The influence of hydroxypropyl-β-cyclodextrin on the enantioselective hydrolysis of 2-amino phenylpropionitrile catalyzed by recombinant nitrilase
© Li and Wang; licensee Springer 2014
Received: 30 January 2014
Accepted: 19 May 2014
Published: 24 July 2014
Hydrolysis of 2-amino phenylpropionitrile by nitrilase is a fundamental biochemical reaction that produces chiral phenylalanine. For practical application of this biochemical reaction, researchers have attempted to improve enzyme enantioselectivity and the reaction rate.
The substrate concentration was increased from 100 to 200 mM without substrate inhibition because of the formation of a substrate-hydroxypropyl-β-cyclodextrin (HP-β-CD) complex. Meanwhile, the activity of recombinant nitrilase increased 2.5 times because the addition of HP-β-CD solubilized hydrophobic substrates in the aqueous system. Furthermore, the formation of the substrate-HP-β-CD inclusion improved the enantioselectivity of the enzymatic reaction toward producing l-phenylalanine (l-Phe). The enantiomeric excess (e.e.) value of l-Phe increased from 65% to 83% when the conversion rate reached 50%.
The recombinant nitrilase enantioselectively hydrolyzed 2-amino phenylpropionitrile to produce l-Phe. The addition of HP-β-CD to the reaction system enhanced the solubility and bioavailability of hydrophobic substrates as well as the enantioselectivity. The results showed that this additive has potential advantages in biochemical reactions of hydrophobic substrates, particularly for enantioselective biosynthesis.
KeywordsHP-β-CD Recombinant nitrilase 2-Amino phenylpropionitrile Enantioselectivity e.e. value
l-Phenylalanine (l-Phe) is an essential amino acid generally used in food industries, human nutrients, and pharmaceuticals. For example, it is a precursor of some anticancer drugs and the dipeptide sweetener aspartame []. In early industrial processes, l-Phe was mainly produced by chemical synthesis. Because of the specific demand for the stereospecific form and the consideration of eco-friendly chemical synthesis, this approach was gradually replaced with bioprocesses, such as microbial fermentation and enzymatic transformation [].
Nitrilase (EC 220.127.116.11) is an enzyme that converts nitrile to its carboxylic acid or amide []. Some important chiral pharmaceutical intermediates and bulk products, such as acrylic acid [], (R)-(−)-mandelic acid [], 3-hydroxyvaleric acid [], and nicotinic acid [], are produced by nitrile hydrolysis. For example, (R)-(−)-mandelic acid, widely used for the production of semisynthetic cephalosporins and anti-obesity agents, is produced by hydrolyzing mandelonitrile [,]. In addition, other studies focused on strategic optimizations for increasing the reaction rate, reducing substrate inhibition, and improving enzyme enantioselectivity [–].
Cyclodextrin (CD) is the generic term for cyclic oligosaccharides that are used frequently as host molecules in supramolecular chemistry. Despite the outside of the CD molecule being hydrophilic, CD contains a hydrophobic cavity that entraps most hydrophobic molecules to form inclusion complexes []. Thus, it improves the solubility and bioavailability of hydrophobic compounds. Its use is of interest for reactions in which hydrophobic compounds are to be delivered. In previous reports, CD has been proven to increase the availability of insoluble substrates, reduce substrate inhibition, and enhance the efficiency of catalysis by increasing the reaction rate in other catalytic reactions []. It has also been used in an enzymatic enantioselective reaction to increase the enantiomeric excess (e.e.) value of the product [].
In our previous study, a novel nitrilase from Rhodobacter sp. LHS-305 was cloned and expressed in Escherichia coli []. This nitrilase displayed high activity toward both aliphatic and aromatic nitriles, similar to the nitrilase from Rhodococcus rhodochrous ATCC 33278 []. It also showed regioselectivity toward dinitriles to produce cyanocarboxylic acids. Because this nitrilase shows properties different from those of typical nitrilases, it potentially has industrial applications in the future.
In this study, the stereoselective hydrolysis of 2-amino phenylpropionitrile by this novel nitrilase was investigated. The influences of CD on this hydrolysis in terms of substrate inhibition and the e.e. value of the product were found to improve the catalytic reaction. The investigations comprehensively increased our knowledge of this unique nitrilase for its application.
Materials and methods
Hydroxypropyl-β-cyclodextrin (HP-β-CD) (≥98.5%) was purchased from Shanghai Lingfeng Chemical Reagent Company (Shanghai, China), biochemical-grade l-Phe from Sigma-Aldrich (St. Louis, MO, USA), and glucose and other chemicals from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
Microorganism and culture media
Recombinant E. coli expressing a novel nitrilase gene from Rhodobacter sphaeroides LHS-305 were used in this study.
Seed medium (g/L) contained peptone 10, yeast extract 5, and NaCl 10, pH 7.0 to 7.5.
Fermentation medium (g/L) contained yeast extract 10, peptone 5, NaCl 5, glucose 3, and MgSO4·7H2O 3, pH 7.0 to 7.5. Media were sterilized for 20 min at 115°C.
Precultures cultured in the seed medium for 3 to 4 h at 37°C were inoculated into 100 mL of fermentation medium in a 500-mL Erlenmeyer flask, with the addition of α-lactose (1 g/L), and cultured on a shaker at 200 rpm and 20°C for 12 h.
Preparation of recombinant nitrilase
The recombinant nitrilase was purified by affinity chromatography as described in our previous report [].
Preparation of the 2-amino phenylpropionitrile/HP-β-CD complex
2-Amino phenylpropionitrile (73 mg) was added to 100 μL methanol to obtain a 5 M substrate stock solution. The stock solution was added to 50 mM PB solution (pH 7.0) with HP-β-CD and stirred for 20 min at 40°C. This yielded the 2-amino phenylpropionitrile/HP-β-CD inclusion complex.
Assay of enzyme activity toward bioconversion of 2-amino phenylpropionitrile
The catalytic reaction was performed in 1 mL of sodium phosphate buffer (50 mM, pH 7.0) containing the substrate 2-amino phenylpropionitrile (20 mM, final concentration) and nitrilase (1 mg/mL, final concentration). Reaction mixtures were incubated at 40°C for 10 min, and reactions were quenched by the addition of 10% (v/v) 1 mol/L HCl. Enzyme activity (U) was defined as the amount of enzyme required for the hydrolysis of l μmol of the 2-amino phenylpropionitrile substrate to the corresponding acid within 1 min. All experiments were performed in triplicate.
Analysis of l-Phe
The enantiomeric purity of Phe was determined by reversed-phase HPLC (Agilent, Santa Clara, CA, USA) equipped with a Chirobiotic T column (Sigma-Aldrich Co.) at a flow rate of 0.5 mL/min with a solvent system (75:25, v/v) of phosphate buffer (25 mM, adjusted to pH 3.5 with H3PO4) and methanol. Peaks were detected using an ultraviolet detector at 210 nm.
Results and discussion
Effects of HP-β-CD addition on the catalytic reaction
Effect of HP-β-CD inclusions on substrate inhibition
Effect of HP-β-CD inclusion on the e.e. value of the product
The recombinant nitrilase enantioselectively hydrolyzed 2-amino phenylpropionitrile to produce l-Phe. Using the HP-β-CD-substrate reaction system, the reaction rate was greatly improved by enhancing the solubility and bioavailability of the hydrophobic substrate. Meanwhile, the e.e. value of the product was also improved significantly because of the formation of inclusion complexes. In this manner, HP-β-CD enhanced both the efficiency of the catalytic reaction and the optical purity of the product. The properties of CDs to form inclusion complexes with hydrophobic molecules led to their practical application in a biochemical reaction with a hydrophobic substrate. In particular, they were used to promote enantioselective biosynthesis.
We thank Guinan Li and Hualei Wang who provided the fundamental works, such as clone and expression of the novel nitrilase in E. coli.
- Zhou H, Liao X, Wang T, Du G, Chen J: Enhanced L-phenylalanine biosynthesis by co-expression of pheAfbr and aroFwt. Bioresour Technol 2010, 101: 4151–4156. 10.1016/j.biortech.2010.01.043View ArticleGoogle Scholar
- Liu DX, Fan CS, Tao JH, Liang GX, Gao SE, Wang HJ, Li X, Song DX: Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on L-phenylalanine biosynthesis. World J Gastroenterol 2004, 10: 3683–3687.Google Scholar
- Fernandes BCM, Mateo C, Kiziak C, Chmura A, Wacker J, van Rantwijk F, Stolz A, Sheldon RA: Nitrile hydratase activity of a recombinant nitrilase. Adv Synth Catal 2006, 348: 2597–2603. 10.1002/adsc.200600269View ArticleGoogle Scholar
- Kamal A, Kumar MS, Kumar CG, Shaik TB: Bioconversion of acrylonitrile to acrylic acid by Rhodococcus ruber strain AKSH-84. J Microbiol Biotechnol 2011,21(1):37–42. 10.4014/jmb.1006.06044View ArticleGoogle Scholar
- Banerjee A, Dubey S, Kaul P, Barse B, Piotrowski M, Banerjee UC: Enantioselective nitrilase from Pseudomonas putida : cloning, heterologous expression, and bioreactor studies. Mol Biotechnol 2009,41(1):35–41. 10.1007/s12033-008-9094-zView ArticleGoogle Scholar
- Wu SJ, Fogiel AJ, Petrillo KL, Hann EC, Mersinger LJ, DiCosimo R, O'Keefe DP, Ben-Bassat A, Payne MS: Protein engineering of Acidovorax facilis 72W nitrilase for bioprocess development. Biotechnol Bioeng 2007, 97: 689–693. 10.1002/bit.21289View ArticleGoogle Scholar
- Yang CS, Wang XD, Wei DZ: A new nitrilase-producing strain named Rhodobacter sphaeroides LHS-305: biocatalytic characterization and substrate specificity. Appl Biochem Biotechnol 2011, 165: 1556–1567. 10.1007/s12010-011-9375-zView ArticleGoogle Scholar
- Zhang ZJ, Xu JH, He YC, Ouyang LM, Liu YY: Cloning and biochemical properties of a highly thermostable and enantioselective nitrilase from Alcaligenes sp. ECU0401 and its potential for (R)-(−)-mandelic acid production. Bioproc Biosyst Eng 2013,34(3):315–322. 10.1007/s00449-010-0473-zView ArticleGoogle Scholar
- Sosedov O, Matzer K, Bürger S, Kiziak C, Baum S, Altenbuchner J, Chmura A, van Rantwijk F, Stolz A: Construction of recombinant Escherichia coli catalysts which simultaneously express an (S)-oxynitrilase and different nitrilase variants for the synthesis of (S)-mandelic acid and (S)-mandelic amide from benzaldehyde and cyanide. Adv Synth Catal 2009, 351: 1531–1538. 10.1002/adsc.200900087View ArticleGoogle Scholar
- Zhang ZJ, Pan J, Liu JF, Xu JH, He YC, Liu YY: Significant enhancement of (R)-mandelic acid production by relieving substrate inhibition of recombinant nitrilase in toluene-water biphasic system. J Biotechnol 2011, 152: 24–29. 10.1016/j.jbiotec.2011.01.013View ArticleGoogle Scholar
- Qiu J, Su EZ, Wang W, Wei DZ: High yield synthesis of D-phenylglycine and its derivatives by nitrilase mediated dynamic kinetic resolution in aqueous-1-octanol biphasic system. Tetrahedron Lett 2014,55(8):1448–1451. 10.1016/j.tetlet.2014.01.044View ArticleGoogle Scholar
- Liu L, Guo QX: Use of quantum chemical methods to study cyclodextrin chemistry. J Incl Phenom Macro Chemistry 2004,50(1–2):95–103. 10.1007/s10847-003-8847-3View ArticleGoogle Scholar
- Yue HY, Yuan QP, Wang WH: Enhancement of L-phenylalanine production by β-cyclodextrin. J Food Eng 2007, 79: 878–884. 10.1016/j.jfoodeng.2006.03.007View ArticleGoogle Scholar
- Mine Y, Zhang L, Fukunaga K, Sugimura Y: Enhancement of enzyme activity and enantioselectivity by cyclopentyl methyl ether in the transesterification catalyzed by Pseudomonas cepacia lipase co-lyophilized with cyclodextrins. Biotechnol Lett 2005,27(6):383–388. 10.1007/s10529-005-1527-1View ArticleGoogle Scholar
- Wang HL, Li GN, Li MY, Wei DZ, Wang XD: A novel nitrilase from Rhodobacter sphaeroides LHS-305: cloning, heterologous expression and biochemical characterization. World J Microbiol Biotechnol 2014, 30: 245–252. 10.1007/s11274-013-1445-7View ArticleGoogle Scholar
- Yeom SJ, Kim HJ, Lee JK, Kim DE, Oh DK: An amino acid at position 142 in nitrilase from Rhodococcus rhodochrous ATCC 33278 determines the substrate specificity for aliphatic and aromatic nitriles. Biochem J 2008, 415: 401–407. 10.1042/BJ20080440View ArticleGoogle Scholar
- Shen Y, Wang M, Zhang L, Ma Y, Ma B, Zheng Y, Liu H, Luo JM: Effects of hydroxypropyl-β-cyclodextrin on cell growth, activity, and integrity of steroid-transforming Arthrobacter simplex and Mycobacterium sp . Appl Microbiol Biotechnol 2011,90(6):1995–2003. 10.1007/s00253-011-3214-6View ArticleGoogle Scholar
- Xue YP, Liu ZQ, Xu M, Wang YJ, Zheng YG, Shen YC: Enhanced biotransformation of (R, S)-mandelonitrile to (R)-(−)-mandelic acid with in situ production removal by addition of resin. Biochem Eng J 2010, 53: 143–149. 10.1016/j.bej.2010.10.009View ArticleGoogle Scholar
- Gröger H: Enzymatic routes to enantiomerically pure aromatic α-hydroxy carboxylic acids: a further example for the diversity of biocatalysis. Adv Synth Catal 2001, 343: 547–558. 10.1002/1615-4169(200108)343:6/7<547::AID-ADSC547>3.0.CO;2-AView ArticleGoogle Scholar
- Tang K, Miao JB, Zhou T, Liu YB, Song LT: Reaction kinetics in reactive extraction for chiral separation of α-cyclohexyl-mandelic acid enantiomers with hydroxypropyl-β-cyclodextrin. Chem Eng Sci 2011,66(3):397–404. 10.1016/j.ces.2010.10.044View ArticleGoogle Scholar
- Aree T, Arunchai R, Koonrugsa N: Fluorometric and theoretical studies on inclusion complexes of β-cyclodextrin and D-, L-phenylalanine. Spectrochim Acta A 2012, 96: 736–743. 10.1016/j.saa.2012.07.049View ArticleGoogle Scholar
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