Green Chemical Syntheses and Processes - ACS Publications

Chapter 10

Biocatalytic Production of 5-Cyanovaleramide from Adiponitrile Downloaded by NORTH CAROLINA STATE UNIV on September 20, 2012 | http://pubs.acs.org Publication Date: August 15, 2000 | doi: 10.1021/bk-2000-0767.ch010

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Robert DiCosimo , Eugenia C. Hann , Amy Eisenberg, Susan K. Fager, Neal E. Perkins , F. Glenn Gallagher, Susan M. Cooper , John E. Gavagan, Barry Stieglitz, and Susan M. Hennessey 1

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DuPont Central Research and Development, DuPont Agricultural Products, Experimental Station, P.O. Box 80328, Wilmington, DE 19880-0328 A biocatalytic process for the hydration of adiponitrile to 5cyanovaleramide has been demonstrated which can be run to higher conversion, produces more product per weight of catalyst, and generates significantly less waste products than alternate chemical processes. Immobilization of Pseudomonas chlororaphis B23 cells in calcium alginate beads produced a catalyst having high nitrile hydratase activity, and excellent stability when recycled in consecutive batch reactions. A total of 13.6 metric tons of 5cyanovaleramide was produced in 93% yield and 96% selectivity, with a catalyst productivity of 3,150 kg of 5-cyanovaleramide per kg P. chlororaphis B23 cells (dry cell weight).

The first step in the manufacture* of a new crop protection chemical required the conversion of adiponitrile (ADN) to 5-cyanovaleramide (5CVAM). The hydration of nitriles to amides can be accomplished using a variety of chemical catalysts (7-5), and manganese dioxide (5) produced 5CVAM with the highest yield and regioselectivity. When a stoichiometric amount of water was reacted with neat ADN at 130 °C using manganese dioxide as catalyst, 5CVAM could be produced at 80 % selectivity if the reaction was run to only ca. 25 % conversion. At higher conversions, the selectivity to 5CVAM decreased significantly as the selectivity to byproduct adipamide (ADAM) increased. Product isolation required dilution of the hot product mixture with toluene and filtration using a filter aid, where the insoluble A D A M remained in the spent manganese dioxide filter cake. Cooling the resulting hot toluene filtrate precipitated 5CVAM, and the large amount of unconverted ADN could subsequently be recovered for recycle by concentration of the remaining toluene solution. Approximately 1.25 kg of catalyst waste (manganese dioxide and filter aid) was generated for each 1 kg cf 5CVAM produced, so only small quantities of 5CVAM were prepared for pilot studies using this procedure. The cost and environmental impact of disposal or

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American Chemical Society

In Green Chemical Syntheses and Processes; Anastas, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2000.

Downloaded by NORTH CAROLINA STATE UNIV on September 20, 2012 | http://pubs.acs.org Publication Date: August 15, 2000 | doi: 10.1021/bk-2000-0767.ch010

115 recycle of the spent, deactivated manganese dioxide catalyst led to the development cf an alternate, green process for 5CVAM production. The nitrile hydratase (EC 4.2.1.84) produced by a variety of bacteria and fungi can readily catalyze the hydration of aliphatic nitriles to the corresponding amides (6-11). For the conversion of ADN to 5C V A M , a regioselective nitrile hydratase was required to limit the further conversion of 5CVAM to ADAM. The absence of amidase activity in the microbial cell catalyst was also important to prevent the subsequent hydrolysis of the amide to a carboxylic acid. Bacterial strains of Bacillus (12), Bacteridium (12), Brevibacterium (12), Micrococcus (12), Pseudomonas (13), and Acinetobacter (14) each produce a nitrile hydratase capable of regioselective hydration of aliphatic nitriles, and a variety of these strains were evaluated for the conversion of ADN to 5CVAM. The biocatalytic process which has been developed and commercialized for the production of 5CVAM (15) can be run to higher conversion, produces more product per weight of catalyst, eliminates the use of toluene for purification of C V A M , and generates less than 1 % of the catalyst waste produced in the chemical process.

Results and Discussion Microbial catalysts having nitrile hydratase activity were obtainedfrompubliclyheld culture collections, or were isolated from soil samples using one of several aliphatic nitriles or amides as carbon and/or nitrogen source. Of the whole-cell catalysts that were initially screened, two which exhibited high regioselectivity for the hydrolysis of ADN to 5CVAM (Scheme 1) were Pseudomonas putida 3LG-1-5-1A (ATCC 55736; 97% regioselectivity) (75), and Rhodococcus sp. A4 (formerly Brevibacterium sp. R312 strain A4, Technische Universiteit Delft, LMD#79.2; 97% regioselectivity) (12,16). The stability of the nitrile hydratase activity of each of these whole-cell catalysts was temperature dependent, with the greatest stability under reaction conditions occurring at 5 °C; increasing reaction temperatures above 5 °C resulted in a more rapid loss of nitrile hydratase activity. Whole cells of 3LG (ca. 4,000 ADN IU/g wet cell weight (wcw)) or A4 (ca. 24,000 ADN IU/g wcw) were initially immobilized in polyacrylamide gel (PAG) (17) and the resulting catalyst used in consecutive batch reactions with catalyst recycle. Under identical reaction conditions, the 3LG/PAG catalyst lost a higher percentage of nitrile hydratase activity in consecutive batch reactions than the A4/PAG catalyst, therefore A4 was chosen for further development. Scheme 1 97 % (ca. 4 h initial reaction time), the stirring was stopped, the catalyst beads immediately settled to the bottom of the reactor, and ca. 90 % of the product mixture was decanted away from the catalyst. The reactor was immediately charged with 1007 L of reaction buffer and 218 kg (2,015 mol) cf adiponitrile, and the reaction repeated. After running thirteen consecutive batch reactions to establish a reaction baseline, a final addition of 20.4 kg of B23/alginate beads was added to the fourteenth consecutive batch reaction (5.8 wt % final catalyst weight /weight reaction mixture). Each decanted product mixture was briefly heated to 40 °C to deactivate any catalyst activity that was present. The reaction time for reaction number 56 was allowed to extend for four hours past the endpoint of the reaction, then two more reactions were run to determine if the increased production and precipitation of A D A M in reaction number 56 produced a loss of catalyst activity in subsequent reactions; no increase in reaction time for the final two batch reactions was observed. A total offifty-eightconsecutive 400-gallon batch reactions were run, using 12.7 metric tons of ADN; the reaction times and concentrations of 5CVAM, ADN and ADAM for each reaction are illustrated in Figure 4. The yields of 5CVAM and A D A M from the combined product mixtures were 93 % and 4 % respectively, with 3 % recovery of unconverted ADN. The reaction time increased from 3.5 h to 6 h over the course of the fifty-eight reactions. At 97 % overall conversion of ADN, the combined yield of recovered 5CVAM was 13.6 metric tons. The catalyst productivity was 3,150 kg of 5CVAM produced per kg of B23 cells (dry cell weight). The total weight of product mixture produced was 70.7 metric tons, which contained 19.2 wt % 5CVAM, 0.99 wt % ADAM, and 0.52 wt % ADN. The 5CVAM was recoveredfromthe product mixture by removal of water by distillation under reduced pressure, then dissolution of the resulting oil at > 65 °C in methanol, which precipitated the byproduct A D A M as well as calcium and butyrate salts. After removal of the byproducts byfiltration,the methanolic 5CVAM solution was used directly in the next process step for herbicide synthesis.

Conclusions As an alternative to chemical catalytic methods for the production of 5CVAM from ADN, a biocatalytic process has been developed and commercialized. Immobilization of Pseudomonas chlororaphis B23 cells in calcium alginate beads produced a catalyst having high nitrile hydratase activity, and excellent stability when recycled in consecutive batch reactions. The biocatalytic reaction was run to higher conversion, produced more product per weight of catalyst, and generated significantly smaller amounts of byproducts and waste products than alternate chemical processes which were examined. In particular, the ratio of catalyst waste to 5CVAM produced in the biocatalytic process was very low; only 0.006 kg of catalyst waste was produced per kg 5CVAM, and this catalyst waste was 93% water by weight. The


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Figure 4. Reaction time (A) and wt % 5CVAM (A), ADAM (·) and ADN (β) for fifty-eight consecutive batch reactions with catalyst recycle to produce a total of 13.6 metric tons of 5CVAM. Reactions were run in two-phase reactions containing ADN and 5.8 wt % (final weight catalyst/weight reaction mixture) B23/alginate beads in 23 mM sodium butyrate/5 mM calcium chloride (pH 7.0) at 5 °C.


124 reaction produced ca. 190 g 5CVAM per liter of reaction volume, and purification and recovery of the product for use in the next process step was readily performed.


Acknowledgments The authors gratefully acknowledge the contributions of L. Winnie Wagner, Robert D. Fallon, Mark J. Nelson, David L. Anton, Ron Grosz, Umesh Hattikadur, John Freudenberger, Rafael Shapiro, Onorato Campopiano, and the Agricultural Products Semiworks (DuPont), Coréen R. B. Reed (Pronova Biopoylmer, Inc.), and Tetsuro Horinouchi and Katsumi Nakamura (Mitsubishi Rayon Co.). Sections of the Results and Discussion which describe optimization of reaction conditions using immobilized B23 cells, and commercial-scale preparation of 5CVAM, are reprinted from reference 15 (Copyright 1999) with permissionfromElsevier Science Ltd.

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