wherein the organic acid comprises not more than 3 carboxyl groups.

8. The method of claim 1, wherein the organic acid comprises not more than 6 carbon atoms.

9. The method of claim 1, wherein the organic acid is at least one selected from the group consisting of lactic acid, tartaric acid, citric acid, malic acid, and succinic acid.

10. The method of claim 1, wherein a ratio of the saccharide to the organic acid is between 3:7 and 7:3 by weight.

11. The method of claim 1, wherein the microorganism belongs to the species Rhodococcus rhodochrous or Rhodococcus pyridinovorans.

12. The method of claim 1, wherein the nitrile hydratase is encoded by a polynucleotide comprising a nucleotide sequence which is at least 70% identical to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or both.

13. The method of claim 1, wherein the nitrile hydratase has an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, or both.

14. A microorganism, obtained by the method of claim 1.

15. The method of claim 1, further comprising: drying the microorganism after the cultivating.

16. The method of claim 1, wherein the number of carbon atoms contained in the saccharide is at least 6.

17. The method of claim 1, wherein the organic acid comprises not more than 1 carboxyl group and not more than 3 carbon atoms.

18. The method of claim 1, wherein the nitrile hydratase is encoded by a polynucleotide comprising a nucleotide sequence which is at least 95% identical to the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, or both.

19. The method of claim 1, wherein the nitrile hydratase has an amino acid sequence which is at least 95% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO. 4, or both.

20. The method of claim 1, wherein the cultivating is performed at a temperature of from 30° C. to 40° C. and at a pH value of from 6 and 8.

21. The method of claim 1, wherein the saccharide comprises glucose and the organic acid comprises lactic acid.

Description

EXAMPLES

Example 1

Cultivation of Rhodococcus ssp.

[0034] Different sugar/organic acid ratios were evaluated for different strains of Rhodococcus (R. rhodochrous “CIBA” (NCIMB 41164) and R. rhodochrous “J1” (FERM BP-1478)), in a microfermentation system.

[0035] The basic cultivation medium contained yeast extract (Biospringer) (1.25 g/l), KH.sub.2PO.sub.4 (10 g/l), K.sub.2HPO.sub.4 (10 g/l), (NH.sub.4).sub.2SO.sub.4 (1 g/l), MgSO.sub.4*7H.sub.2O (0.375 g/l), CaCl.sub.2*2H.sub.2O (25 mg/l), Trace element solution (2.5 g/l) (see below), Co(NO.sub.3).sub.2 (31.2 mg/l), anti-foam P2000 (BASF) (62.5 mg/l), urea (7 g/l), and ammonium glutamate (2.5 g/l) in H.sub.2O.

[0036] Trace Element Solution: Citric acid mono hydrate (40 g/l), ZnSO.sub.4*7H.sub.2O (11 g/l), (NH.sub.4).sub.2Fe(SO.sub.4).sub.2*6H.sub.2O (8.5 g/l), MnSO.sub.4*H.sub.2O (3 g/l), and CuSO.sub.4*5H.sub.2O (0.8 g/l) in H.sub.2O.

[0037] The medium was then supplemented with 10 g/l of carbon source (saccharide, organic acid or mixture of both as indicated in the Tables below). pH was adjusted to 6.6 using H.sub.3PO.sub.4 or NaOH. The medium was inoculated directly from cryo stocks to OD 0.05 (measured at 600 nm). Cultivation was carried out at 37° C. for 64 h at 1,100 rpm in a BioLector micro fermentation system (M2P Labs, Baesweiler, Germany) using 48-well flower plates with a working volume of 1.5 ml. After cultivation, samples were drawn, quenched with hydrochloric acid and nitrile hydratase activity was determined as described in Example 2.

[0038] In the tables below, relative nitrile hydratase activities for different strains and saccharide/acid ratios are provided. Nitrile hydratase activities achieved with the saccharide as sole carbon source were set to 100%.

TABLE-US-00001 TABLE 1 R. rhodochrous “CIBA” Saccharide/Acid ratio by weight Saccharide Acid Only acid 1/9 3/7 1/1 7/3 1/9 Only saccharide Glucose Lactic N/A  24% 1037% 1242% 748% 422% 100% Tartaric  173%  244% 1354% 1044% 737% 191% 100% Malic  197%  392% 1199% 2596% 1709%  801% 100% Citric  164%  190%  161% 1433% 635%  57% 100% Succinic  150%  510% 1365% 2303% 1228%  547% 100% Fructose Lactic N/A   1%  93%  156% 225% 160% 100% Tartaric  34%  46%  128%  106% 148%  60% 100% Malic  39%  94%  278%  333% 363% 399% 100% Citric  32%   0%  134%  291% 251%  81% 100% Succinic  30%  16%   0%  182% 152%  93% 100% Mannose Lactic N/A  188% 8947% 2141% 2888%  363% 100% Tartaric 1388% 1519% 3543% 2415% 333% 178% 100% Malic 1579%  911%  633% 1185% 1326%  503% 100% Citric 1315%  81%  92%  857% 754% 272% 100% Succinic 1204% 2477% 2658% 2660% 1395%  226% 100% Ribose Lactic N/A  99% 5939% 7655% 9628%  957% 100% Tartaric 1487% 1039% 4630% 14304%  11834%  170% 100% Malic 1693% 2022% 7187% 15682%  17555%  434% 100% Citric 1409% 1748% 3172% 12115%  5503%  1663%  100% Succinic 1291% 3378% 9647% 17868%  14895%  444% 100%

TABLE-US-00002 TABLE 2 R. rhodochrous “J1” Saccharide/Acid ratio by weight Saccharide Acid Only acid 1/9 3/7 1/1 7/3 1/9 Only saccharide Glucose Lactic 1963% 2092% 5069% 4934% 3731% 1836% 100% Succinic N/A 1920% 1270% 4110% 5548% 1668% 100% Fructose Lactic  73%  76%  79%  134%  118%  55% 100% Succinic N/A  33%  47%  130%  145%  51% 100%

Example 2

Measurement of Nitrile Hydratase Activity

[0039] Samples were taken from the cultivation medium of Example 1 and diluted 1:10 (v/v) with 50 mmol/l KH.sub.2PO.sub.4 buffer (pH 7.0). The solution obtained is further diluted 1:20 (v/v) with 50 mmol/l KH.sub.2PO.sub.4 buffer (pH 7.0) to achieve a final dilution factor of 1:200 for the cultivation medium.

[0040] Reaction:

[0041] 875 μl of KH.sub.2PO.sub.4 buffer (pH 7.0) were pipetted in a 2 ml Eppendorf tube. 100 μl of the diluted cultivation medium were added and the mixture was pre-incubated in a thermomixer at 25° C. for 5 min at 500 rpm. After pre-incubation, the reaction was started by addition of 25 μl acrylonitrile. The reaction was carried out at 25° C. and 1,000 rpm for 10 min.

[0042] Preparation of Samples:

[0043] After 10 min of reaction time, the reaction was quenched by transferring 300 μl of the reaction solution to a 1.5 ml Eppendorf tube which contained 300 μl of 1.4% (m/v) hydrochloric acid. The mixture was vortexed briefly and centrifuged in a table top centrifuge for 1 min at 10,000 rpm to separate the cells. 100 μl of the clear supernatant were mixed with 900 μl of water. The mixture was then analyzed by HPLC.

[0044] HPLC Analysis: [0045] Column: Aqua 5μ C18 125A, 250×4.60 mm (Phenomenex) [0046] Oven Temperature: 45° C. [0047] Injection Volume: 1 μl [0048] Detection: UV 210 nm [0049] Flow: 1.0 ml/min [0050] Solvent A: 25 mmol/l KH.sub.2PO.sub.4 pH 2.5 [0051] Solvent B: Acetonitrile [0052] Seperation: 10% B (isocratic) [0053] Retention: Acrylamide: ˜3.6 min, [0054] HPLC-value should be between 0.25 and 1.25 mmol/l

[0055] The concentration of acrylamide determined by HPLC should be between 0.25 and 1.25 mmol/l. If this is not achieved directly, the cell concentration used in the reaction needs to be adjusted accordingly.

[0056] Determination of Nitrile Hydratase Activity: [0057] c: amount of substance [mM] product (HPLC-value) [0058] t: incubation time [min], in this test 10 min [0059] k: dilution factor—total dilution from start sample to HPLC sample

[0060] calculation of activity in kU/l

[00001]$\mathrm{Activity}.Math..Math.\left(\mathrm{in}.Math..Math.\mathrm{kU}.Math.\text{/}.Math.I\right)=\frac{c}{t}.Math..Math.k$

[0061] One activity unit [U] is defined as 1 μmol generated acrylamide during one minute reaction time.