摘要
光学纯的α-羟基酸是多种药物合成的通用中间体及重要的精细化学品。其中,光学纯的扁桃酸及其取代衍生物作为最重要的代表,在工业领域已得到非常广泛的应用。(R)-(-)-扁桃酸被广泛用于头孢菌素、青霉素、抗肿瘤药物及减肥药物等的合成中。(R)-(-)-扁桃酸还可以作为酸性手性拆分剂用于外消旋醇类和胺类的手性拆分。(R)-(-)-邻氯扁桃酸可用于合成新型安全高效的抗血小板聚集药物氯吡格雷(clopidogrel)。在各种化学和酶法合成芳香族α-羟基酸的途径中,腈水解酶因其能动力学拆分廉价易得的α-羟基腈(氰醇)生成光学纯的α-羟基酸,具有100%的理论产率,且反应无副产物生成,使其在工业上具有重要的应用价值。
本课题引入了基于系统进化分析的底物特异性预测方法,结合传统的基因组探矿技术,高效的从数据库中挖掘到了一个来源于Burkholderia cenocepacia J2315的新型芳基乙腈类腈水解酶BCJ2315。酶学性质研究发现,该腈水解酶对扁桃腈具有较高的活力、高度对映体选择性及高的底物耐受性,在水解扁桃腈生成光学纯的(R)-(-)-扁桃酸方面展现了较高的潜力。通过对反应条件、反应模式的优化及对BCJ2315进行蛋白质工程改造,本课题实现了(R)-(-)-扁桃酸和(R)-(-)-邻氯扁桃酸的高对映体选择性及高浓度的生产,为实现其工业化生产打下了坚实的基础。主要工作结果如下：
第一,在传统的基因组探矿技术上,本课题引入了基于系统进化分析的底物特异性预测方法,从基因组数据库中特异性地挖掘到7个新型扁桃腈腈水解酶。通过水解扁桃腈试验,7个腈水解酶都表现出了较高的对映体选择性。其中,来源于Burkholderia cenocepacia J2315的腈水解酶BCJ2315对扁桃腈表现出了最高的比活(27.79U/mg)和最高的对映体选择性(98.4%),且反应过程中无副产物生成,展现出了较高的应用潜力。氨基酸序列分析显示,BCJ2315与别的酶序列一致性最高只有71%,是一个新型的腈水解酶。由于其对扁桃腈的高活力及高对映体选择性,BCJ2315被选定做进一步的研究。
第二,本节对腈水解酶BCJ2315的酶学性质进行了系统的研究。SDS-PAGE显示BCJ2315由一个分子量约为38kDa的单体组成,通过凝胶过滤层析测定腈水解酶BCJ2315全酶的分子量为450kDa,表明BCJ2315由12个相同的单体构成。BCJ2315的最适温度和pH分别为45℃和8.0。BCJ2315具有非常宽的底物谱,能够水解绝大多数的腈类底物。在所有24种被检测的底物中,BCJ2315表现出了对芳基乙腈类底物的偏好性,且对扁桃腈展现了最高的水解活力,是一个芳基乙腈类的腈水解酶。与已报道的以苯乙腈为最适底物的高对映体选择性的芳基乙腈类水解酶不同,BCJ2315水解扁桃腈的活力为芳基乙腈类水解酶模式底物苯乙腈的8.8倍,因此是一个真正意义上的扁桃腈腈水解酶。BCJ2315对脂肪腈和杂环腈也表现出了较低的水解活力,但对芳香腈(苯甲腈)类底物没有活性。动力学参数研究表明,BCJ2315对扁桃腈具有很强的亲和力,Km值仅有0.14mM,低于已报道的对扁桃腈具有高度对映体选择性的腈水解酶1-3个数量级。BCJ2315同样对扁桃腈具有很高的催化效率,Kcat和Kcat/Kmm值分别为15.4s-1和1.1×105M-1s-1。这些结果表明,腈水解酶BCJ2315在水解扁桃腈生成光学纯的(R)-(-)-扁桃酸方面具有非常高的应用潜力。
第三,本节考察了重组腈水解酶BCJ2315细胞水解扁桃腈生成(R)-(-)-扁桃酸的应用潜力。在底物耐受性方面,腈水解酶BCJ2315重组菌M15/BCJ2315对扁桃腈表现出了较高的底物耐受性。它可以以较低的菌体量(10mg/mL湿菌体)在1小时内将100mM的底物扁桃腈完全转化完,产物ee值为97.6%。在重复使用批次方面,重组菌M15/BCJ2315可以重复使用14个批次而没有任何活力的丧失,表现出了非常好的批次稳定性。为减少单一水相反应体系中高浓度底物扁桃腈对反应的抑制,实现高浓度产物(R)-(-)-扁桃酸的生产,本节建立了水/乙酸乙酯两相体系。在水/乙酸乙酯(90：10)两相体系中,4h时间里累积产生了500mM的(R)-(-)-扁桃酸,产物ee值为97.1%,有效地减轻了底物抑制效应并提高了产物浓度。同时本节也考察了单一水相体系中批次补料反应模式在生产高浓度(R)-(-)-扁桃酸时的效果。终浓度100mM的扁桃腈底物分8批加入反应体系中,18h的反应时间里共产生了744mM的产物(R)-(-)-扁桃酸。在随后改进的连续补料反应模式中,通过连续流加底物和控制反应pH使酶的活力始终保持在一个较高的水平。在300mL的反应体系中,21h的反应时间里共累积产生了2.1M的扁桃酸,产物ee值为97.6%。反应规模提高到20L后,由于底物与酶的混合效果提高了,累积生成2.1M扁桃酸的反应时间缩短到18.5h,产物ee值为97.6%。通过对反应条件及反应模式的优化,(R)-(-)-扁桃酸的累计生产浓度达到了2.1M,为目前文献所报道的腈水解酶生产(R)-(-)-扁桃酸的最高水平,为进一步实现其工业化奠定了良好的基础。
第四,本节以邻氯扁桃腈作为底物,从酶库的筛选、新酶挖掘、溶剂工程及蛋白质工程四个方面最终实现了腈水解酶对映体选择性催化邻氯扁桃腈生成光学纯的(R)-(-)-邻氯扁桃酸。首先对筛选扁桃腈腈水解酶时得到的7个腈水解酶及10个新筛选的腈水解酶进行邻氯扁桃腈水解试验,检测它们对邻氯扁桃腈的对映体选择性。结果显示,来源于Labrenzia aggregata的腈水解酶LaN表现出了最高的对映体选择性(96.3%),但活力较低。且在本课题进行的时候,LaN已经被报道。其余的腈水解酶对邻氯扁桃腈都表现出了较低的对映体选择性,产物ee值均低于90%。在水解邻氯扁桃腈生成邻氯扁桃酸的过程中,虽然BCJ2315的对映体选择性较低(89.2%),但其比活力是所有被检测的腈水解酶中最高的,因此本节选择BCJ2315作为催化邻氯扁桃腈的候选催化剂,并通过溶剂工程和蛋白质工程从反应条件和基因水平两个方面提高其对邻氯扁桃腈的对映体选择性。在反应条件方面,反应温度和pH的提高都有利于提高腈水解酶BCJ2315的对映体选择性。7种水溶性有机溶剂的加入也有效的提高了BCJ2315的对映体选择性,且同时提高了酶的活力。通过向反应中添加30%乙醇做助溶剂,产物ee值可提高到98.2%。因此本节采用底物连续流加的方法,通过连续流加溶解在乙醇中的邻氯扁桃腈来不断提高反应体系中乙醇的浓度,以达到提高反应对映体选择性的目的。在100mL反应体系中,反应8个小时,共累计生成了415mM的(R)-(-)-邻氯扁桃酸,产物的ee值提高到96.7%。在蛋白质工程方面,通过随机突变技术结合定点突变技术,本节成功得到了相对活力及对映体选择性都有很大提高的腈水解酶BCJ2315的突变体。突变体含有两个突变位点,分别是1113M和Y199G。突变体水解邻氯扁桃腈的相对酶活为野生型的3.76倍,产物ee值从野生型的89.2%提高到98.7%。利用乙酸乙酯/水两相体系,完全水解500mM的邻氯扁桃腈的反应时间从野生型的7h缩短到3h,产物ee值为97.2%。本节成功实现了(R)-(-)-邻氯扁桃酸的高对映体选择性和高浓度生产,为其工业化生产奠定了良好的基础。本节同时构建了影响腈水解酶BCJ2315对映体选择性的四个关键位点的饱和突变体文库,有利于后续腈水解酶的立体选择性机制研究工作的开展。
Optically pure a-hydroxylcarboxylic acids are versatile intermediates for the production of various pharmaceuticals and fine chemicals. Among them, optically pure mandelic acid and substituted derivatives are regarded as the most important representatives.(R)-(-)-mandelic acid is widely used for the production of semisynthetic cephalosporins, penicillins, antitumor agents and antiobesity agents. It is also used as an universal acidic chiral resolving agent for the resolution of racemic alcohols and amines.(R)-o-chloromandelic acid is the key precursor for the synthesis of Clopidogrel, a platelet aggregation inhibitor widely administered to atherosclerotic patients with the risk of a heart attack or stroke that are caused by the formation of a clot in the blood. Many chemical and enzymatic approaches to produce optically pure aromatic a-hydroxy carboxylic acid have been developed. Among them, nitrilase-mediated pathway becomes increasingly popular because of the absence of the cofactor involvement, cheap starting material in the form of mandelonitrile, high enantioselectivity and theoretically100%of the product.
In this thesis, a novel arylacetonitrilase (BCJ2315) was efficiently discovered from Burkholderia cenocepacia J2315by phylogeny-based enzymatic substrate specificity prediction (PESSP) combination with traditional genome mining technology. BCJ2315demonstrated higher specificity, enantioselectivity and substrate tolarence based on the study of enzymatic properties. Thus BCJ2315showed great potential in the production of (R)-(-)-mandelic acid by hydrolysis of mandelonitrile. The optimization of the reaction conditions and reaction mode were succefully performed to achieve the production of (R)-(-)-mandelic acid and (R)-o-chloromandelic acid in a high concentration and enantioselectivity. Protein engineering of BCJ2315was also used to achieve the production (R)-o-chloromandelic acid in a high concentration and enantioselectivity. The obtained results laid a solid foundation for the industrial production of (R)-(-)-mandelic acid and (R)-o-chloromandelic acid. The results were as fllows:
First, seven mandelonitrile hydrolases were discovered from GenBank by phylogeny-based enzymatic substrate specificity prediction (PESSP) combination with traditional genome mining technology. All of them showed relatively high enantioselectivity toward mandelonitrile. Among them, the nitrilase BCJ2315from Burkholderia cenocepacia J2315demonstrated the highest specificity (27.79U/mg) and the highest enantioselectivity (98.4%). No byproduct was observed in the biotransformation. The highest identity was71%compared with other enzymes in amino acid sequence to demonstrate that BCJ2315is a novel nitrilase. BCJ2315was chosen for further study because of its high specific activity and enantioselectivity.
Second, We carried out a systematic study on the enzyme properties of the BCJ2315. BCJ2315showed one single band on the SDS-PAGE with a molecular weight of38kDa. The molecular weight of the purified native BCJ2315estimated by conducting a gel filtration chromatography was about450kDa. This result indicated that the native BCJ2315consisted of12subunits with identical size. The optimum temperature and pH of the purified BCJ2315were45℃and8.0, respectively. BCJ2315showed a very broad substrate spectrum, it could hydrolyze most of the24assayed nitriles. A clear preference of BCJ2315for arylacetonitriles as substrates indicated that this enzyme is an arylacetonitrilase. A lower activity was also observed with the aliphatic and heterocyclic nitriles. No detectable activities were observed with the aromatic nitriles. Different from other reported highly enantioselctive arylacetonitrilases which had phenylacetonitrile as their optimal substrate, BCJ2315showed the highest activity toward mandelonitrile (8.8times more than that of phenylacetonitrile), indicating that BCJ2315is a highly active mandelonitrile hydrolase. The low value of Km indicated that BCJ2315had high affinity toward mandelonitrile. The Km-values of other highly enantioselective mandelonitrile hydrolases were one to three orders of magnitude higher than that of BCJ2315. BCJ2315also had a high catalytic efficiency. The Kcat and Kcat/Km were15.4s-1and1.1×105M-1s-1, respectively. These results demonstrated great potential of BCJ2315in hydrolyzing mandelonitrile to produce optically pure (R)-(-)-mandelic acid.
Third, the catalytic efficiency of the recombinant E. coli M15/BCJ2315was also tested in hydrolyzing mandelonitrile to produce (R)-(-)-mandelic acid to further investigate the potential of BCJ2315. M15/BCJ2315had a strong substrate tolerance and could completely hydrolyze mandelonitrile (100mM) with fewer amounts of wet cells (10mg/ml) within1h. The ee value of the product was97.6%. M15/BCJ2315could be reused up to14cycles without any loss of activity and thus was very stable. An ethyl acetate-water biphasic system was built to relieve the substrate inhibition and improve the yield of (R)-(-)-mandelic acid. A total of500mM (R)-(-)-mandelic acid was produced in ethyl acetate-water biphasic system (10%v/v) within4h with a high enantiomeric excess of97.1%, which efficiently relieved the substrate inhibition and improved the yield of (R)-(-)-mandelic acid. To achieve production of (R)-(-)-mandelic acid in high concentration, a fed-batch mode was also used. Mandelonitrile was fed into the reaction system to a final concentration of100mM when the mandelonitrile in the reaction was exhausted. After8batches, the cumulative concentration of744mM (R)-(-)-mandelic acid was produced in18h. In an modified fed-batch mode, we make the activity of BCJ2315in a high level by continuous substrate feeding and control of reaction pH. In300ml of reaction scale, the accumulation concentration of (R)-(-)-mandelic acid reached2.1M after18h of biotrasformation with97.6%ee value. When the reaction was scaled up to30L, the reaction time was shortened to18.5h when the concentration of product reached2.1M as the improved mix effect between enzyme and substrate, giving a ee value of97.6%. Through the optimization of the reaction conditions and reaction mode, the concentration of (R)-(-)-mandelic acid has been raised to2.1M, which is higer than that of other reports. The obtained results had laid a good foundation for further industry applications.
Fourth, screening the existing enzyme library, mining new enzymes, solvent engineering and protein engineering were used to achieve high enantioselective production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile. Seven mandelonitrile hydrolases from the existing enzyme library and ten new enzymes were used to measure their enantioselectivity toward o-chloromandelonitrile. The nitrilase from Labrenzia aggregata showed the highest enantioselectivity (96.3%).Unfortunately, when our work was in progress, this nitrilase was reported. The nitrilase showed a relative low specific activity toward o-chloromandelonitrile. The rest of the nitrilases demonstated low enantioselectivity toward o-chloromandelonitrile with ee values no more than90%. The nitrilase BCJ2315still showed the highest specific activity in the hydrolysis of o-chloromandelonitrile to (R)-o-chloromandelic acid, thus it was chosen as the candidate catalyst. Solvent and protein engineering were used to improve the BCJ2315's enantioselectivity toward o-chloromandelonitrile from the reaction conditions and amino acid level. Improving the reaction temperature and pH was conducive to improving the BCJ2315's enantioselectivity. Seven kinds of water-soluble organic solvents were great helpful to improving the BCJ2315's enantioselectivity and activity when added into the reaction mixture. When30%of ethanol as co-solvents was added into the reaction mixture, the ee value of product could be increased to98.2%. A continuous substrate feeding mode was used to produce high concentration of (R)-o-chloromandelic acid. The substrate o-chloromandelonitrile dissolved in ethanol was fed continuously to improve the concentration of ethanol in the reaction system in order to increase the enantioselectivity of the reaction. The cumulative concentration of product was415mM (R)-o-chloromandelic acid after8h of hydrolysis with ee value of96.7%in100mL reaction system. Through the random mutations technology combined with site-directed mutagenesis technology, we have successfully obtained the mutant of the nitrilase BCJ2315which had a higher relative activity and enantioselectivity toward o-chloromandelonitrile compared with those of the nitrilase BCJ2315. The mutant contained two mutations, I113M and Y199G. The relative activity of the mutant was three times more than that of wild type. The ee value of the product was increased to98.7%from89.2%of the wild-type. Using ethyl acetate/water two-phase system, the reaction time of fully hydrolyzing500mM o-chloromandelonitrile was shortened to3h, compated with7h of the wild-type.The ee value of the final product was97.2%. The mutant could produce (R)-(-)-chloromandelic acid in high enantioselectivity and concentration, therefore had great potential in the industrial production of (R)-(-)-chloromandelic acid. Four key' hot spots' had been identified that had great influence on the enantioselectivity of BCJ2315and saturated mutant library has been constructed at these four sites. They offered great opportunity to study the mechanism of the enantioselectivity of the nitrilase BCJ2315.
引文
[1]Groger H. Enzymatic routes to enantiomerically pure aromatic alpha-hydroxy carboxylic acids:A further example for the diversity of biocatalysis. Advanced Synthesis & Catalysis.2001,343(6-7):547-558.
[2]Yamamoto K, Oishi K, Fujimatsu I, Komatsu K. Production of R-(-)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750. Applied and environmental microbiology.1991,57(10):3028-3032.
[3]Wang H, Sun H, Wei D. Discovery and characterization of a highly efficient enantioselective mandelonitrile hydrolase from Burkholderia cenocepacia J2315 by phylogeny-based enzymatic substrate specificity prediction. BMC biotechnology. 2013,13:14.
[4]Mateo C, Chmura A, Rustler S, van Rantwijk F, Stolz A, Sheldon RA. Synthesis of enantiomerically pure (S)-mandelic acid using an oxynitrilase-nitrilase bienzymatic cascade:a nitrilase surprisingly shows nitrile hydratase activity. Tetrahedron-Asymmetry.2006,17(3):320-323.
[5]Zhang CS, Zhang ZJ, Li CX, Yu HL, Zheng GW, Xu JH. Efficient production of (R)-o-chloromandelic acid by deracemization of o-chloromandelonitrile with a new nitrilase mined from Labrenzia aggregata. Applied microbiology and biotechnology. 2012,95(1):91-99.
[6]Hummel W. New alcohol dehydrogenases for the synthesis of chiral compounds. Advances in biochemical engineering/biotechnology.1997,58:145-184.
[7]Takao M., Masakatsu F. Process for producing D-mandelic acid from benzoylformic acid.US5441888.
[8]郭黛苹,许小平等.R-扁桃酸的不对称生物合成.医药工业杂志.2007,8(7)：484-488
[9]肖美添,黄雅燕等.生物不对称合成R-扁桃酸的影响因素[J].过程工程学报.2004,4(1)：32-36.
[10]Hummel, W., Schutte, H., Kula, M.-R. D-(-)-Mandelic acid dehydrogenase from Lactobacillus curvatus. Appl. Microbiol Biotechnol.1988,28:433-439.
[11]高静,贺莹,姜艳军.扁桃酸的酶促光学拆分[J].化工学报.2006,57(12)：12926-2932.
[12]韦晗宁,申佩弘,石玲玲,武波对映选择性Geotrichum sp. GXU33脂肪酶的筛选、产酶及酶学性质研究.工业微生物.2008,38(1)：30-36.
[13]Glieder A, Weis R, Skranc W, Poechlauer P, Dreveny I, Majer S, Wubbolts M, Schwab H, Gruber K. Comprehensive step-by-step engineering of an (R)-hydroxynitrile lyase for large-scale asymmetric synthesis. Angewandte Chemie-International Edition.2003, 42(39):4815-4818.
[14]Effenberger F, Forster S, Wajant H. Hydroxynitrile lyases in stereoselective catalysis. Current opinion in biotechnology.2000, 11(6):532-539.
[15]Kato Y, Ooi R, Asano Y. Distribution of aldoxime dehydratase in microorganisms. Applied and environmental microbiology.2000,66(6):2290-2296.
[16]Asano Y, Kato Y. Z-Phenylacetaldoxime degradation by a novel aldoxime dehydratase from Bacillus sp.strain OxB-1. FEMS microbiology letters.1998,158:185-190.
[17]Kato Y, Ooi R, Asano Y. Isolation and characterization of a bacterium possessing a novel aldoximedehydration activity and nitrile-degrading enzymes. Arch Microbiology. 1998,170:85-90.
[18]Kato Y, Ooi R, Asano Y. A new enzymatic method of nitrile synthesis by Rhodococcus sp. strain YH3-3. Journal of Molecular Catalysis B-Enzymatic.1999,6(3):249-256.
[19]Kato Y, Ooi R, Asano Y. Distribution of aldoxime dehydratase in microorganisms. Applied and environmental microbiology.2000,66(6):2290-2296.
[20]Kato Y, Nakamura K, Sakiyama H, Mayhew SG, Asano Y. Novel heme-containing lyase, phenylacetaldoxime dehydratase from Bacillus sp, strain OxB-1:Purification, characterization, and molecular cloning of the gene. Biochemistry.2000, 39(4):800-809.
[21]Asano Y. Overview of screening for new microbial catalysts and their uses in organic synthesis-selection and optimization of biocatalysts. Journal of biotechnology.2002, 94(1):65-72.
[22]Brenner C. Catalysis in the nitrilase superfamily. Current opinion in structural biology. 2002,12(6):775-782.
[23]Pace HC, Brenner C. The nitrilase superfamily:classification, structure and function. Genome biology.2001,2(1).
[24]Raczynska JE, Vorgias CE, Antranikian G, Rypniewski W. Crystallographic analysis of a thermoactive nitrilase. Journal of structural biology.2011,173(2):294-302.
[25]Thuku RN, Weber BW, Varsani A, Sewell BT. Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous Jl yields a stable, active helical form. The FEBS journal.2007,274(8):2099-2108.
[26]Kim JS, Tiwari MK, Moon HJ, Jeya M, Ramu T, Oh DK, Kim IW, Lee JK. Identification and characterization of a novel nitrilase from Pseudomonas fluorescens Pf-5. Applied microbiology and biotechnology.2009,83(2):273-283.
[27]O'Reilly C, Turner PD. The nitrilase family of CN hydrolysing enzymes-a comparative study. Journal of applied microbiology.2003,95(6):1161-1174.
[28]Martinkova L, Kren V. Biotransformations with nitrilases. Current opinion in chemical biology.2010,14(2):130-137.
[29]Fernandes BCM, Mateo C, Kiziak C, Chmura A, Wacker J, van Rantwijk F, Stolz A, Sheldon RA. Nitrile hydratase activity of a recombinant nitrilase. Advanced Synthesis & Catalysis.2006,348(18):2597-2603.
[30]Gong JS, Lu ZM, Li H, Shi JS, Zhou ZM, Xu ZH. Nitrilases in nitrile biocatalysis: recent progress and forthcoming research. Microbial cell factories.2012,11:142.
[31]Thuku RN, Brady D, Benedik MJ, Sewell BT. Microbial nitrilases:versatile, spiral forming, industrial enzymes. Journal of applied microbiology.2009,106(3):703-727.
[32]Chmura A, Shapovalova AA, van Pelt S, van Rantwijk F, Tourova TP, Muyzer G, Sorokin DY. Utilization of arylaliphatic nitriles by haloalkaliphilic Halomonas nitrilicus sp. nov. isolated from soda soils. Applied microbiology and biotechnology. 2008.81(2):371-378.
[33]Yamamoto K, Oishi K, Fujimatsu 1, Komatsu K. Purification and characterization of the nitrilase from Alcaligcnes fuecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile..1 Ferment Bioeng.1992,73:425-430.
[34]Nagasawa T, Mauger J, Yamada H. A novel nitrilase, arylacetonitrilase, of Alcaligenes faecalis JM3. Purification and characterization. European journal of biochemistry/ FEBS.1990,194(3):765-772.
[35]Nageshwar YVD, Sheelu G, Shambhu RR, Muluka H, Mehdi N, Malik MS, Kamal A. Optimization of nitrilase production from Alcaligenes faecalis MTCC 10757 (IICT-A3):effect of inducers on substrate specificity. Bioprocess and biosystems engineering.2011,34(5):515-523.
[36]Xue YP, Xu SZ, Liu ZQ, Zheng YG, Shen YC. Enantioselective biocatalytic hydrolysis of (R,S)-mandelonitrile for production of (R)-(-)-mandelic acid by a newly isolated mutant strain. Journal of industrial microbiology & biotechnology.2011, 38(2):337-345.
[37]Choi SY, Goo YM. Hydrolysis of the nitrile group in a-aminophenylacetonitrile by nitrilase:Development of a new biotechnology for stereospecific production of S-a-phenylglycine. Arch Pharm Res.1986,9:45-47.
[38]Kaplan O, Vejvoda V, Plihal O, Pompach P, Kavan D, Bojarova P, Bezouska K, Mackova M, Cantarella M, Jirku V et al. Purification and characterization of a nitrilase from Aspergillus niger K10. Applied microbiology and biotechnology.2006, 73(3):567-575.
[39]Chauhan S, Wu S, Blumerman S, Fallon RD, Gavagan JE, DiCosimo R, Payne MS. Purification, cloning, sequencing and over-expression in Escherichia coli of a regioselective aliphatic nitrilase from Acidovorax facilis 72W. Applied microbiology and biotechnology.2003,61(2):118-122.
[40]Yamamoto K, Komatsu K. Purification and characterization of nitrilase responsible for the enantioselective hydrolysis from Acinetobacter sp. AK 226. Agricultural and biological chemistry.1991,55(6):1459-1466.
[41]Makadam AM, Knowles CJ. The stereospecific bioconversion of a-aminopropionitrile to L-alanine by an immobilised bacterium isolated from soil. Biotechnology letters. 1985,7:865-870.
[42]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. Bioprocess and biosystems engineering.2011,34(3):315-322.
[43]Shen M, Zheng YG, Shen YC. Isolation and characterization of a novel Arthrobacter nitroguajacolicus ZJUTB06-99, capable of converting acrylonitrile to acrylic acid. Process Biochemistry.2009,44(7):781-785.
[44]Almatawah QA, Cramp R, Cowan DA. Characterization of an inducible nitrilase from a thermophilic bacillus. Extremophiles:life under extreme conditions.1999, 3(4):283-291.
[45]Zheng YG, Chen J, Liu ZQ, Wu MH, Xing LY, Shen YC. Isolation, identification and characterization of Bacillus subtilis ZJB-063, a versatile nitrile-converting bacterium. Applied microbiology and biotechnology.2008,77(5):985-993.
[46]Zhu D, Mukherjee C, Biehl ER, Hua L. Discovery of a mandelonitrile hydrolase from Bradyrhizobium japonicum USDA110 by rational genome mining. Journal of biotechnology.2007,129(4):645-650.
[47]Gradledy ML, Deverson CJF, Knowles CJ. Asymmetric hydrolysis of R-(-), S(+)-2-methylbutyronitrile by Rhodococcus rhodochrous NCIMB 11216. Archives of microbiology.1994,161:246-251.
[48]Levy-Schil S, Soubrier F, Crutz-Le Coq AM, Faucher D, Crouzet J, Petre D. Aliphatic nitrilase from a soil-isolated Comamonas testosteroni sp.:gene cloning and overexpression, purification and primary structure. Gene.1995,161(1):15-20.
[49]Goldlust A, Bohak Z. Induction, purification, and characterization of the nitrilase of Fusarium oxysporum f sp. melonis. Biotechnology and applied biochemistry.1989, 11:581-601.
[50]Vejvoda V, Kubac D, Davidova A, Kaplan O, Sulc M, Sveda O, Chaloupkova R, Martinkova L. Purification and characterization of nitrilase from Fusarium solani IMI196840. Process Biochemistry.2010,45(7):1115-1120.
[51]Vejvoda V, Kaplan O, Bezouska K, Pompach P, Sulc M, Cantarella M, Benada O, Uhnakova B, Rinagelova A, Lutz-Wahl S et al. Purification and characterization of a nitrilase from Fusarium solani 01. Journal of Molecular Catalysis B-Enzymatic.2008, 50(2-4):99-106.
[52]Williamson DS, Dent KC, Weber BW, Varsani A, Frederick J, Thuku RN. Cameron RA, van Heerden JH, Cowan DA, Sewell BT. Structural and biochemical characterization of a nitrilase from the thermophilic bacterium, Geobacillus pallidus RAPc8. Applied microbiology and biotechnology.2010,88(1):143-153.
[53]Stalker DM, Malyj LD, McBride KE. Purification and properties of a nitrilase specific for the herbicide bromoxynil and corresponding nucleotide sequence analysis of the bxn gene. The Journal of biological chemistry.1988,263(13):6310-6314.
[54]Bhalla TC, Kumar H. Nocardia globerula NHB-2:a versatile nitrile-degrading organism. Canadian journal of microbiology.2005,51(8):705-708.
[55]Sharma NN, Sharma M, Kumar H, Bhalla TC. Nocardia globerula NHB-2:Bench scale production of nicotinic acid. Process Biochemistry.2006,41(9):2078-2081.
[56]Harper DB. Microbial metabolism of aromatic nitriles. Enzymology of C-N cleavage by Nocardia sp. (Rhodochrous group) N.C.I.B.11216. The Biochemical journal.1977, 165(2):309-319.
[57]Alonso FOM, Oestreicher EG, Antunes OAC. Production of enantiomerically pure D-phenylglycine using Pseudomonas aeruginosa 10145 as biocatalyst. Brazilian Journal of Chemical Engineering.2008,25(1):1-8.
[58]Layh N, Parratt J, Willetts A. Characterization and partial purification of an enantioselective arylacetonitrilase from Pseudomonas fluorescens DSM 7155. Journal of Molecular Catalysis B-Enzymatic.1998,5(5-6):467-474.
[59]Banerjee A, Kaul P, Banerjee UC. Purification and characterization of an enantioselective arylacetonitrilase from Pseudomonas putida. Archives of microbiology.2006,184(6):407-418.
[60]Yanase H, Sakai T, Tonomuro K. Purification, crystallization and some properties of [3-cyano-L-alanine-degrading enzyme in Pseudomonas sp.13. Agricultural and biological chemistry.1983,47:473-482.
[61]Mueller P, Egorova K, Vorgias CE, Boutou E, Trauthwein H, Verseck S, Antranikian G. Cloning, overexpression. and characterization of a thermoactive nitrilase from the hyperthermophilic archacon Pyrococcus abyssi. Protein expression and purification. 2006.47(2):672-681.
[62]Dong HP. Liu ZQ, Zheng YG. Shen YC. Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemie ethyl 4-cyano-3-hydroxybutyatc by Rhodococcus erythropolis. Applied microbiology and biotechnology. 2010, 87(4): 1335-1345.
163] Mathew CD, Nagasawa I, Kobayashi M, Yamada IF Nitrilase-Catalyzed Production of Nicotinic Acid from 3-Cyanopyridine in Rhodococcus rhodochrous H. Applied and environmental microbiology. 1988, 54(4): 1030-1032.
[64]Kobayashi M, Nagasawa T. Yamada H. Nitrilase of Rhodococcus rhodochrous .11. Purification and characterization. European journal of biochemistry / FKBS. 1989, 182(2):349-356.
[65]Nagasawa T, Wieser M. Nakamura T, Iwahara 11, Yoshida T, Gekko K. Nitrilase of Rhodococcus rhodochrous Jl. Conversion into the active form by subunit association. European journal of biochemistry / FEBS. 2000, 267(1):138-144.
[66]Kobayashi M, Yanaka N. Nagasawa T. Yamada 11. Purification and characterization of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles. Journal of bacteriology. 1990. 172(9):4807-4815.
[67]Harper DB. Purification and properties of an unusual nitrilase from Nocardia N.C.I.B. 11216. Biochemical Society transactions. 1976, 4(3):502-504.
[68]TC. B, A. M, A. W. Y. O, K. F. Asymmetric hydrolysis of a-aminonitriles to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA-34. Applied microbiology and biotechnology. 1992. 37:184-190.
[69]Prasad S, Misra A, Jangir VP, Awasthi A, Raj .1, Bhalla TC. A propionitrile-induced nitrilase of Rhodococcus sp NDB 1165 and its application in nicotinic acid synthesis. World journal of microbiology & biotechnology. 2007. 23(3):345-353.
[70]Kaplan O, Vejvoda V. Charvatova-Pisvejcova A, Martinkova J. Ilyperinduction of nitrilases in filamentous fungi. Journal oi' industrial microbiology & biotechnology. 2006, 33(11):891-896.
[71]Kaplan O, Nikolaou K, Pisvejcova A. Martinkova 1,. Hydrolysis of nitriles and amides by Illamentous fungi. Lnzyme and microbial technology. 2006. 38(1-2):260-264.
[72]Harper DB. Fungal degradation of aromatic nitriles. Lnzymology of C-N cleavage by Fusariuin solani. The Biochemical journal. 1977, 167(3):685-692.
[73]Snajdrova R, Kristova-Mylerova V, Crestia D, Nikolaou K. Kuzma M, Femaire M. Gallienne K. Bolte J. Bezouska K. Kren V el cil. Nitrile biotranslbrmation by Aspergillus niger. Journal of Molecular Catalysis B-Fnzymatic. 2004. 29(1-6):227-232.
[74]Sewell BT, Herman MN. Meyers PR. Jandhyala D. Benedik M.l. The cyanide degrading nitrilase from Pseudomonas slulzeh AK61 is a two-fold symmetric, 14-subunil spiral. Structure. 2003. 11 (11): 1413-1422.
[75]Beslwick LA, Groning LM, James DC, Bones A. Rossiter JT. Purification and characterization of a nitrilase from lirassicti ncipus. Physiologia plantarum. 1993. 89:811-816.
[76]Dohmoto M. Sano J. Tsunoda IF Yamaguchi K. Structural analysis of the TNIT4 genes encoding nitrilase-like protein from tobacco. DNA research : an international journal for rapid publication of reports on genes and genomes. 1999. 6(5):313-317.
[77]Park W.I. Kriechbaumer V. Moller A. Piotrowski M. Meelev RB. Gierl A. Glawischni" E. The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid. Plant physiology.2003,133(2):794-802.
[78]Osswald S, Wajant H, Effenberger F. Characterization and synthetic applications of recombinant AtNIT1 from Arabidopsis thaliana. European Journal of Biochemistry. 2002,269(2):680-687.
[79]Heinemann U, Engels D, Burger S, Kiziak C, Mattes R, Stolz A. Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp strain PCC6803 and heterologous expression and characterization of the encoded protein. Applied and environmental microbiology.2003,69(8):4359-4366.
[80]Kiziak C, Conradt D, Stolz A, Mattes R, Klein J. Nitrilase from Pseudomonas fluorescens EBC191:cloning and heterologous expression of the gene and biochemical characterization of the recombinant enzyme. Microbiology-Sgm.2005, 151:3639-3648.
[81]Chuck R. A catalytic green process for the production of niacin. Chimia.2000, 54(9):508-513.
[82]Almatawah QA, Cowan DA. Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine. Enzyme and microbial technology.1999,25(8-9):718-724.
[83]Prasad S, Misra A, Jangir VP, Awasthi A, Raj J, Bhalla TC. A propionitrile-induced nitrilase of Rhodococcus sp NDB 1165 and its application in nicotinic acid synthesis. World journal of microbiology & biotechnology.2007,23(3):345-353.
[84]Shaw NM, Robins KT, Kiener A. Lonza:20 years of biotransformations. Advanced Synthesis & Catalysis.2003,345(4):425-435.
[85]Straathof AJ, Sie S, Franco TT, van der Wielen LA. Feasibility of acrylic acid production by fermentation. Applied microbiology and biotechnology.2005, 67(6):727-734.
[86]Xu XB, Lin JP, Cen PL. Advances in the research and development of acrylic acid production from biomass. Chinese J Chem Eng.2006,14(4):419-427.
[87]Nagasawa T, Nakamura T, Yamada H. Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase. Applied microbiology and biotechnology. 1990,34:322-324.
[88]Luo H, Wang T, Yu H, Yang H, Shen Z. Expression and catalyzing process of the nitrilase in Rhodococcus rhodochrous tg1-A6. Mod Chem Ind.2006,26:109-111.
[89]Shi YH, Sun HY, Lu DM, Le QH, Chen DX, Zhou YC. Separation of glycolic acid from glycolonitrile hydrolysate by reactive extraction with tri-n-octylamine. Sep Purif Technol.2006,49(1):20-26.
[90]He YC, Xu JH, Su JH, Zhou L. Bioproduction of glycolic acid from glycolonitrile with a new bacterial isolate of Alcaligenes sp. ECU0401. Applied biochemistry and biotechnology.2010,160(5):1428-1440.
[91]Wu SJ, Fogiel AJ, Petrillo KL, Jackson RE, Parker KN, DiCosimo R, Ben-Bassat A, O'Keefe DP, Payne MS. Protein engineering of nitrilase for chemoenzymatic production of glycolic acid. Biotechnology and bioengineering.2008,99(3):717-720.
[92]Panova A, Mersingera LI, Liu Q. Foo T, Roe DC, Spillan WL, Sigmund AE, Ben-Bassat A, Wagner LW, O'Keefe DP el al. Chemoenzymatic synthesis of glycolic acid. Advanced Synthesis & Catalysis.2007,349(8-9):1462-1474.
[93]薛建萍,罗积杏,李还宝,于军华,朱健,许弘,沈寅初.生物催化生产羟 基乙酸.中国专利,CN1772912A,2006.
[94]Kaul P, Banerjee A, Mayilraj S, Banerjee UC. Screening for enantioselective nitrilases: kinetic resolution of racemic mandelonitrile to (R)-(-)-mandelic acid by new bacterial isolates. Tetrahedron-Asymmetry.2004,15(2):207-211.
[95]Banerjee A, Kaul P, Banerjee UC. Enhancing the catalytic potential of nitrilase from Pseudomonas putida for stereoselective nitrile hydrolysis. Applied microbiology and biotechnology.2006,72(1):77-87.
[96]Zhang ZJ, Xu JH, He YC, Ouyang LM, Liu YY, Imanaka T. Efficient production of (R)-(-)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp. Process Biochemistry.2010,45(6):887-891.
[97]Zhang ZJ, Pan JA, 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. Journal of biotechnology.2011,152(1-2):24-29.
[98]Robertson DE, Chaplin JA, DeSantis G, Podar M, Madden M, Chi E, Richardson T, Milan A, Miller M, Weiner DP et al. Exploring nitrilase sequence space for enantioselective catalysis. Applied and environmental microbiology.2004, 70(4):2429-2436.
[99]DeSantis G, Zhu Z, Greenberg WA, Wong K, Chaplin J, Hanson SR, Farwell B, Nicholson LW, Rand CL, Weiner DP et al. An enzyme library approach to biocatalysis: development of nitrilases for enantioselective production of carboxylic acid derivatives. Journal of the American Chemical Society.2002,124(31):9024-9025.
[100]Kaul P, Stolz A, Banerjee UC. Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis. Advanced Synthesis & Catalysis.2007, 349(13):2167-2176.
[101]Rossi JC, Rey P, Taillades J, Gros G, Nore O. Hydrolysis of nitriles using an immobilized nitrilase:Applications to the synthesis of methionine hydroxy analogue derivatives. Journal of agricultural and food chemistry.2004,52(26):8155-8162.
[102]DeSantis G, Wong K, Farwell B, Chatman K, Zhu ZL, Tomlinson G, Huang HJ, Tan XQ, Bibbs L, Chen P et al. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). Journal of the American Chemical Society.2003,125(38):11476-11477.
[103]Kretz KA, Richardson TH, Gray KA, Robertson DE, Tan XQ, Short JM. Gene site saturation mutagenesis:A comprehensive mutagenesis approach. Method Enzymol. 2004,388:3-11.
[104]Bergeron S, Chaplin DA, Edwards JH, Ellis BSW, Hill CL, Holt-Tiffin K, Knight JR, Mahoney T, Osborne AP, Ruecroft G. Nitrilase-catalysed desymmetrisation of 3-hydroxyglutaronitrile:Preparation of a statin side-chain intermediate. Organic Process Research & Development.2006,10(3):661-665.
[105]Hann EC, Sigmund AE, Hennessey SM, Gavagan JE, Short DR, Ben-Bassat A, Chauhan S, Fallon RD, Payne MS, DiCosimo R. Optimization of an immobilized-cell biocatalyst for production of 4-cyanopentanoic acid. Organic Process Research & Development.2002,6(4):492-496.
[106]Cooling FB, Fager SK, Fallon RD, Folsom PW, Gallagher FG, Gavagan JE, Hann EC, Herkes FE, Phillips RL, Sigmund A et al. Chemoenzymatic production of 1,5-dimethyl-2-piperidone. Journal of Molecular Catalysis B-Enzymatic.2001, 11(4-6):295-306.
[107]Jennings RA, Johnson DR, Seamans RE, Zeller JR. U.S. Patent 5362883. 1994.
[108]Burns MP, Wong JW. U.S. Patent Appl. 2005009154 A1. 2005.
[109]Zhu D, Mukherjee C, Biehl ER, Hua L. Nitrilase-catalyzed selective hydrolysis of dinitriles and green access to the cyanocarboxylic acids of pharmaceutical importance. Advanced Synthesis & Catalysis. 2007, 349(10): 1667-1670.
[110]Xie ZY, Feng JL, Garcia E, Bernett M, Yazbeck D, Tao JH. Cloning and optimization of a nitrilase for the synthesis of (3S)-3-cyano-5-methyl hexanoic acid. Journal of Molecular Catalysis B-Enzymatic. 2006, 41(3-4):75-80.
[111]Burns MP, Weaver JK, Wong JW. Stereoselective bioconversion of aliphatic dinitriles into cyano carboxylic acids: US, 0196905 Al. 2007.
[112]Chen J, Zheng RC, Zheng YG, Shen YC. Microbial transformation of nitriles to high-value acids or amides. Advances in biochemical engineering/biotechnology. 2009, 113:33-77.
[113]Zhou XF, Zhang YL, Xu DQ, Cao WH, Dai CM, Qiang ZM, Yang Z, Zhao JF. Treatment of succinonitrile wastewater by immobilized high efficiency microorganism strains. Water Sci Technol. 2008, 58:911-918.
[114]McBride KE, Kenny JW, Stalker DM. Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae. Applied and environmental microbiology. 1986,52:325-330.
[115]Vesela AB, Franc M, Pelantova H, Kubac D, Vejvoda V, Sulc M, Bhalla TC, Mackova M, Lovecka P, Janu P et al. Hydrolysis of benzonitrile herbicides by soil actinobacteria and metabolite toxicity. Biodegradation. 2010, 21(5):761-770.
[116]Martinkova L, Uhnakova B, Patek M, Nesvera J, Kren V. Biodegradation potential of the genus Rhodococcus. Environment international. 2009, 35(1): 162-177.
[117]Battistel E, Morra M, Marinetti M. Enzymatic surface modification of acrylonitrile fibers. Appl Surf Sci. 2001, 177(1-2):32-41.
[118]Matama T, Carneiro F, Caparros C, Gubitz GM, Cavaco-Paulo A. Using a nitrilase for the surface modification of acrylic fibres. Biotechnology journal. 2007, 2(3):353-360.
[119]Fischer-Colbrie G, Matama T, Heumann S, Martinkova L, Cavaco Paulo A, Guebitz G. Surface hydrolysis of polyacrylonitrile with nitrile hydrolysing enzymes from Micrococcus luteus BST20. Journal of biotechnology. 2007, 129(1):62-68.
[120]Feng LL, Zhang JF, Luo H, Li Z. Surface modification of acrylonitrile fibers and membrane by nitrilase from Escherichia coli BL21 (DE3)/pET-Nit. Silk: Inheritance and Innovation - Modern Silk Road. 2011, 175-176:651-655.
[121]Brady D, Beeton A, Zeevaart J, Kgaje C, van Rantwijk F, Sheldon RA. Characterisation of nitrilase and nitrile hydratase biocatalytic systems. Applied microbiology and biotechnology. 2004, 64(1):76-85.
[122]Andexer JN, Langermann JV, Kragl U, Pohl M. How to overcome limitations in biotechnological processes - examples from hydroxynitrile lyase applications. Trends Biotechnol. 2009, 27(10):599-607.
[123]Martinkova L, Vejvoda V, Kren V. Selection and screening for enzymes of nitrile metabolism. Journal of biotechnology. 2008. 133(3):318-326.
[124]Martinkova L. Vejvoda V. Kaplan O, Kubac D. Malandra A. Cantarella M, Bezouska K. Kren V. Fungal nilrilases as biocatalysts: Recent developments. Biotechnology advances. 2009. 27(6):661-670.
[125]Watanabe I. Satoh Y, Knomoto K. Screening, isolation and taxonomical properties of microorganisms having acrylonitrile-hydrating activity. Agricultural and biological chemistry. 1987. 51:3193-3199.
[126]Asano Y.Tani Y.Yamada H. A new enzyme nitrile hydralase which degrades acetonitrile in combination with amidase. Agricultural and biological chemistry. 1980. 44:2151-2152.
[127]Mann KC. Kisenberg A, F'ager SK, Perkins NK. Gallagher FG. Cooper SM. Gavagan JK, Stieglitz B. Hennessey SM, DiCosimo R. 5-eyanovaleramide production using immobilized Pseudomonas chlororaphis B23. Bioorganic & medicinal chemistry. 1999. 7(10):2239-2245.
[128]Nagasawa T, Mathew C.D, Mauger J, Yamada H. Nitrile Ilydratase-Catalyzed Production of Nicotinamide from 3-Cyanopyridine in Rhodococcus rhodochrous .11. Applied and environmental microbiology. 1988, 54(7): 1766-1769.
[129]Nagasawa T, Shimizu H, Yamada H. The superiority of the third generation catalyst. Rhodococcus rhodochrous H nitrile hydratase, for the industrial production of acrylamide. 1993, 40:189-195.
[130]Ress-Loschke M. Friedrich T. Ilauer B, Mattes R, Kngels D. Method for producing chiral carboxylie acids from nitriles with the assistance of a nitrilase or microorganisms which contain a gene for the nitrilase:PC Ipatent application WO/2000/023577. 2000.
[131]Kndo T, Tamura K. Process lor producing R(-)-mandelic acid and derivatives thereof. Kuropean Pateni KP0449648. 1991.
[132]Kndo T. Tamura K. Process for producing R(-)-mandelic acid or a derivative thereof from a mandelonitrile using Rhodococcus. US Patent 5296373. 1994.
[133]Kndo T, Yamagami T. Tamura K. Biological process for producing alpha-hydroxyamide or alphahydroxy acid. US Pateni 5326702. 1994.
[134]Yamamoto K. Olsubo K, Oishi K. Process lor producing optically active alpha-substituted organic acid and microorganism and enzyme used therefore. US Patent 5283193. 1991.
1135] Favre-Bulle O. Pierrard .1, David C, Morel P. I lorbez D. Industrial scale process for the preparation of 2-hydroxy-4-methylbutyric acid using a nitrilase: US Patent 6180359. 2001.
[136]Pierrard J, Favre-Bulle O. lourdat C. Method for modulating nitrilase selectivity, nitrilases obtained by said method and use thereof: PCT patent application WO/2001/034786. 2001.
[137]Podar M, Kads JR. Richardson 111. Involution of a microbial nitrilase gene family: a comparative and environmental genomics study. BMC evolutionary biology. 2005. 5:
[138]Seffernick JK, Samanta SK. Louie I'M. Wacketl LP. Subramanian M. Investigative mining of sequence data for novel enzymes: A ease study with nitrilases. Journal of biotechnology. 2009, 143(1): 1 7-26.
[139]Zhu DM, Mukherjee C. Yang Y. Rios BK. Gallagher DT. Smith NN. Biehl KR. Ilua I, A new nitrilase from Bradyrhizohium japonicum USDA 110 -Gene cloning, biochemical characterization and substrate specificity. Journal of biotechnology. 2008. 133(3):327-333.
[140]Petrickova A, Vesela AB, Kaplan O, Kubac D, Uhnakova B, Malandra A, Felsberg J, Rinagelova A, Weyrauch P, Kren V et al. Purification and characterization of heterologously expressed nitrilases from filamentous fungi. Applied microbiology and biotechnology.2012,93(4):1553-1561.
[141]Streit WR, Daniel R, Jaeger KE. Prospecting for biocatalysts and drugs in the genomes of non-cultured microorganisms. Current opinion in biotechnology.2004, 15(4):285-290.
[142]Cowan D, Meyer Q, Stafford W, Muyanga S, Cameron R, Wittwer P. Metagenomic gene discovery:past, present and future. Trends Biotechnol.2005,23(6):321-329.
[143]Green BD, Keller M. Capturing the uncultivated majority. Current opinion in biotechnology.2006,17(3):236-240.
[144]Schmeisser C, Steele H, Streit WR. Metagenomics, biotechnology with non-culturable microbes. Applied microbiology and biotechnology.2007,75(5):955-962.
[145]Knietsch A, Bowien S, Whited G, Gottschalk G, Daniel R. Identification and characterization of coenzyme B12-dependent glycerol dehydratase-and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures. Applied and environmental microbiology.2003, 69(6):3048-3060.
[146]Kiziak C, Klein J, Stolz A. Influence of different carboxy-terminal mutations on the substrate-, reaction-and enantiospecificity of the arylacetonitrilase from Pseudomonas fluorescens EBC191. Protein Engineering Design & Selection.2007,20(8):385-396.
[147]Kiziak C, Stolz A. Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191. Applied and environmental microbiology.2009, 75(17):5592-5599.
[148]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. Biotechnology and bioengineering.2007,97(4):689-693.
[149]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. Biochemical Journal.2008, 415:401-407.
[150]Petrickova A, Sosedov O, Baum S, Stolz A, Martinkova L. Influence of point mutations near the active site on the catalytic properties of fungal arylacetonitrilases from Aspergillus niger and Neurospora crassa. Journal of Molecular Catalysis B-Enzymatic.2012,77:74-80.
[151]Chen J, Zheng YG, Shen YC. Biosynthesis of p-methoxyphenylacetic acid from p-methoxyphenylacetonitrile by immobilized Bacillus subtil is ZJB-063. Process Biochemistry.2008,43(9):978-983.
[152]Sheldon RA. Cross-linked enzyme aggregates (CLEAs):stable and recyclable biocatalysts. Biochemical Society transactions.2007,35(Pt 6):1583-1587.
[153]Swartz JD, Miller SA, Wright D. Rapid Production of Nitrilase Containing Silica Nanoparticles Offers an Effective and Reusable Biocatalyst for Synthetic Nitrile Hydrolysis. Organic Process Research & Development.2009,13(3):584-589.
[154]Chaplin JA, Levin MD, Morgan B, Farid N, Li J, Zhu ZL, McQuaid J, Nicholson LW, Rand CA, Burk MJ. Chemoenzymatic approaches to the dynamic kinetic asymmetric synthesis of aromatic amino acids. Tetrahedron-Asymmetry.2004,15(18):2793-2796.
[155]Winkler M, Knall AC, Kulterer MR, Klempier N. Nitrilases catalyze key step to conformationally constrained GABA analogous gamma-amino acids in high optical purity. Journal of Organic Chemistry.2007,72(19):7423-7426.
[156]Winkler M, Meischler D, Klempier N. Nitrilase-catalyzed enantioselective synthesis of pyrrolidine- and piperidinecarboxylic acids. Advanced Synthesis & Catalysis.2007, 349(8-9):1475-1480.
[157]Winkler M, Kaplan O, Vejvoda V, Klempier N, Martinkova L. Biocatalytic application of nitrilases from Fusarium solani 01 and Aspergillus niger K10. Journal of Molecular Catalysis B-Enzymatic.2009,59(4):243-247.
[158]Kielbasinski P, Rachwalski M, Mikolajczyk M, Szyrej M, Wieczorek MW, Wijtmans R, Rutjes FPJT. Enzyme-promoted desymmetrisation of prochiral bis(cyanomethyl) sulfoxide. Advanced Synthesis & Catalysis.2007,349(8-9):1387-1392.
[159]Kielbasinski P, Rachwalski M, Wieczorek WM, Kwiatkowska MSA, Mikolajczyk M, Sieron L, Rutjes FPJT. Enzyme-promoted desymmetrisation of prochiral bis(cyanomethyl)phenylphosphine oxide. Tetrahedron-Asymmetry.2007, 18(17):2108-2112.
[160]Kielbasinski P, Rachwalski M, Mikolajczyk M, Rutjes FPJT. Nitrilase-catalysed hydrolysis of cyanomethyl p-tolyl sulfoxide:stereochemistry and mechanism. Tetrahedron-Asymmetry.2008,19(5):562-567.
[161]Banerjee A, Dubey S, Kaul P, Barse B, Piotrowski M, Banerjee U. Enantioselective Nitrilase from Pseudomonas putida:Cloning, Heterologous Expression, and Bioreactor Studies. Molecular biotechnology.2009,41(1):35-41.
[162]Kaul P, Banerjee UC. Predicting enzyme behavior in nonconventional media: correlating nitrilase function with solvent properties. Journal of industrial microbiology & biotechnology.2008,35(7):713-720.
[163]Rustler S, Motejadded H, Altenbuchner J, Stolz A. Simultaneous expression of an arylacetonitrilase from Pseudomonas fluorescens and a (S)-oxynitrilase from Manihot esculenta in Pichia pastoris for the synthesis of (S)-mandelic acid. Applied microbiology and biotechnology.2008,80(l):87-97.
[164]Sosedov O, Matzer K, Burger 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. Advanced Synthesis & Catalysis.2009,351(10):1531-1538.
[165]Sheldon RA. Cross-Linked Enzyme Aggregates as Industrial Biocatalysts. Organic Process Research & Development.2011,15(1):213-223.
[166]Ankati H, Zhu DN, Yang Y, Biehl ER, Hua L. Asymmetric Synthesis of Both Antipodes of beta-Hydroxy Nitriles and beta-Hydroxy Carboxylic Acids via Enzymatic Reduction or Sequential Reduction/Hydrolysis. Journal of Organic Chemistry.2009,74(4):1658-1662.
[167]Pollock JA, Clark KM, Martynowicz BJ, Pridgeon MG, Rycenga MJ, Stolle KE, Taylor SK. A mild biosynthesis of lactones via enantioselective hydrolysis of hvdroxvnitriles. Tetrahedron-Asvmmetrv.2007.18(16):1888-1892.
[168]Piotrowski M, Schonfelder S, Weiler EW. The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode beta-cyano-L-alanine hydratase/nitrilase. Journal of Biological Chemistry.2001,276(4):2616-2621.
[169]Vorwerk S, Biernacki S, Hillebrand H, Janzik I, Muller A, Weiler EW, Piotrowski M. Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster. Planta.2001, 212(4):508-516.
[170]Liu ZQ, Dong LZ, Cheng F, Xue YP, Wang YS, Ding JN, Zheng YG, Shen YC. Gene Cloning, Expression, and Characterization of a Nitrilase from Alcaligenes faecalis ZJUTB10. Journal of agricultural and food chemistry.2011,59(21):11560-11570.
[171]Kobayashi M, Izui H, Nagasawa T, Yamada H. Nitrilase in biosynthesis of the plant hormone indole-3-acetic acid from indole-3-acetonitrile:cloning of the Alcaligenes gene and site-directed mutagenesis of cysteine residues. Proceedings of the National Academy of Sciences of the United States of America.1993,90(1):247-251.
[172]Zelinski T, Keβeler M, Hauer B, Friedrich T. Modified nitrilases and their use in methods for the production of carboxylic acids:US7985572.2004.
[173]He YC, Zhang ZJ, Xu JH, Liu YY. Biocatalytic synthesis of (R)-(-)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of Alcaligenes faecalis ECU0401. Journal of industrial microbiology & biotechnology. 2010,37(7):741-750.
[174]Kaul P, Banerjee A, Banerjee UC. Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst. Biomacromolecules.2006,7(5):1536-1541.
[175]Xue YP, Xu M, Chen HS, Liu ZQ, Wang YJ, Zheng YG. A Novel Integrated Bioprocess for Efficient Production of (R)-(-)-Mandelic Acid with Immobilized Alcaligenes faecalis ZJUTB10. Organic Process Research & Development.2013, 17(2):213-220.
[176]Nagasawa T, Yoshida T, Matsuyama A. Method for producing the carboxylic acid:JP2006223246.2006.
[177]Muller A, Weiler EW. Indolic constituents and indole-3-acetic acid biosynthesis in the wild-type and a tryptophan auxotroph mutant of Arabidopsis thaliana. Planta.2000, 211(6):855-863.
[178]Kazlauskas RJ, Weissfloch ANE, Rappaport AT, Cuccia LA. A rule to predict which enantiomer of a secondary alcohol reacts faster in reactions catalyzed by cholesterol esterase, lipase from Pseudomonas cepacia, and lipase from Candida rugosa. Journal of Organic Chemistry.1991,56:2656-2665.
[179]Wu ZL, Li ZY. Enantioselective hydrolysis of various racemic a-substituted arylacetonitriles using Rhodococcus sp. CGMCC 0497. Tetrahedron: Asymmetry.2001,12(23):3305-3312
[180]Schreiner, U., Hecher, B., Obrowsky, S., Waich, K., Klempier, N., Steinkellner, G., Gruber, K., Rozzell, J. D., Glieder, A., Winkler, M. Directed evolution of Alcaligenes faecalis nitrilase. Enzyme and Microbial Technology.2010,47(4):140-146