Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of ( R )- o -Chloromandelic Acid

Abstract

The nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic ␣-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzing o-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained from Burkholderia cenocepacia strain J2315 (BCJ2315) in hydrolyzing o-chloromandelonitrile. O ptically pure aromatic ␣-hydroxy carboxylic acids, such as mandelic acid and its derivatives, are widely used as intermediates and resolving agents for the production of many pharmaceutical and agricultural products (1, 2). Among them, (R)-ochloromandelic acid is one of the most preferred chiral building blocks for industrial synthesis of the antithrombotic agent (S)-clopidogrel, commercialized under the brand name Plavix (3, 4).Considering the great importance of (R)-o-chloromandelic acid in pharmaceuticals and the ever-growing demand for green catalytic processes, tremendous efforts have been made to establish enantioselective routes of production (1). Among them, the nitrilase-mediated pathway is a simple and practical approach for the commercial production of (R)-o-chloromandelic acid and its derivatives because it does not involve a cofactor, uses a less-expensive starting material, and theoretically yields 100% of the desired product.However, few nitrilases have been identified with high enantioselectivity and activity toward o-chloromandelonitrile (2, 3, 5-7). This severely reduces the practical applications of nitrilasemediated production of optically pure (R)-o-chloromandelic acid. A nitrilase with high enantioselectivity and specific activity is crucial to fit the manufacturing process and reduce industrial production costs. These requirements may be met by discovering new enzymes or by applying protein engineering to enhance the performance of the existing nitrilases, and convincing successes have been achieved by both methods (8).In our previous study, we identified and characterized a novel arylacetonitrilase, BCJ2315, from Burkholderia cenocepacia strain J2315 (9). BCJ2315 demonstrated high activity toward o-chloromandelonitrile and tolerated up to 50 mM o-chloromandelonitrile, though the o-chloromandelonitrile was highly toxic to the enzyme, demonstrating great potential for production of (R)-ochloromandelic acid under high o-chloromandelonitrile concentrations (4). Unfortunately, BCJ2315 showed relatively low enantioselectivity toward o-chloromandelonitrile, providing the desired product with only 89.2% enantiomeric excess (ee), which reduces its value for industrial production of (R)-o-chloromandelic acid (4).In the present study, we aspired to improve the fitness of BCJ2315 for the hydrolysis of o-chloromandelonitrile to efficiently produce (R)-o-chloromandelic acid. A protein engineering approach was successfully used to improve the enantioselectivity and activity of BCJ2315 by changing the enzyme structu...

Biokatalyse: Enzymatische Synthese für industrielle Anwendungen

“…[114] Angewandte Chemie Aufsätze die In-situ-Racemisierung des Cyanhydrins und ermçglicht den Zugang zum Endprodukt in 100 %t heoretischer Ausbeute (Schema 22 a). [107,120] Eines der produktivsten Verfahren wurde von der Wei-Gruppe berichtet, die E.-coli-Zellen einsetzten, die eine Nitrilase aus Burkholderia cenocepacia J2315 (BCJ2315) exprimieren. [107] Durch eine kontinuierliche Bedingt durch die sterische Hinderung durch den ortho-Chlorsubstituenten sind die meisten natürlichen Nitrilasen aber ungeeignet bezüglich ihrer Aktivität und/oder Enantioselektivität.…”

“…Eine Doppelmutante (I113M/ Y199G) der gleichen Nitrilase (BCJ2315) mit hoher Aktivität und Enantioselektivitätwurde daher generiert. [120] Mittels E.coli-Zellen (3.9 gBTM L À1 ), die diese Mutante exprimieren, wurde (R)-63 b bei einer Konzentration von 500 mm (93 gL À1 ) mit 98.7 % ee in 3h erzeugt. Die RZA erreicht 31 gL À1 h À1 (Tabelle 5).…”

“…[123a] Die Anwendung der gerichteten Evolution führte zur Identifizierung der A190G-Mutante mit hçherer Enantioselektivität. [108] Mit dieser verbesserten Nitrilase (10 gL À1 )u nd hoher Konzentration von 65 (3 m,330 gL À1 )wurde (R)-66 in 96 %A usbeute [107,120] und b) ein ähnliches Hydroxynitrillyase-Verfahren [113,121] zur Synthese von (R)-Mandelsäure und (R)-o-Chlormandelsäure zur Herstellung von Clopidogrel 64. Nitrilasen kçnnen zur Herstellung chiraler Carbonsäuren über kinetische Racematspaltung genutzt werden, wie kürzlich füre in Verfahren zur Synthese von Pregabalin gezeigt wurde (Schema 24).…”

Bioengineering of Nitrilases Towards Its Use as Green Catalyst: Applications and Perspectives

“…Further, certain fungal nitrilases from Gibberella, Neurospora, and Aspergillus were expressed in E. coli [47]. Recently, one study was carried out where enantio-selectivity and activity of nitrilase from Burkholderia cenocepacia BCJ2315 enhanced by the use of site-directed mutagenesis [34]. In another recent study, the recombinant nitrilase of Fusarium proliferatum AUF-2 catalysed aliphatic, heterocyclic and aromatic nitriles respectively [48].…”

Development of a robust nitrilase by fragment swapping and semi‐rational design for efficient biosynthesis of pregabalin precursor

“…Among these efficient strategies, numerous successful examples have been reported in the past few decades using protein engineering. Desired mutants are usually generated by directed evolution, semi‐rational or rational design approach (Gong et al, ; Luo, Ma, Chang, Yu, & Shen, ; Wang et al, ). However, directed evolution is often too time‐consuming to be implemented in process development because it requires construction and screening of large libraries that mostly contain variants with reduced or even no activity.…”