Abstract
A nitrilase from Hoeflea phototrophica DFL-43 (HpN) demonstrating excellent catalytic activity towards benzoylacetonitrile was identified from a nitrilase tool-box, which was developed previously in our laboratory for (R)-o-chloromandelic acid synthesis from o-chloromandelonitrile. The HpN was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity by nickel column affinity chromatography, and its biochemical properties were studied. The HpN was very stable at 30–40 °C, and highly active over a wide range of pH values (pH 6.0–10.0). In addition, the HpN could tolerate against several hydrophilic organic solvents. Steady-state kinetics indicated that HpN was highly active towards benzoylacetonitrile, giving a KM of 4.2 mM and a kcat of 170 s−1, the latter of which is ca. fivefold higher than the highest record reported so far. A cascade reaction for the synthesis of optically pure (S)-β-phenylalanine from benzoylacetonitrile was developed by coupling HpN with an ω-transaminase from Polaromonas sp. JS666 in toluene-water biphasic reaction system using β-alanine as an amino donor. Various (S)-β-amino acids could be produced from benzoylacetonitrile derivatives with moderate to high conversions (73–99%) and excellent enantioselectivity (> 99% ee). These results are significantly advantageous over previous studies, indicating a great potential of this cascade reaction for the practical synthesis of (S)-β-phenylalanine in the future.
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Ankati H, Zhu D, Yang Y, Biehl ER, Hua L (2009) Asymmetric synthesis of both antipodes of β-hydroxy nitriles and β-hydroxy carboxylic acids via enzymatic reduction or sequential reduction/hydrolysis. J Org Chem 74:1658–1662
Banerjee A, Kaul P, Banerjee UC (2006) Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida. Arch Microbiol 184:407–418
Bea HS, Park HJ, Lee SH, Yun H (2011) Kinetic resolution of aromatic β-amino acids by transaminase. Chem Commun 47:5894–5896
Bornscheuer UT, Huisman GW, Kazlauskas RJ, Lutz S, Moore JC, Robins K (2012) Engineering the third wave of biocatalysis. Nature 485:185–194
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Chen S, Yuan F, Zhao H, Li B (2013) tert-BuOK-Catalyzed condensation of ethyl diazoacetate to aldehydes and palladium-catalyzed 1,2-hydrogen migration for the synthesis of β-ketoesters under solvent-free conditions. RSC Adv 3:12616–12620
DeSantis G, Wong K, Farwell B, Chatman K, Zhu Z, Tomlinson G, Huang H, Tan X, Bibbs L, Chen P, Kretz K, Burk MJ (2003) Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). J Am Chem Soc 125(38):11476–11477
Dong HP, Liu ZQ, Zheng YG, Shen YC (2010) Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyrate by Rhodococcus erythropolis. Appl Microbiol Biotechnol 87:1335–1345
Fawcett JK, Scott JE (1960) A rapid and precise method for the determination of urea. J Clin Pathol 13:156–159
Galliford CV, Scheidt KA (2008) An unusual dianion equivalent from acylsilanes for the synthesis of substituted β-keto esters. Chem Commun 16:1926–1928
Gavagan JE, Fager SK, Fallon RD, Folsom PW, Herkes FE, Eisenberg A, Hann EC, DiCosimo R (1998) Chemoenzymatic production of lactams from aliphatic α,ω-dinitriles. J Org Chem 63:4792–4801
Gong JS, Shi JS, Lu ZM, Li H, Zhou ZM, Xu ZH (2017) Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis: recent insights and promises. Crit Rev Biotechnol 37:69–81
Ito S, Ota A, Yamamoto K, Kawashima Y (1992) Resolution of the enantiomers of thiol compounds by reversed-phase liquid chromatography using chiral derivatization with 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate. J Chromatogr A 626:187–196
Jin M, Fischbach MA, Clardy J (2006) A biosynthetic gene cluster for the acetyl-CoA carboxylase inhibitor andrimid. J Am Chem Soc 128:10660–10661
Kim J, Kyung D, Yun H, Cho BK, Seo JH, Cha M, Kim BG (2007) Cloning and characterization of a novel β-transaminase from Mesorhizobium sp. strain LUK: a new biocatalyst for the synthesis of enantiomerically pure β-amino acids. Appl Environ Microbiol 73:1772–1782
Koszelewski D, Tauber K, Faber K, Kroutil W (2010) ω-Transaminases for the synthesis of non-racemic α-chiral primary amines. Trends Biotechnol 28:324–332
Li D, Ji L, Wang X, Wei D (2013) Enantioselective acylation of β-phenylalanine acid and its derivatives catalyzed by penicillin G acylase from Alcaligenes faecalis. Prep Biochem Biotechnol 43:207–216
Martinkova L, Kren V (2010) Biotransformations with nitrilase. Curr Opin Chem Biol 14:130–137
Martinkova L, Mylerova V (2003) Synthetic applications of nitrile-converting enzymes. Curr Org Chem 7:1279–1295
Mathew S, Jeong SS, Chung T, Lee SH, Yun H (2016a) Asymmetric synthesis of aromatic β-amino acids using ω-transaminase: Optimizing the lipase concentration to obtain thermodynamically unstable β-keto acids. Biotechnol J 11:185–190
Mathew S, Nadarajan SP, Chung T, Park HH, Yun H (2016b) Biochemical characterization of thermostable ω-transaminase from Sphaerobacter thermophilus and its application for producing aromatic β- and γ-amino acids. Enzyme Microb Technol 87–88:52–60
Mathew S, Nadarajan SP, Sundaramoorthy U, Jeon H, Chung T, Yun H (2017) Biotransformations of β-keto nitriles to chiral (S)-β-amino acids using nitrilase and ω-transaminase. Biotechnol Lett 39:535–543
Nagasawa T, Nakamura T, Yamada H (1990) Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase. Appl Microbiol Biotechnol 34:322–324
Ress-Loschke M, Friedrich T, Hauer B, Mattes R, Engels D (2005) Method for producing chiral carboxylic acids from nitriles with the assistance of a nitrilase or microorganisms which contain a gene for the nitrilase. US Patent, US 6,869,783 B1
Robertson DE, Chaplin JA, DeSantis G, Podar M, Madden M, Chi E, Richardson T, Milan A, Miller M, Weiner DP, Wong K, McQuaid J, Farwell B, Preston LA, Tan X, Snead MA, Keller M, Mathur E, Kretz PL, Burk MJ, Short JM (2004) Exploring nitrilase sequence space for enantioselective catalysis. Appl Environ Microbiol 70:2429–2436
Ruf S, Buning C, Schreuder H, Horstick G, Linz W, Olpp T, Pernerstorfer J, Hiss K, Kroll K, Kannt A, Kohlmann M, Linz D, Hubschle T, Rutten H, Wirth K, Schmidt T, Sadowski T (2012) Novel β-amino acid derivatives as inhibitors of cathepsin A. J Med Chem 55:7636–7649
Steer DL, Lew RA, Perlmutter P, Smith AI, Aguilar MI (2002) β-Amino acids: versatile peptidomimetics. Curr Med Chem 9:811–822
Stepanek E, Kubicek M, Marek M, Adler PM (1999) Optimal design and operation of a separating microreactor. Chem Eng Sci 54:1493–1498
Weiner B, Szymanski W, Janssen DB, Minnaard AJ, Feringa BL (2010) Recent advances in the catalytic asymmetric synthesis of β-amino acids. Chem Soc Rev 39:1656–1691
Weise NJ, Parmeggiani F, Ahmed ST, Turner NJ (2015) The bacterial ammonia lyase EncP: a tunable biocatalyst for the synthesis of unnatural amino acids. J Am Chem Soc 137:12977–12983
Weise NJ, Ahmed ST, Parmeggiani F, Turner NJ (2017) Kinetic resolution of aromatic β-amino acids using a combination of phenylalanine ammonia lyase and aminomutase biocatalysts. Adv Synth Catal 359:1–8
Wu S, Fogiel AJ, Petrillo KL, Jackson RE, Parker KN, DiCosimo R, Ben-Bassat A, O’Keefe DP, Payne MS (2008) Protein engineering of nitrilase for chemoenzymatic production of glycolic acid. Biotechnol Bioeng 99:717–720
Wu B, Szymanski W, de Wildeman S, Poelarends GJ, Feringa BL, Janssen DB (2010) Efficient tandem biocatalytic process for the kinetic resolution of aromatic β-amino acids. Adv Synth Catal 352:1409–1412
Xie Z, Feng J, Garcia E, Bernett M, Yazbeck D, Tao J (2006) Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid. J Mol Catal B-Enzym 41:75–80
Xu P, Zheng GW, Zong MH, Li N, Lou WY (2017) Recent progress on deep eutectic solvents in biocatalysis. Bioresour Bioprocess 4:34
Yamamoto K, Ueno Y, Otsubo K, Kawakami K, Komatsu K (1990) Production of S-(+)-ibuprofen from a nitrile compound by Acinetobacter sp. strain AK226. Appl Environ Microbiol 56:3125–3129
You P, Qiu J, Su E, Wei D (2013) Carica papaya lipase catalyzed resolution of β-amino esters for the highly enantioselective synthesis of (S)-Dapoxetine. Eur J Org Chem 2013:557–565
Zhang ZJ, Xu JH, He YC, Ouyang LM, Liu YY, Imanaka T (2010) Efficient production of (R)-(–)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp. Process Biochem 45:887–891
Zhang ZJ, Xu JH, He YC, Ouyang LM, Liu YY (2011a) Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. and its potential for (R)-(–)-mandelic acid production. Bioprocess Biosyst Eng 34:315–322
Zhang ZJ, Pan J, Liu JF, Xu JH, He YC, Liu YY (2011b) Significant enhancement of (R)-mandelic acid production by relieving substrate inhibition of recombinant nitrilase in toluene-water biphasic system. J Biotechnol 152:24–29
Zhang CS, Zhang ZJ, Li CX, Yu HL, Zheng GW, Xu JH (2012) Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata. Appl Microbiol Biotechnol 95:91–99
Zhang D, Chen X, Zhang R, Yao P, Wu Q, Zhu D (2015a) Development of β-amino acid dehydrogenase for the synthesis of β-amino acids via reductive amination of β-keto acids. ACS Catal 5:2220–2224
Zhang ZJ, Yu HL, Imanaka T, Xu JH (2015b) Efficient production of (R)-(–)-mandelic acid by isopropanol-permeabilized recombinant E. coli cells expressing Alcaligenes sp. nitrilase. Biochem Eng J 95:71–77
Zhu D, Mukherjee C, Biehl ER, Hua L (2007) Discovery of a mandelonitrile hydrolase from Bradyrhizobium japonicum USDA110 by rational genome mining. J Biotechnol 129:645–650
Funding
This study was funded by National Natural Science Foundation of China (nos. 21406067 and 21536004), Natural Science Foundation of Shanghai (no. 18ZR1408400), Fundamental Research Funds for the Central Universities (no. 22221818014) and Shanghai Commission of Science and Technology (no. 15JC1400403).
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Zhang, ZJ., Cai, RF. & Xu, JH. Characterization of a new nitrilase from Hoeflea phototrophica DFL-43 for a two-step one-pot synthesis of (S)-β-amino acids. Appl Microbiol Biotechnol 102, 6047–6056 (2018). https://doi.org/10.1007/s00253-018-9057-7
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DOI: https://doi.org/10.1007/s00253-018-9057-7