Biocatalytic Hydrolysis of 3‐Hydroxyalkanenitriles to 3‐Hydroxyalkanoic Acids

Abstract

Microbial catalysts having a combination of nitrile hydratase and amidase activities had a significantly-higher specific activity for hydrolysis of 3-hydroxyalkanenitriles than microbial nitrilase catalysts. Comamonas testosteroni 22 ± 1, Dietzia sp. ADL1 and Comamonas testosteroni 5-MGAM-4D nitrile hydratase/amidase biocatalysts each hydrolyzed 3-hydroxyvaleronitrile to 3-hydroxyvaleric acid (as the ammonium salt) in 99 ± 100% yields, but in consecutive batch reactions with catalyst recycle, alginate-immobilized C. testosteroni 5-MGAM-4D had superior enzyme stability and volumetric productivity. In a series of 85 consecutive batch reactions with biocatalyst recycle for the production of 1.0 M 3-hydroxyvaleric acid, the recovered nitrile hydratase and amidase activities in the final reaction were 29% and 40%, respectively, of the initial activities. The catalyst productivity for this series of reactions was 670 g 3-hydroxyvaleric acid/g dry cell weight (50 g 3-hydroxyvaleric acid/g biocatalyst bead), and the volumetric productivity of the initial reaction in the series was 44 g 3-HVA/L/h. Similar results were obtained with alginate-immobilized C. testosteroni 5-MGAM-4D for the hydrolysis of 3-hydroxybutryonitrile and 3-hydroxypropionitrile to the corresponding 3-hydroxyalkanoic acid ammonium salts.

Protein engineering of Acidovorax facilis 72W nitrilase for bioprocess development

“…The use of immobilized whole cells in a bioreactor, particularly with recycle capability, represented a significant advance for biological transformations, often producing nearly pure product at high yield and high concentration. Examples include immobilized C. testosteroni 5-MGAM-4D (nitrile hydratase/amidase) for hydrolysis of 3-hydroxyalkanenitriles to 3-hydroxyalkanoic acids with >98% yield (Hann et al, 2003), and immobilized A. facilis 72W (nitrilase) for the regioselective hydrolysis of (E,Z)-2-methyl-2-butenenitrile for production of (E)-2-methyl-2-butenoic acid at 100% yield (Hann et al, 2004). Despite the sensitivity of nitrilase enzymes to various inactivating mechanisms, the A. facilis 72W nitrilase appears quite robust, losing little activity, even after numerous recycle reactions.…”

“…coli cells expressing nitrilase were immobilized in alginate beads at a fixed cell loading as previously described (Hann et al, 2003). Six grams of catalyst beads was continuously mixed with 13.4 mL of distilled, deionized water, 0.2 mL of 0.2 M calcium acetate (pH 7.0), and 0.4 mL (0.386 g) of 3-HVN (0.4 M final) at 358C.…”

“…The Acidovorax facilis 72W nitrilase converts 3-hydroxynitriles to the corresponding 3-hydroxycarboxylic acids in high yield (Hann et al, 2003). However, this enzyme has a relatively low specific activity for these substrates.…”

Chemoenzymatic Approach to Enantiomerically Pure (R)‐3‐Hydroxy‐3‐methyl‐4‐pentenoic Acid Ester and Its Application to a Formal Total Synthesis of Taurospongin A

“…While amidases had in many cases worked enantioselectively on b-alkoxy-and acyloxy-substituted nitriles, [16][17][18][19] the loss of stereoselection had also been observed when the substituent was altered from alkoxy and/or acyloxy to a hydroxy group. [16,[20][21] In this case, as the antipodal substrate, (S)-5 can be prepared from the enantiomerically pure form of (R)-epoxide 3, [3] this total procedure in Scheme 2 and Scheme 3 would also work well to provide (S)-1b.…”

Exploring the Synthetic Applicability of a Cyanobacterium Nitrilase as Catalyst for Nitrile Hydrolysis

“…[6] However, chemical hydrolysis often results in the undesirable elimination of OH for nitriles with β-hydroxy group, yielding unsaturated by-products. [7] Cyano carboxylic acids are also important intermediates for a variety of applications. Because dinitriles are readily available both on bench scale and in technical quantities, selective hydrolysis of dinitriles to cyano carboxylic acids is particularly interesting.…”