Efficient production of (R)-(−)-mandelic acid in biphasic system by immobilized recombinant E. coli

Ni, Kefeng; Wang, Hualei; Zhao, Li; Zhang, Minjie; Zhang, Siyuan; Ren, Yuhong; Wei, Dongzhi

doi:10.1016/j.jbiotec.2013.07.024

Cited by 33 publications

(19 citation statements)

References 29 publications

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“…Nevertheless, this system was found to exhibit the highest volumetric productivity of 571 g/(L d) compared to those reported in this study or in studies of the Alcaligenes nitrilase [1,2,8]. Only the nitrilase from B. cepacia gave higher volumetric productivities [up to 982 g/(L d)]; this value was achieved after recycling the catalyst [10] ( Table 3).…”

Section: Batch Production Of (R)-mandelic Acid In Biphasic Systemsmentioning

confidence: 42%

“…However, this problem was solved by adding toluene (10 %, v/v) which decreased the substrate concentration in the aqueous phase [1]. Alternatively, ethylacetate (30 % v/v) was used as a cosolvent in a process catalyzed by the whole-cell catalyst based on the nitrilase from B. cepacia [10]. The hydrolysis of 500 mM (R,S)-mandelonitrile by E. coli whole-cell catalysts producing the fungal enzymes was first examined at pH 8.0 (near the activity optimum of the enzymes).…”

Section: Resultsmentioning

confidence: 99%

“…The productivity of the latter catalyst was increased to 55 g/g by cell recycling [1]. The highest catalyst productivity (156 g/g) was achieved with the nitrilase from B. cepacia when the catalyst (immobilized whole cells of E. coli) was recycled [10] (Table 3).…”

Section: Batch Production Of (R)-mandelic Acid In Biphasic Systemsmentioning

confidence: 99%

“…A number of nitrilases suitable for the production of (R)-mandelic acid were also found by screening a metagenomic library [5,6]. The process was recently improved using recombinant strains [1,2,[7][8][9][10] or enzyme mutants [2]. A similar process was also developed for the production of (R)-o-chloromandelic acid, a precursor of the anti-coagulant drug Clopidogrel Ò [11].…”

Section: Introductionmentioning

confidence: 99%

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Exploring the Potential of Fungal Arylacetonitrilases in Mandelic Acid Synthesis

2015

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The application of arylacetonitrilases from filamentous fungi to the hydrolysis of high concentrations of (R,S)-mandelonitrile (100-500 mM) was demonstrated for the first time. Escherichia coli strains expressing the corresponding genes were used as whole-cell catalysts. Nitrilases from Aspergillus niger, Neurospora crassa, Nectria haematococca, and Arthroderma benhamiae (enzymes NitAn, NitNc, NitNh, and NitAb, respectively) exhibited different degrees of enantio- and chemoselectivity (amide formation). Their enantio- and chemoselectivity was increased by increasing pH (from 8 to 9-10) and adding 4-10% (v/v) toluene as the cosolvent. NitAn and NitNc were able to convert an up to 500 mM substrate in batch mode. NitAn formed a very low amount of the by-product, amide (<1% of the total product). This enzyme produced up to >70 g/L of (R)-mandelic acid (e.e. 94.5-95.6%) in batch or fed-batch mode. Its volumetric productivities were the highest in batch mode [571 ± 32 g/(L d)] and its catalyst productivities in fed-batch mode (39.9 ± 2.5 g/g of dcw). NitAb hydrolyzed both enantiomers of 100 mM (R,S)-mandelonitrile at pH 5.0 and is therefore promising for the enantioretentive transformation of (S)-mandelonitrile. Sequence analysis suggested that fungal arylacetonitrilases with similar properties (enantioselectivity, chemoselectivity) were clustered together.

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Section: Batch Production Of (R)-mandelic Acid In Biphasic Systemsmentioning

confidence: 42%

Section: Resultsmentioning

confidence: 99%

Section: Batch Production Of (R)-mandelic Acid In Biphasic Systemsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Exploring the Potential of Fungal Arylacetonitrilases in Mandelic Acid Synthesis

2015

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“…Fermentation of the I113M/ Y199G (MG) mutant (encoding the replacement of I with M at position 113 and Y with G at position 199) was performed as described previously (13) with modified fermentation medium (10 g/liter peptone, 5 g/liter yeast extract, 5 g/liter NaCl, 2 g/liter MgSO 4 , and 8 g/liter glycerol) and supplementary medium (10 g/liter peptone, 5 g/liter yeast extract, 5 g/liter NaCl, 2 g/liter MgSO 4 , and 20 g/liter glycerol).…”

Section: Methodsmentioning

confidence: 99%

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

Wang

Gao

Sun

et al. 2015

Appl Environ Microbiol

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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...

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Nitrile‐Converting Enzymes and their Synthetic Applications

Martı́nková

2016

Green Biocatalysis

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Efficient production of (R)-(−)-mandelic acid in biphasic system by immobilized recombinant E. coli

Cited by 33 publications

References 29 publications

Exploring the Potential of Fungal Arylacetonitrilases in Mandelic Acid Synthesis

Exploring the Potential of Fungal Arylacetonitrilases in Mandelic Acid Synthesis

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

Nitrile‐Converting Enzymes and their Synthetic Applications

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