MEANS AND METHODS FOR PRODUCING AMIDE COMPOUNDS WITH LESS ACRYLIC ACID

Abstract

The present invention relates to means and methods for producing an amide compound from a nitrile compound with less acrylic acid as by-product using a Nitrile hydratase (NHase) and Amidase producing microorganism as biocatalyst. Also provided is an aqueous amide compound obtained by the methods of the invention as well as a composition comprising acrylamide or polyacrylamide as well as a dried microorganism exhibiting a NHase/Amidase activity ratio of at least 400 when being brought into contact with a nitrile compound to convert said nitrile compound into an amide compound.

Claims

1. A method for producing an amide compound from a nitrile compound, comprising contacting a nitrile compound with a Nitrile hydratase (NHase) and Amidase producing microorganism, wherein said microorganism is pre-treated by a drying step before being contacted with said nitrile compound.

2. The method of claim 1, wherein the ratio of the NHase activity to Amidase activity of said microorganism is increased, when compared to a reference microorganism.

3. The method of claim 1, wherein the ratio of the NHase activity to Amidase activity of said microorganism is increased by a factor of at least 1.4 when compared to a reference microorganism.

4. The method of claim 1, wherein the ratio of the Amidase activity to NHase activity of said microorganism is decreased by a factor of at least 0.7 when compared to a reference microorganism.

5. The method of claim 1, wherein the microorganism exhibits an NHase/Amidase activity ratio of at least 400.

6. A method for producing an amide compound from a nitrile compound, comprising the steps of: a) drying a NHase and Amidase producing microorganism; and b) contacting a nitrile compound with said microorganism.

7. The method of claim 6, wherein step a) increases the NHase/Amidase activity ratio of said microorganism.

8. The method of claim 1, wherein said drying is mediated by spray drying, freeze-drying, heat drying, air drying, vacuum drying, fluidized-bed drying and/or spray granulation.

9. The method of claim 1, wherein the contacting step is conducted with a dried microorganism.

10. The method of claim 1, wherein the contacting step is conducted with a reconstituted microorganism.

11. The method of claim 10, wherein the microorganism is suspended in an aqueous composition.

12. The method of claim 1, wherein the contacting step is conducted with a microorganism that is in the form of a powder, granule, suspension, and/or matrix bound microorganism.

13. The method of claim 1, wherein the microorganism is a bacterium.

14. The method of claim 13, wherein the microorganism is selected from the group consisting of Rhodococcus, Aspergillus, Acidovorax, Agrobacterium, Bacillus, Bradyrhizobium, Burkholderia, Escherichia, Geobacillus, Klebsiella, Mesorhizobium, Moraxella, Pantoea, Pseudomonas, Rhizobium, Rhodopseudomonas, Serratia, Amycolatopsis, Arthrobacter, Brevibacterium, Corynebacterium, Microbacterium, Micrococcus, Nocardia, Pseudonocardia, Trichoderma, Myrothecium, Aureobasidium, Candida, Cryptococcus, Debaryomyces, Geotrichum, Hanseniaspora, Kluyveromyces, Pichia, Rhodotorula, Comomonas, and Pyrococcus.

15. The method of claim 14, wherein the microorganism is selected from the group consisting of Rhodococcus, Pseudomonas, Escherichia and Geobacillus.

16. The method of claim 14, wherein the microorganism is selected from the group consisting of Rhodococcus rhodochrous, Rhodococcus pyridinovorans, Rhodococcus erythropolis, Rhodococcus equi, Rhodococcus ruber, Rhodococcus opacus, Aspergillus niger, Acidovorax avenae, Acidovorax facilis, Agrobacterium tumefaciens, Agrobacterium radiobacter, Bacillus subtilis, Bacillus pallidus, Bacillus smithii, Bacillus sp BR449, Bradyrhizobium oligotrophicum, Bradyrhizobium diazoefficiens, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia gladioli, Escherichia coli, Geobacillus sp. RAPc8, Klebsiella oxytoca, Klebsiella pneumonia, Klebsiella variicola, Mesorhizobium ciceri, Mesorhizobium opportunistum, Mesorhizobium sp F28, Moraxella, Pantoea endophytica, Pantoea agglomerans, Pseudomonas chlororaphis, Pseudomonas putida, Rhizobium, Rhodopseudomonas palustris, Serratia liquefaciens, Serratia marcescens, Amycolatopsis, Arthrobacter, Brevibacterium sp CH1, Brevibacterium sp CH2, Brevibacterium sp R312, Brevibacterium imperiale, Corynebacterium nitrilophilus, Corynebacterium pseudodiphteriticum, Corynebacterium glutamicum, Corynebacterium hoffmanii, Microbacterium imperiale, Microbacterium smegmatis, Micrococcus luteus, Nocardia globerula, Nocardia rhodochrous, Pseudonocardia thermophila, Trichoderma, Myrothecium verrucaria, Aureobasidium pullulans, Candida famata, Candida guilliermondii, Candida tropicalis, Cryptococcus flavus, Cryptococcus sp UFMG-Y28, Debaryomyces hanseii, Geotrichum candidum, Geotrichum sp JR1, Hanseniaspora, Kluyveromyces thermotolerans, Pichia kluyveri, Rhodotorula glutinis, Comomonas testosteroni, Pyrococcus abyssi, Pyrococcus furiosus, Pyrococcus horikoshii, Brevibacterium casei, and Nocardia sp. 163.

17. The method of claim 16, wherein the microorganism is Rhodococcus rhodochrous or Rhodococcus pyridinovorans.

18. The method of claim 17, wherein the microorganism is Rhodococcus rhodochrous (NCIMB 41164), Rhodococcus rhodochrous (FERM BP-1478) or Rhodococcus rhodochrous M33.

19. The method of claim 1, wherein the nitrile compound is acrylonitrile.

20. The method of claim 1, wherein the amide compound is acrylamide.

21. The method of claim 1, the method comprising: (a) a pre-treatment of the biocatalyst microorganism by a drying step; (b) an activation step comprising mixing the dried microorganism obtained in step (a) with an aqueous solution to give an activation mixture, wherein the activation mixture comprises a buffer, and (c) converting the nitrile compound to the amide compound using the dried microorganism in a reaction mixture, wherein the reaction mixture comprises said buffer of step (b), wherein the ratio of the molar concentration of the buffer in the activation mixture to the molar concentration of said buffer in the reaction mixture, optionally before the end of the conversion, is about 2:1 or more.

22. A method for producing a microorganism with an increased NHase/Amidase activity ratio, comprising drying a NHase and Amidase producing microorganism.

23. A method for producing a microorganism with a decreased Amidase activity, comprising drying a NHase and Amidase producing microorganism.

24. A method for reducing the formation of acrylic acid when producing an amide compound from a nitrile compound, comprising contacting acrylonitrile with a NHase and Amidase producing microorganism, wherein said microorgansims is a microorganism obtained by the method of claim 22.

25. The method of claim 1, wherein said NHase and Amidase producing microorganism is not immobilized before being dried.

26. An aqueous amide compound solution obtained by the method of claim 1.

27. A composition comprising acrylamide or polyacrylamide and a NHase and Amidase producing microorganism, said microorganism exhibiting a NHase/Amidase activity ratio of at least 400 and/or a ratio of the NHase activity to Amidase activity which is increased by a factor of at least 1.7, when compared to a reference microorganism.

28-30. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0097] FIG. 1: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41164 as biocatalyst. The dried biocatalyst has been re-suspended in water after spray drying. A total amount of 3.36 g of the dried biocatalyst (batch Ch10) was employed, which had an NHase activity of 116 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L=liter) at 26° C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 2.4 g dried biocatalyst was added to the reactor. The ACN concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding ACN to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.96 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the ACN concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (<100 ppm residual ACN) was 13.78 h.

[0098] FIG. 2: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41164 as biocatalyst. The dried biocatalyst has been re-suspended in 33 mL of 100 mM phosphate buffer (pH 7.0) after spray drying, which corresponds to the activation step as disclosed herein. The activation step was conducted for 1.0 h. A total amount of 3.36 g of the dried biocatalyst (batch Ch10) was employed, which had an NHase activity of 116 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L=liter) at 26° C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 2.4 g dried biocatalyst was added to the reactor. The ACN concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding ACN to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.96 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the ACN concentration was maintained at 0.8% w/w, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (<100 ppm residual ACN) was 2.31 h.

[0099] FIG. 3: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41164 as biocatalyst. The dried biocatalyst has been re-suspended in water after spray drying. A total amount of 3.36 g of the dried biocatalyst (batch Ch10) was employed, which had an NHase activity of 116 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L=liter) at 26° C. Directly prior to biocatalyst addition, 33 mL of 100 mM phosphate buffer (pH 7.0) was added to the reactor, which corresponds to the amount of buffer that was used in the activation step of the experiment depicted in FIG. 2. At the beginning of the reaction, re-suspended biocatalyst corresponding to 2.4 g dried biocatalyst was added to the reactor. The ACN concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding ACN to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.96 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the ACN concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (<100 ppm residual ACN) was 11.98 h.

[0100] FIG. 4: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 mL of 100 mM phosphate buffer (pH 7.0), which corresponds to the activation step disclosed herein. The activation step was conducted for 0.5 h. 1.8 g of the dried biocatalyst (batch Ch10) was employed, which had an NHase activity 116 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L=liter) at 23° C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 1.8 g dried biocatalyst was added to the reactor. The ACN concentration after beginning of the bioconversion was maintained at 1 w/w % by feeding ACN to the reactor, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (<100 ppm residual ACN) was 7.13 h.

[0101] FIG. 5: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41164 as biocatalyst. The dried biocatalyst has been re-suspended in water. A total amount of 1.29 g of the dried biocatalyst (batch V3) was employed, which had an NHase activity 172 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L=liter) at 26° C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 0.92 g dried biocatalyst was added to the reactor. The ACN concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding ACN to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.37 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the ACN concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. No full conversion (<100 ppm residual ACN) was reached after 20 h.

[0102] FIG. 6: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 mL of 100 mM phosphate buffer (pH 8.0), which corresponds to the activation step. The activation step was conducted for 0.5 h. A total amount of 1.29 g of the dried biocatalyst (batch V3) was employed, which had an NHase activity 172 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L=liter) at 26° C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 0.92 g dried biocatalyst was added to the reactor. The ACN concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding ACN to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.37 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the ACN concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (<100 ppm residual ACN) was 4.4 h.

[0103] FIG. 7: Time course of acrylamide (ACM, depicted in dark grey) [%] and acrylonitrile (ACN, depicted in light grey) [%] concentrations in w/w % in a bioconversion of acrylonitrile to acrylamide applying spray dried Rhodococcus rhodochrous NCIMB 41164 as biocatalyst. The dried biocatalyst has been re-suspended in 30 mL of 100 mM citrate buffer (pH 7.0), which corresponds to the activation step. The activation step was conducted for 0.5 h. A total amount of 1.29 g of the dried biocatalyst (batch V3) was employed, which had an NHase activity 172 kU/g as measured before the beginning of the bioconversion. The reaction was conducted at a 4 L scale (L=liter) at 26° C. At the beginning of the reaction, re-suspended biocatalyst corresponding to 0.92 g dried biocatalyst was added to the reactor. The ACN concentration from 0 h to 1 h after beginning of the bioconversion was maintained at 2 w/w % by feeding ACN to the reactor. At 1 h after beginning of the bioconversion, re-suspended biocatalyst corresponding to 0.37 g dried biocatalyst was added to the reactor. After 1 h after beginning of the bioconversion, the ACN concentration was maintained at 0.8 w/w %, until a total amount of 1553 g acrylonitrile has been added to the reactor. Total reaction time until full conversion (<100 ppm residual ACN) was 7.25 h.

EXAMPLES

[0104] The following examples further describe and exemplify the invention provided herein without limiting the invention to any specifications or embodiments defined therein.

[0105] Three experiments in connection with the production of an amide compound from a nitrile compound were conducted. The inoculum for production was either fermentation broth containing a biocatalyst, a concentrate of said biocatalyst, a spray-dried biocatalyst or a freeze-dried biocatalyst. The concentrate is the form of the biocatalyst prior to its pre-treating by a drying step before being contacted with said nitrile compound to be converted to an amide compound. Concentrate means that the fermentation broth is concentrated by reducing liquid fermentation broth, e.g. by centrifugation. Thus, the fermentation broth, the concentrate and the dried powder as used in this Example contain the same biocatalyst. The activity ratio between NHase and Amidase as well as the NHase activity were determined in line with commonly known procedures. NHase activity in the setting up means that all setting ups contain the same amount of biocatalyst as is reflected by the almost identical NHase activity. Hence, the conditions were the same for the fermentation broth, concentrate and dried powder. In addition, the concentration of acrylic acid was determined. These data are summarized in Table 1, below, as well as the end of the bio-conversion reaction.

[0106] Exp 1: Water and 20 g of ACN were placed in a reactor. The amount of water was adjusted so that the total amount of water +biocatalyst was 2447 g. Three different forms of a biocatalyst were used in independent runs:

[0107] (i) a fermentation broth containing Rhodococcus rhodochrous NCIMB 41164. Water content: 88.2% (w/w).

[0108] (ii) a concentrate, for which the fermentation broth from (i) has been concentrated by centrifugation. Water content: 83.5% (w/w).

[0109] (iii) dry powder obtained by spray drying of the concentrate from (ii). Residual water content of the dry powder: 8.05% (w/w). Spray drying was operated at 115° C. gas inlet temperature and 65° C. gas outlet temperature.

[0110] The biocatalyst was added to the reactor, whereby the reaction started. During the reaction, 1533 g of additional acrylonitrile was added so that the overall reaction batch size at the end was 4000 g. The temperature was kept constant at 26° C. during the reaction. The ACN concentration was measured by on-line FTIR, and the rate of addition of ACN was adjusted so that the ACN concentration in the reaction mixture was kept constant at 0.8±0.1% (w/w) until the entire ACN has been added to the reaction. The reaction was stopped after ACN concentration had decreased to <100 ppm due to conversion. At the end of the reaction, the acrylamide (ACM)-concentration in every run was 51% (w/w).

[0111] Exp 2: Water and 60 g of ACN were placed in a reactor. The amount of water was adjusted so that the total amount of water +biocatalyst was 2447 g. Three different forms of a biocatalyst were used in independent runs:

[0112] (i) a fermentation broth containing Rhodococcus rhodochrous NCIMB 41164. Water content: 91.8% (w/w).

[0113] (ii) a concentrate, for which the fermentation broth of (i) has been concentrated by centrifugation. Water content: 85.3% (w/w).

[0114] (iii) dry powder obtained by spray drying of the concentrate from (ii). Residual water content of the dry powder: 6.8% (w/w). Spray drying was operated at 115° C. gas inlet temperature and 60 ° C. gas outlet temperature.

[0115] The biocatalyst was added to the reactor, whereby the reaction started. During the reaction, 1493 g of additional acrylonitrile was added so that the overall reaction batch size at the end was 4000 g. The temperature was kept constant at 26° C. during the reaction. The ACN concentration was measured by on-line FTIR, and the rate of addition of ACN was adjusted so that ACN concentration in the reaction mixture was controlled. During the first hour of the reaction, ACN concentration was kept constant at 2.0%±0.15% (w/w), thereafter, it was kept constant at 0.8%±0.15% (w/w) until the entire ACN has been added to the reaction. The reaction was stopped after ACN concentration had decreased to <100 ppm due to conversion. At the end of the reaction, the ACM-concentration in every run was 50% (w/w).

[0116] Exp 3: Water and 60 g of ACN were placed in a reactor. The amount of water was adjusted so that the total amount of water +biocatalyst was 2447 g. Two different forms of a biocatalyst were used in independent runs:

[0117] (i) a concentrate, for which a fermentation broth containing Rhodococcus rhodochrous NCIMB 41164 has been concentrated by centrifugation. Water content: 81% (w/w).

[0118] (ii) dry powder obtained by freeze drying of the concentrate from (i). Residual water content of the dry powder: 6.8% (w/w).

[0119] The biocatalyst was added to the reactor, whereby the reaction started. During the reaction, 1493 g of additional acrylonitrile was added so that the overall reaction batch size at the end was 4000 g. The temperature was kept constant at 26° C. during the reaction. The ACN concentration was measured by on-line FTIR, and the rate of addition of ACN was adjusted so that the ACN concentration in the reaction mixture was controlled. During the first hour of reaction, ACN concentration was kept constant at 2.0%±0.15% (w/w), thereafter, it was kept constant at 0.8%±0.15% (w/w) until the entire ACN has been added to the reaction. The reaction was stopped after ACN concentration had decreased to <100 ppm due to conversion. At the end of the reaction, the ACM-concentration in every run was 50% (w/w).

TABLE-US-00001 TABLE 1 NHase Activity activity ratio in the End of Acrylic NHase/ setting reaction acid No. Form of biocatalyst Amidase up [kU] [h] [ppm] Exp. 1 fermentation broth 209 220 4.9 844 concentrate 96 227 4.5 758 spray-dried powder 330 220 5.47 260 Exp. 2 fermentation broth 108 247 4.9 849 concentrate 81 247 6.4 856 spray-dried powder 418 246 7.5 281 spray-dried powder 418 270 5.9 258 Exp. 3 concentrate N/A 247 5.22 516 Freeze-dried powder N/A 247 5.35 297

[0120] It is apparent that a microorganism which was pre-treated by a drying step before being contacted with a nitrile compound which is then subject to bio-conversion by said microorganism has the highest value as regards NHase/Amidase activity. Given the fact that in each reaction (setting up) almost the same amount of biocatalyst was used (as determined by the NHase activity of each of the employed biocatalyst forms, namely fermentation/concentrate/spray-dries/freeze-dried), it is apparent that the drying step, i.e. subjecting a biocatalyst to a drying step before bringing it into contact with a nitrile compound significantly influences the amount of the by-product acrylic acid. This means that because of the drying step, the Amidase activity is reduced to such an extent that such dried microorganisms produce an amide compound with less acrylic acid as by-product which is apparent from the outermost right column. In sum, since reaction parameters are kept equal between the different setting ups, it is apparent that the improvement in reducing the amount of acrylic acid can be ascribed to the drying step.

[0121] Exp 4: Freeze dried powder was obtained by lyophilisation of the concentrated fermentation broth in a Christ Alpha 2-4 LSCplus laboratory freeze dryer. The concentrate was first frozen overnight at −20° C. and subsequently dried. During drying, the shelf temperature was −25° C., the condensator temperature was −82° C. and the chamber pressure was 0,25 mbar.

[0122] Water and 18 g of ACN were placed in a reactor. The amount of water was adjusted so that the total amount of water +biocatalyst was 1835 g. Two different forms of a biocatalyst were used in independent runs:

[0123] (i) a fermentation broth containing Rhodococcus rhodochrous J1. Water content: 96.1% (w/w).

[0124] (ii) a dry powder obtained by concentration of (i) by centrifugation up to a water content of 83.6% (w/w) and freeze drying of the concentrate.

[0125] The biocatalyst was added to the reactor, whereby the reaction started. During the reaction, 1147 g of additional acrylonitrile was added so that the overall reaction batch size at the end was 3000 g. The temperature was kept constant at 23° C. during the reaction. The ACN concentration was measured by on-line FTIR, and the rate of addition of ACN was adjusted so that the ACN concentration in the reaction mixture was kept constant at 1.0±0.1% (w/w) until the entire ACN had been added to the reaction. The reaction was stopped after ACN concentration had decreased to <100 ppm due to conversion. At the end of the reaction, the ACM concentration in every run was 51% (w/w).

[0126] Exp 5: The experiments were performed as in Exp 4 above, except that the ACN concentration in the reaction mixture was controlled at 0.3±0.1% (w/w) during the reaction.

[0127] The results from Exp 4-5 are shown in table 2 below.

TABLE-US-00002 TABLE 2 NHase activity Activity loading ratio [kU/kg End of Acrylic NHase/ batch reaction acid No. Form of biocatalyst Amidase size] [h] [ppm] Exp. 4 Fermentation broth 138 64.8 5.7 604 Concentrate 112 Not performed Freeze-dried powder 211 62.3 5.0 305 Exp. 5 Fermentation broth 138 64.8 7.5 885 Concentrate 112 Not performed Freeze-dried powder 211 62.3 6.6 568

[0128] Exp. 6: Activation of a Spray Dried Biocatalyst

[0129] Spray dried biocatalyst is weighed out in a centrifuge tube (Falcon®) and suspended in 30 ml buffer for the activation step as disclosed herein. Unless indicated otherwise, said buffer was 100 mM phosphate buffer, pH 7.0. The biocatalyst is buffer-treated for 0.5 h at room temperature. Then the biomass (biocatalyst) suspension is transferred to the reactor and further incubated for 1 h. After addition of the biomass suspension to the reactor, the centrifuge tube is rinsed with water and the solvent is transferred as well to the reactor. This amount of water is considered for the water weighing into the reactor.

[0130] Exp. 7: General Protocol for Bioconversion

[0131] The hydration of acrylonitrile is generally carried out in a stirred tank reactor (rpm=250, volume V=4 L) with an external circulating loop for cooling. For this purpose 2.4 L of water is filled in the reactor as well as the biocatalyst. Biomass is added as spray dried cells of Rhodococcus rhodochrous, which has been previously suspended into water. As described herein, the spray dried cells can also directly be suspended in buffer according to the activation step disclosed herein. In order to start the reaction, acrylonitrile is dosed into the stirred tank reactor employing a process control system. A constant concentration of acrylonitrile of 0.5 to 5 w/w % is adjusted by the use of an online Fourier Transform Infrared (FTIR) analysis, which directly communicates with the process control unit (Labview). The reaction temperature is constantly kept at 20 to 29° C. The dosage of acrylonitrile is stopped after the addition of 1553 g acrylonitrile. After the complete conversion of residual acrylonitrile, i.e. when a residual ACN concentration of <100 ppm is reached, and obtaining 52 w/w % acrylamide, the reaction is finished.

[0132] Exp. 8: Determination of the concentration of acrylic acid, acrylamide, acrylic acid and acrylonitrile in the obtained aqueous acrylamide solutions by HPLC

[0133] The following conditions were applied in order to determine the contents of acrylamide, acrylic acid and acrylonitrile:

[0134] Column: Aqua C18, 250*4.6 mm (Phenomenex)

[0135] Guard column: C18 Aqua

[0136] Temperature: 40° C.

[0137] Flow rate: 1.00 ml/min

[0138] Injection volume: 1.0 μl

[0139] Detection: UV detector, wavelength 210 nm

[0140] Stop time: 8.0 minutes

[0141] Post time: 0.0 minutes

[0142] Maximum pressure: 250 bar

[0143] Eluent A: 10 mM KH2PO4, pH 2.5

[0144] Eluent B: Acetonitrile

TABLE-US-00003 Gradient: Time [min] A [%] B [%] Flow [ml/min] 0.0 90.0 10.0 1.00 8.0 90.0 10.0 1.00

[0145] Matrix: Fermentation broths, bioconversion mixtures Sample is filtered through 0.22 μm

TABLE-US-00004 Analytes: Retention time [min] Acrylamide 3.29 Acrylic acid 3.91 Acrylonitrile 4.35

[0146] Exp 9

[0147] Spray dried Rhodococcus rhodochrous (NCIMB 41164) of batch Ch10 was used for bioconversion reactions of acrylonitrile to acrylamide. The bioconversion reactions were carried out according to the protocol of Exp 7.

[0148] In Run 1 (depicted in FIG. 1), 3.36 g biocatalyst that has been resuspended in water was used (2.4 g biocatalyst was added at the beginning of the bioconversion, 0.96 g biocatalyst was added after 1 h). In Run 2 (depicted in FIG. 2), 3.36 g biocatalyst was used (2.4 g biocatalyst was added at the beginning of the bioconversion, 0.96 g biocatalyst was added after 1 h) that has been activated with 100 mM phosphate buffer (pH 7.0), according to the invention. Activation was conducted according to Exp. 6. In Run 3 (depicted in FIG. 3), 3.36 g biocatalyst that has been resuspended in water was used (2.4 g biocatalyst was added at the beginning of the bioconversion, 0.96 g biocatalyst was added after 1 h), but the same amount of phosphate buffer that has been used in the activation step of Run 2 was directly added to the reactor prior to the addition of the biocatalyst. In Run 4 (depicted in FIG. 4), 1.8 g biocatalyst was used (1.8 g biocatalyst was added at the beginning of the bioconversion) that has been buffer-treated with 100 mM phosphate buffer (pH 7.0) according to Exp. 6. The results are outlined in the table below.

TABLE-US-00005 Total reaction Run # Biocatalyst Activation time 1 3.36 g Rhodococcus no/resuspension in 13.78 h rhodochrous (NCIMB water 41164) of batch Ch10 2 3.36 g Rhodococcus 100 mM phosphate  2.31 h rhodochrous (NCIMB buffer (pH 7.0) 41164) of batch Ch10 3 3.36 g Rhodococcus no/resuspension in 11.98 h rhodochrous (NCIMB water 41164) of batch addition of 33 mL Ch10 phosphate buffer (100 mM, pH 7.0) to the reactor 4 1.8 g Rhodococcus 100 mM phosphate  7.13 h rhodochrous (NCIMB buffer (pH 7.0) 41164) of batch Ch10

[0149] As can be seen from Run 2 (FIG. 2), activation of the dried biocatalyst using phosphate buffer reduces the total reaction time from 13.78 h (Run 1, FIG. 1) to 2.31 h. Run 3 (FIG. 3) shows that addition of phosphate buffer to the reaction mixture without activation of the dried biocatalyst has almost no influence on the reaction time compared to Run 1. Run 4 (FIG. 4) demonstrates that if the dried biocatalyst is activated using phosphate buffer, the amount of the biocatalyst can be reduced from 3.36 g to 1.8 g while the total reaction time is still less than using 3.36 g of non-buffer-treated biocatalyst as in Run 1.

[0150] Exp 10:

[0151] Spray dried Rhodococcus rhodochrous (NCIMB 41164) of batch V3 was used for bioconversion reactions of acrylonitrile to acrylamide. The bioconversion reactions were carried out according to the protocol of Exp 7.

[0152] In Run 5 (depicted in FIG. 5), 1.29 g biocatalyst that has been resuspended in water was used (0.92 g biocatalyst was added at the beginning of the bioconversion, 0.37 g biocatalyst was added after 1 h). In Run 6 (depicted in FIG. 6), 1.29 g biocatalyst was used (0.92 g biocatalyst was added at the beginning of the bioconversion, 0.37 g biocatalyst was added after 1 h) that has been activated with 100 mM phosphate buffer (pH 8.0) as disclosed herein. Activation was conducted according to Exp. 6. In Run 7 (depicted in FIG. 7), 1.29 g biocatalyst was used (0.92 g biocatalyst was added at the beginning of the bioconversion, 0.37 g biocatalyst was added after 1 h) that has been activated with 100 mM citrate buffer (pH 7.0). The results are outlined in the table below.

TABLE-US-00006 Total Run # Biocatalyst Activation reaction time 5 1.29 g Rhodococcus no/resuspension in Incomplete rhodochrous (NCIMB water conversion 41164) of batch V3 after 20 h 6 1.29 g Rhodococcus 100 mM phosphate 4.39 h rhodochrous (NCIMB buffer (pH 8.0) 41164) of batch V3 7 1.29 g Rhodococcus 100 mM citrate buffer 7.25 h rhodochrous (NCIMB (pH 7.0) 41164) of batch V3

[0153] As can be seen from Runs 6 and 7 (FIGS. 6 and 7), both the activation step of Rhodococcus rhodochrous (NCIMB 41164) of batch V3 with phosphate buffer (100 mM, pH 8.0) and citrate buffer (100 mM, pH 7.0) leads to a dramatic reduction of total reaction time from an incomplete conversion after 20 h to a complete conversion after 4.39 h and 7.25 h, respectively.