恶臭假单胞菌中腈水合酶钴离子摄取机制和热稳定性改造研究
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摘要
腈水合酶（Nitrile hydratase,简写NHase, EC4.2.1.84）是一种能够催化腈类化合物水合生成相应酰胺的金属酶。目前国际上已经广泛应用第三代工业用菌玫瑰红红球菌Rhodococcus rhodochrous J1实现酰胺类物质的工业生产。此法具有选择性好，回收率高，副产物少，反应可在常温常压下进行，省去了产品提浓和丙烯腈回收等步骤的优势，使生产工艺过程得到简化。我国利用腈水合酶生产丙烯酰胺的生物技术虽然起步较晚，但发展很快。
腈水合酶的广泛应用推动了其基础研究的发展。本研究室长期从事腈水合酶机理的研究，前期发现R. rhodochrous J1来源的腈水合酶金属离子摄取机理是一种称为“亚基自身交换”的新型蛋白质翻译后修饰体系。尽管如此，这种新型的蛋白质翻译后修饰体系是否具有普遍性还需要进一步探讨。然而，腈水合酶不稳定的酶学性质限制了体外研究酶分子翻译后修饰的机制，因此，提高其稳定性对进一步系统探讨酶催化的分子机制以及广泛应用于工业生产具有重要的意义。
本论文以恶臭假单胞菌Pseudomonas putida NRRL-18668中腈水合酶为研究对象，系统探讨了其Co离子摄取机理，并对酶分子进行了热稳定性改造研究，主要结果如下：
（1）利用N端原则在大肠杆菌中成功表达了P. putida腈水合酶调控蛋白P14K。P14K对腈水合酶的功能性表达是必要的，但在大肠杆菌中很难表达。利用N端原则，将P14K的N端的第二位Lys突变成Ala、截断N端前16个氨基酸后使新的N端第二位氨基酸是Gly、在N端融合strep标签，都能实现P14K在大肠杆菌中成功表达。此外，通过在P14K的N端融合pelB信号肽也可以提高P14K的稳定性。
（2）P. putida菌中腈水合酶（α亚基àβ亚基à调控蛋白）的Co离子摄取机制遵循亚基自身交换机制。首先分离了调控蛋白复合物，发现该复合物是由P14K与腈水合酶自身α亚基形成的蛋白质三聚体α(P14K)2。设计表达了含有两种不同分子量α亚基（α和HisT7-α）的腈水合酶(HisT7-α)2β2和调控蛋白α(P14K)2，把纯化的不含Co离子的(HisT7-α)2β2与含Co离子的α(P14K)2混合4小时后发现，原来的腈水合酶(HisT7-α)2β2变成了α2β2，说明这个过程中两者的α亚基相互发生了交换。由此证明了P. putida菌中腈水合酶的Co离子摄取机制是遵循亚基自身交换机制的。本实验不仅说明新型蛋白质翻译后修饰亚基自身交换机制存在于P. putida腈水合酶中，结合本实验室以前的研究结果也说明亚基自身交换机制具有一定的普遍性。
（3）P14K上C端Leu85-Ala144（C-domain）高度柔性带正电的结构特征在腈水合酶Co离子摄取的过程中具有重要作用。分子动力学模拟显示C-domain具有高度柔性的结构特征。突变该结构域氨基酸序列上保守的并与结构柔性相关的Gly86，发现腈水合酶只保留了野生酶10%的酶活。说明C-domain高度柔性的结构特征对于腈水合酶的激活有重要的作用。突变C-domain上的带正电的氨基酸为中性氨基酸导致腈水合酶失活，说明C-domain带正电的特征也同样对于腈水合酶的激活有重要的作用。同时发现这些突变体的Co离子含量很低。另外，量子化学计算结果显示腈水合酶摄取Co离子时需要克服203kcal×mol-1的能垒，结合上述实验结果和分析，我们认为C-domain高度柔性带正电的结构特征恰好就是为了克服这个能垒而辅助α亚基摄取Co离子的。
（4）在腈水合酶β亚基N端融合自组装肽ELK16（SAP-NHase-2）能诱导腈水合酶形成活性包涵体。在腈水合酶β亚基C端融合ELK16（SAP-NHase-10）或是融合自组装肽EAK16（SAP-NHase-1）虽都不能诱导腈水合酶形成活性包涵体，但能提高腈水合酶的稳定性，其中SAP-NHase-1, SAP-NHase-2和SAP-NHase-10的热稳定性较原始酶分别提高了45%,30%和50%。
Nitrile hydratase (NHase, EC4.2.1.84) is a metalloenzyme that can catalyze thehydration of a nitrile to the corresponding amide. It is widely used in the industrial application.Millions of acrylamide were produced by NHase. NHase catalysis possesses advantages suchas good selectivity, productivity, less side product, under common temperature and pressure,without product concentration and recycle of acrylnitrile and so on. A rapid improvement ofNHase catalysis was conducted in China.
A wide range use of NHase induces its fundamental research. A long research of NHasecatalysis was conducted in our laboratory. Recently, a cobalt-containing NHase inRhodococcus rhodochrous J1, which uses a novel mode of post-translational maturation, wasdiscovered. This novel mode of post-translational maturation was called self-subunitswapping. This not only enhances the research of NHase catalysis but also raises theknowledge of people, which lays the foundation for new method. However, further study wasimperative.
The mechanism of cobalt incorporation into NHase in Pseudomonas putidaNRRL-18668was discovered. The stability of NHase was also researched. The main resultswere conducted as follows:
(1) P14K was successfully expressed in Escherichia coli. Nitrile hydratase (NHase)activators are essential for functional NHase biosynthesis. However, the activator P14K of thecobalt-containing NHase from P. putida is difficult to heterogeneously express in E. coli,which has retarded the clarification of the mechanism underlying the involvement of P14K inthe maturation of NHase. We here successfully expressed P14K through genetic modificationsaccording to N-end rule and analyze the mechanism underlying the instability of this protein.We found that mutation of the second N-terminal amino acid lysine to alanine or truncatingthe N-terminal16amino-acid sequence resulted in successful expression of P14K. Moreover,either addition of a strep tag alone (strep-P14K) or fusion of a pelB leader and srtep tagtogether (pelB-strep-P14K) at the N-terminus increased the expression of P14K.
(2) The incorporation of cobalt into another type of Co-NHase, with a gene organizationof <α-subunit><β-subunit>, was also dependent on self-subunit swapping.We successfully isolated a recombinant NHase activator protein (P14K) of P. putida. ThisP14K was found to form a complex α(P14K)2with the α-subunit of the NHase. Theincorporation of cobalt into the NHase of P. putida was confirmed to be dependent on theα-subunit substitution between cobalt-containing α(P14K)2and cobalt-free NHase. Theseresults expand on the general features of self-subunit swapping maturation..
(3) The flexibility and positive charge of the C-terminal domain (C-domain) of theself-subunit swapping chaperone (P14K) of NHase from P. putida might play an importantrole in the cobalt incorporation for NHase activation. We first proposed a flexible C-domainfrom L85to A144using molecular dynamic simulation, and found that the C-domaintruncation and the P14K(G86I) mutation, which alter the C-domain flexibility, resulted invery low NHase activity. In addition, elimination of the positive charge in the C-domain also sharply affected NHase activity, and these mutants exhibited low cobalt content. Based on thestructural and energetic analyses, we proposed that the flexible, positively charged C-domainmost likely performs an external action that allows the cobalt-free NHase to overcome theenergetic barrier (203kcal×mol-1), resulting in a cobalt-containing NHase.
(4) The thermo-stability of nitrile hydratase (NHase) was enhanced by fusing with two ofthe SAPs (EAK16and ELK16). When the ELK16was fused to the N-terminus of β-subunit,the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16fused NHasein the N-terminus of β-subunit (SAP-NHase-1) and ELK16fused NHase in the C-terminus ofβ-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the wild typeNHase, the thermal stability of SAP-NHase-1, SAP-NHase-2and SAP-NHase-10wereenhanced by45%,30%and50%, respectively.
腈水合酶的广泛应用推动了其基础研究的发展。本研究室长期从事腈水合酶机理的研究，前期发现R. rhodochrous J1来源的腈水合酶金属离子摄取机理是一种称为“亚基自身交换”的新型蛋白质翻译后修饰体系。尽管如此，这种新型的蛋白质翻译后修饰体系是否具有普遍性还需要进一步探讨。然而，腈水合酶不稳定的酶学性质限制了体外研究酶分子翻译后修饰的机制，因此，提高其稳定性对进一步系统探讨酶催化的分子机制以及广泛应用于工业生产具有重要的意义。
本论文以恶臭假单胞菌Pseudomonas putida NRRL-18668中腈水合酶为研究对象，系统探讨了其Co离子摄取机理，并对酶分子进行了热稳定性改造研究，主要结果如下：
（1）利用N端原则在大肠杆菌中成功表达了P. putida腈水合酶调控蛋白P14K。P14K对腈水合酶的功能性表达是必要的，但在大肠杆菌中很难表达。利用N端原则，将P14K的N端的第二位Lys突变成Ala、截断N端前16个氨基酸后使新的N端第二位氨基酸是Gly、在N端融合strep标签，都能实现P14K在大肠杆菌中成功表达。此外，通过在P14K的N端融合pelB信号肽也可以提高P14K的稳定性。
（2）P. putida菌中腈水合酶（α亚基àβ亚基à调控蛋白）的Co离子摄取机制遵循亚基自身交换机制。首先分离了调控蛋白复合物，发现该复合物是由P14K与腈水合酶自身α亚基形成的蛋白质三聚体α(P14K)2。设计表达了含有两种不同分子量α亚基（α和HisT7-α）的腈水合酶(HisT7-α)2β2和调控蛋白α(P14K)2，把纯化的不含Co离子的(HisT7-α)2β2与含Co离子的α(P14K)2混合4小时后发现，原来的腈水合酶(HisT7-α)2β2变成了α2β2，说明这个过程中两者的α亚基相互发生了交换。由此证明了P. putida菌中腈水合酶的Co离子摄取机制是遵循亚基自身交换机制的。本实验不仅说明新型蛋白质翻译后修饰亚基自身交换机制存在于P. putida腈水合酶中，结合本实验室以前的研究结果也说明亚基自身交换机制具有一定的普遍性。
（3）P14K上C端Leu85-Ala144（C-domain）高度柔性带正电的结构特征在腈水合酶Co离子摄取的过程中具有重要作用。分子动力学模拟显示C-domain具有高度柔性的结构特征。突变该结构域氨基酸序列上保守的并与结构柔性相关的Gly86，发现腈水合酶只保留了野生酶10%的酶活。说明C-domain高度柔性的结构特征对于腈水合酶的激活有重要的作用。突变C-domain上的带正电的氨基酸为中性氨基酸导致腈水合酶失活，说明C-domain带正电的特征也同样对于腈水合酶的激活有重要的作用。同时发现这些突变体的Co离子含量很低。另外，量子化学计算结果显示腈水合酶摄取Co离子时需要克服203kcal×mol-1的能垒，结合上述实验结果和分析，我们认为C-domain高度柔性带正电的结构特征恰好就是为了克服这个能垒而辅助α亚基摄取Co离子的。
（4）在腈水合酶β亚基N端融合自组装肽ELK16（SAP-NHase-2）能诱导腈水合酶形成活性包涵体。在腈水合酶β亚基C端融合ELK16（SAP-NHase-10）或是融合自组装肽EAK16（SAP-NHase-1）虽都不能诱导腈水合酶形成活性包涵体，但能提高腈水合酶的稳定性，其中SAP-NHase-1, SAP-NHase-2和SAP-NHase-10的热稳定性较原始酶分别提高了45%,30%和50%。
Nitrile hydratase (NHase, EC4.2.1.84) is a metalloenzyme that can catalyze thehydration of a nitrile to the corresponding amide. It is widely used in the industrial application.Millions of acrylamide were produced by NHase. NHase catalysis possesses advantages suchas good selectivity, productivity, less side product, under common temperature and pressure,without product concentration and recycle of acrylnitrile and so on. A rapid improvement ofNHase catalysis was conducted in China.
A wide range use of NHase induces its fundamental research. A long research of NHasecatalysis was conducted in our laboratory. Recently, a cobalt-containing NHase inRhodococcus rhodochrous J1, which uses a novel mode of post-translational maturation, wasdiscovered. This novel mode of post-translational maturation was called self-subunitswapping. This not only enhances the research of NHase catalysis but also raises theknowledge of people, which lays the foundation for new method. However, further study wasimperative.
The mechanism of cobalt incorporation into NHase in Pseudomonas putidaNRRL-18668was discovered. The stability of NHase was also researched. The main resultswere conducted as follows:
(1) P14K was successfully expressed in Escherichia coli. Nitrile hydratase (NHase)activators are essential for functional NHase biosynthesis. However, the activator P14K of thecobalt-containing NHase from P. putida is difficult to heterogeneously express in E. coli,which has retarded the clarification of the mechanism underlying the involvement of P14K inthe maturation of NHase. We here successfully expressed P14K through genetic modificationsaccording to N-end rule and analyze the mechanism underlying the instability of this protein.We found that mutation of the second N-terminal amino acid lysine to alanine or truncatingthe N-terminal16amino-acid sequence resulted in successful expression of P14K. Moreover,either addition of a strep tag alone (strep-P14K) or fusion of a pelB leader and srtep tagtogether (pelB-strep-P14K) at the N-terminus increased the expression of P14K.
(2) The incorporation of cobalt into another type of Co-NHase, with a gene organizationof <α-subunit><β-subunit>
(3) The flexibility and positive charge of the C-terminal domain (C-domain) of theself-subunit swapping chaperone (P14K) of NHase from P. putida might play an importantrole in the cobalt incorporation for NHase activation. We first proposed a flexible C-domainfrom L85to A144using molecular dynamic simulation, and found that the C-domaintruncation and the P14K(G86I) mutation, which alter the C-domain flexibility, resulted invery low NHase activity. In addition, elimination of the positive charge in the C-domain also sharply affected NHase activity, and these mutants exhibited low cobalt content. Based on thestructural and energetic analyses, we proposed that the flexible, positively charged C-domainmost likely performs an external action that allows the cobalt-free NHase to overcome theenergetic barrier (203kcal×mol-1), resulting in a cobalt-containing NHase.
(4) The thermo-stability of nitrile hydratase (NHase) was enhanced by fusing with two ofthe SAPs (EAK16and ELK16). When the ELK16was fused to the N-terminus of β-subunit,the resultant NHase (SAP-NHase-2) became an active inclusion body; EAK16fused NHasein the N-terminus of β-subunit (SAP-NHase-1) and ELK16fused NHase in the C-terminus ofβ-subunit (SAP-NHase-10) did not affect NHase solubility. Compared with the wild typeNHase, the thermal stability of SAP-NHase-1, SAP-NHase-2and SAP-NHase-10wereenhanced by45%,30%and50%, respectively.
引文
1Yang H, Wang J, Jia X, et al. Structural insight into the mechanisms of enveloped virus tethering bytetherin[J]. Proc Natl Acad Sci U S A,2010,107(43):18428-18432
2Wu J, Kinoshita K, Khosla C, et al. Biochemical analysis of the substrate specificity of theβ-ketoacyl-acyl carrier protein synthase domain of module2of the erythromycin polyketide synthase[J].Biochemistry,2004,43(51):16301-16310
3Kobayashi M, Shimizu S. Cobalt proteins[J]. Eur J Biochem,1999,261(1):1-9
4Wu J, Bell A F, Luo L, et al. Probing hydrogen-bonding interactions in the active site of medium-chainacyl-CoA dehydrogenase using Raman spectroscopy[J]. Biochemistry,2003,42(40):11846-11856
5Wang K, Wang B, Yang H-L, et al. New strategy for specific activation of recombinant microbialpro-transglutaminase by introducing an enterokinase cleavage site[J]. Biotechnol Lett,2013,35(3):383-388
6Asano Y, Tani Y, Yamada H. A new enzyme "Nitrile hydratase" which degrades acetonitrile incombination with amidase[J]. Agr Biol Chem,1980,44(9):2251-2252
7Kato Y, Nakamura K, Sakiyama H, et al. Novel heme-containing lyase, phenylacetaldoximedehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of thegene[J]. Biochemistry,2000,39(4):800-809
8Foerstner K U, Doerks T, Muller J, et al. A nitrile hydratase in the eukaryote Monosiga brevicollis[J].PLoS One,2008,3(12): e3976
9Marron A O, Akam M, Walker G. Nitrile hydratase genes are present in multiple eukaryoticsupergroups[J]. PloS one,2012,7(4): e32867
10King N, Westbrook M J, Young S L, et al. The genome of the choanoflagellate Monosiga brevicollisand the origin of metazoans[J]. Nature,2008,451(7180):783-788
11Mascharak P K. Structural and functional models of nitrile hydratase[J]. Coordin Chem Rev,2002,225(1):201-214
12董礼,陈敏,李梅,等.柴胡红景天中一个新氰苷类化合物[J].药学学报,2009,44(12):1383-1386
13Komeda H, Kobayashi M, Shimizu S. Characterization of the gene cluster of high-molecular-massnitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1[J]. Proc NatlAcad Sci U S A,1996,93(9):4267-4272
14Battistel E, Bernardi A, Maestri P. Enzymatic decontamination of aqueous polymer emulsionscontaining acrylonitrile[J]. Biotechnol Lett,1997,19(2):131-134
15Schmid A, Dordick J, Hauer B, et al. Industrial biocatalysis today and tomorrow[J]. Nature,2001,409(6817):258-268
16Vejvoda V, Martínková L, Veselá A B, et al. Biotransformation of nitriles to hydroxamic acids via anitrile hydratase amidase cascade reaction[J]. J Mol Catal B-Enzym,2011,71(1):51-55
17Babu V, Choudhury B. Competitive adsorptions of nitrile hydratase and amidase on polyacrylonitrileand its effect on surface modification[J]. Colloid Surface B,2012,89(2):277-282
18Nagasawa T, Shimizu H, Yamada H. The superiority of the third-generation catalyst, Rhodococcusrhodochrou J1nitrile hydratase, for industrial production of acrylamide[J]. Appl Microbiol Biot,1993,40(2-3):189-195
19Yamada H, Kobayashi M. Nitrile hydratase and its application to industrial production ofacrylamide[J]. Biosci Biotech Bioch,1996,60(9):1391-1400
20Chen A C, Damian D L. Nicotinamide and the skin[J]. Australas J Dermatol,2014,7(4):41-45
21Mu G, Luan F, Liu H, et al. Use of experimental design and artificial neural network in optimization ofcapillary electrophoresis for the determination of nicotinic acid and nicotinamide in food compared withhigh-performance liquid chromatography[J]. Food Anal Method,2013,6(1):191-200
22肖志斌,洪挺,陆豫.二氧化锰催化水解3-氰基吡啶生成烟酰胺[J].南昌大学学报:理科版,2011,35(4):365-369
23Prasad S, Bhalla T C. Nitrile hydratases (NHases): At the interface of academia and industry[J].Biotechnol Adv,2010,28(6):725-741
24Yamaki T, Oikawa T, Ito K, et al. Cloning and sequencing of a nitrile hydratase gene fromPseudonocardia thermophila JCM3095[J]. J Ferment Bioeng,1997,83(5):474-477
25Cameron R A, Sayed M, Cowan D A. Molecular analysis of the nitrile catabolism operon of thethermophile Bacillus pallidus RAPc8[J]. Bba-Gen Subjects,2005,1725(1):35-46
26Hourai S, Miki M, Takashima Y, et al. Crystal structure of nitrile hydratase from a thermophilicBacillus smithii[J]. Biochem Bioph Res Co,2003,312(2):340-345
27Chiyanzu I, Cowan D A, Burton S G. Immobilization of Geobacillus pallidus RAPc8nitrile hydratase(NHase) reduces substrate inhibition and enhances thermostability[J]. J Mol Catal B-Enzym,2010,63(3):109-115
28Nagasawa T, Takeuchi K, Nardi-Dei V, et al. Optimum culture conditions for the production ofcobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1[J]. Appl Microbiol Biot,1991,34(6):783-788
29Nagasawa T, Takeuchi K, Yamada H. Characterization of a new cobalt-containing nitrile hydratasepurified from urea-induced cells of Rhodococcus rhodochrous J1[J]. Eur J Biochem,1991,196(3):581-589
30李建军,李纪生.二腈的区域和对映选择性生物水解反应-腈水合酶与二腈分子作用模式初探[J].化学学报,2001,59(10):1827-1830
31Moreau J, Azza S, Bigey F, et al. Application of high-performance liquid chromatography to the studyof the biological transformation of adiponitrile[J]. J Chromatogr B,1994,656(1):197-202
32Shen Y, Wang M, Li X, et al. Highly efficient synthesis of5-cyanovaleramide byRhodococcusruberCGMCC3090resting cells[J]. J Chem Technol Biot,2012,87(10):1396-1400
33Petrillo K L, Wu S, Hann E C, et al. Over-expression in Escherichia coli of a thermally stable andregio-selective nitrile hydratase from Comamonas testosteroni5-MGAM-4D[J]. Appl Microbiol Biot,2005,67(5):664-670
34Shapiro R, DiCosimo R, Hennessey S M, et al. Discovery and development of a commercial synthesisof azafenidin[J]. Org Process Res Dev,2001,5(6):593-598
35Wu J, Zaleski T J, Valenzano C, et al. Polyketide double bond biosynthesis. Mechanistic analysis ofthe dehydratase-containing module2of the picromycin/methymycin polyketide synthase[J]. J Am ChemSoc,2005,127(49):17393-17404
36Wu J, Bell A F, Jaye A A, et al. Ring current effects in the active site of medium-chain Acyl-CoAdehydrogenase revealed by NMR spectroscopy[J]. J Am Chem Soc,2005,127(23):8424-8432
37Wu J, Cheung T, Grande C, et al. Biochemical characterization of human SET and MYNDdomain-containing protein2methyltransferase[J]. Biochemistry,2011,50(29):6488-6497
38Wu J, He W, Khosla C, et al. Chain elongation, macrolactonization, and hydrolysis of natural andreduced hexaketide substrates by the picromycin/methymycin polyketide synthase[J]. Angew Chem-GerEdit,2005,117(46):7729-7732
39Zhou Z, Hashimoto Y, Kobayashi M. Nitrile degradation by Rhodococcus: Useful microbialmetabolism for industrial productions[J].日本放线菌学会志,2005,19(1):18-26
40Zhou Z, Hashimoto Y, Kobayashi M. Self-subunit swapping chaperone needed for the maturation ofmultimeric metalloenzyme nitrile hydratase by a subunit exchange mechanism also carries out theoxidation of the metal ligand cysteine residues and insertion of cobalt[J]. J Biol Chem,2009,284(22):14930-14938
41Zhou Z, Hashimoto Y, Shiraki K, et al. Discovery of posttranslational maturation by self-subunitswapping[J]. Proc Natl Acad Sci U S A,2008,105(39):14849-14854
42Nagashima S, Nakasako M, Dohmae N, et al. Novel non-heme iron center of nitrile hydratase with aclaw setting of oxygen atoms[J]. Nat Struct Mol Biol,1998,5(5):347-351
43Miyanaga A, Fushinobu S, Ito K, et al. Mutational and structural analysis of cobalt‐containing nitrilehydratase on substrate and metal binding[J]. Eur J Biochem,2004,271(2):429-438
44Arakawa T, Kawano Y, Kataoka S, et al. Structure of thiocyanate hydrolase: a new nitrile hydratasefamily protein with a novel five-coordinate cobalt (III) center[J]. J Mol Biol,2007,366(5):1497-1509
45Brodkin H R, Novak W R, Milne A C, et al. Evidence of the participation of remote residues in thecatalytic activity of co-type nitrile hydratase from Pseudomonas putida[J]. Biochemistry,2011,50(22):4923-4935
46Swartz R D, Coggins M K, Kaminsky W, et al. Nitrile Hydration by Thiolate-and Alkoxide-LigatedCo-NHase Analogues. Isolation of Co (III)-Amidate and Co (III)-Iminol Intermediates[J]. J Am Chem Soc,2011,133(11):3954-3963
47Song L, Wang M, Shi J, et al. High resolution X-ray molecular structure of the nitrile hydratase fromRhodococcus erythropolis AJ270reveals posttranslational oxidation of two cysteines into sulfinic acids anda novel biocatalytic nitrile hydration mechanism[J]. Biochem Biophys Res Commun,2007,362(2):319-324
48Mitra S, Holz R C. Unraveling the catalytic mechanism of nitrile hydratases[J]. J Biol Chem,2007,282(10):7397-7404
49Yamanaka Y, Hashimoto K, Ohtaki A, et al. Kinetic and structural studies on roles of the serine ligandand a strictly conserved tyrosine residue in nitrile hydratase[J]. J Biol Inorg Chem,2010,15(5):655-665
50Yu H, Liu J, Shen Z. Modeling catalytic mechanism of nitrile hydratase by semi-empirical quantummechanical calculation[J]. J Mol Graph Model,2008,27(4):522-528
51Kuhn M L, Martinez S, Gumataotao N, et al. The Fe-Type Nitrile Hydratase from Comamonastestosteroni Ni1Does Not Require an Activator Accessory Protein for Expression in Escherichia coli[J].Biochem Biophys Res Commun,2012,424(3):365-370
52Okamoto S, Van Petegem F, Patrauchan M A, et al. AnhE, a metallochaperone involved in thematuration of a cobalt-dependent nitrile hydratase[J]. J Biol Chem,2010,285(33):25126-25133
53Nojiri M, Yohda M, Odaka M, et al. Functional expression of nitrile hydratase in Escherichia coli:requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine[J]. JBiochem,1999,125(4):696-704
54Nishiyama M, Horinouchi S, Kobayashi M, et al. Cloning and characterization of genes responsiblefor metabolism of nitrile compounds from Pseudomonas chlororaphis B23[J]. J Bacteriol,1991,173(8):2465-2472
55Hashimoto Y, Nishiyama M, Horinouchi S, et al. Nitrile hydratase gene from Rhodococcus sp. N-774requirement for its downstream region for efficient expression[J]. Biosci Biotechnol Biochem,1994,58(10):1859-1864
56Wu S, Fallon R D, Payne M S. Over-production of stereoselective nitrile hydratase from Pseudomonasputida5B in Escherichia coli: activity requires a novel downstream protein[J]. Appl Microbiol Biot,1997,48(6):704-708
57Lu J, Zheng Y, Yamagishi H, et al. Motif CXCC in nitrile hydratase activator is critical for NHasebiogenesis in vivo[J]. FEBS Lett,2003,553(3):391-396
58Mitra S, Job K M, Meng L, et al. Analyzing the catalytic role of Asp97in the methionineaminopeptidase from Escherichia coli[J]. FEBS J,2008,275(24):6248-6259
59Deng H, Callender R, Zhu J, et al. Determination of the ionization state and catalytic function ofGlu-133in Peptide deformylase by difference FTIR spectroscopy[J]. Biochemistry,2002,41(33):10563-10569
60Cai J, Han C, Hu T, et al. Peptide deformylase is a potential target for anti-Helicobacter pylori drugs:Reverse docking, enzymatic assay, and X-ray crystallography validation[J]. Protein Sci,2006,15(9):2071-2081
61Chae P S, Kim M, Jeung C S, et al. Peptide-cleaving catalyst selective for peptide deformylase[J]. JAm Chem Soc,2005,127(8):2396-2397
62Waldron K J, Rutherford J C, Ford D, et al. Metalloproteins and metal sensing[J]. Nature,2009,460(7257):823-830
63Schwarz G, Mendel R R, Ribbe M W. Molybdenum cofactors, enzymes and pathways[J]. Nature,2009,460(7257):839-847
64Tottey S, Waldron K J, Firbank S J, et al. Protein-folding location can regulate manganese-bindingversus copper-or zinc-binding[J]. Nature,2008,455(7216):1138-1142
65Hu Y, Fay A W, Ribbe M W. Molecular insights into nitrogenase FeMo cofactor insertion: the role ofHis362of the MoFe protein α subunit in FeMo cofactor incorporation[J]. J Biol Inorg Chem,2007,12(4):449-460
66Fay A W, Hu Y, Schmid B, et al. Molecular insights into nitrogenase FeMoco insertion-The role of His274and His451of MoFe protein [alpha] subunit[J]. J Inorg Biochem,2007,101(11-12):1630-1641
67Hu Y, Fay A W, Schmid B, et al. Molecular Insights into Nitrogenase FeMoco Insertion[J]. J BiolChem,2006,281(41):30534-30541
68Changela A, Chen K, Xue Y, et al. Molecular basis of metal-ion selectivity and zeptomolar sensitivityby CueR[J]. Science,2003,301(5638):1383-1387
69Ribbe M W, Burgess B K. The chaperone GroEL is required for the final assembly of themolybdenum-iron protein of nitrogenase[J]. Proc Natl Acad Sci U S A,2001,98(10):5521-5526
70Lei M, Lu W, Meng W, et al. Structure of PAK1in an autoinhibited conformation reveals a multistageactivation switch[J]. Cell,2000,102(3):387-397
71Meng W, Swenson L L, Fitzgibbon M J, et al. Structure of mitogen-activated protein kinase-activatedprotein (MAPKAP) kinase2suggests a bifunctional switch that couples kinase activation with nuclearexport[J]. J Biol Chem,2002,277(40):37401-37405
72Kataoka S, Arakawa T, Hori S, et al. Functional expression of thiocyanate hydrolase is promoted by itsactivator protein, P15K[J]. FEBS Lett,2006,580(19):4667-4672
73Murakami T, Nojiri M, Nakayama H, et al. Post-translational modification is essential for catalyticactivity of nitrile hydratase[J]. Protein Sci,2000,9(5):1024-1030
74Stevens J M, Belghazi M, Jaouen M, et al. Post-translational modification of Rhodococcus R312andComamonas NI1nitrile hydratases[J]. J Mass Spectrom,2003,38(9):955-961
75Shearer J, Callan P E, Amie J. Use of Metallopeptide Based Mimics Demonstrates That theMetalloprotein Nitrile Hydratase Requires Two Oxidized Cysteinates for Catalytic Activity[J]. Inorg Chem,2010,49(19):9064-9077
76Arakawa T, Kawano Y, Katayama Y, et al. Structural Basis for Catalytic Activation of ThiocyanateHydrolase Involving Metal-Ligated Cysteine Modification[J]. J Am Chem Soc,2009,131(41):14838-14843
77Reddie K G, Carroll K S. Expanding the functional diversity of proteins through cysteine oxidation[J].Curr Opin Chem Biol,2008,12(6):746-754
78Zhou Z, Hashimoto Y, Cui T, et al. Unique biogenesis of high-molecular mass multimericmetalloenzyme nitrile hydratase: intermediates and a proposed mechanism for self-subunit swappingmaturation[J]. Biochemistry,2010,49(44):9638-9648
79高毅,刘铭,曹竹安.生物法生产丙烯酰胺过程中腈水合酶的抑制和失活[J].过程工程学报,2005,5(2):193-196
80罗积杏,薛建萍,沈寅初.我国生物法生产丙烯酰胺的现状及研发概况[J].上海化工,2007,32(2):17-21
81刘铭,李春,高毅,等. Nocardia sp. RS合成腈水合酶过程及其高酶活的表达工艺[J].过程工程学报,2003,3(6):555-559
82Cantarella L, Gallifuoco A, Spera A, et al. Nitrile, amide and temperature effects on amidase-kineticsduring acrylonitrile bioconversion by nitrile-hydratase/amidase in-situ cascade system[J]. BioresourceTechnol,2013,142(3):320-328
83Cantarella L, Gallifuoco A, Malandra A, et al. Application of continuous stirred membrane reactor to3-cyanopyridine bioconversion using the nitrile hydratase–amidase cascade system of Microbacteriumimperiale CBS498-74[J]. Enzyme Microb Tech,2010,47(3):64-70
84孙云鹏,于慧敏,孙旭东,等.游离细胞腈水合酶催化丙烯腈水合反应的双稳态反应动力学[J].化工学报,2010(7):1783-1789
85张蕾.腈水合酶固定化细胞的载体选择及复合固定化的研究[D]:[硕士学位论文].哈尔滨:哈尔滨理工大学,2009
86张云天.利用膜生物反应器微生物转化丙烯酰胺的研究[D]:[硕士学位论文].哈尔滨:哈尔滨理工大学,2008
87黄绣川,曾亮,胡常伟,等.腈水合酶催化水合制备丙烯酰胺反应的研究[J].石油化工,2005,33(10):978-982
88马武生.诺卡氏菌(Nocardia sp.) HD9611发酵优化控制及其游离细胞法转化丙烯酰胺的研究[D]:[硕士学位论文].河南:河南大学,2004
89Kubá D, Kaplan O, Eli áková V, et al. Biotransformation of nitriles to amides using soluble andimmobilized nitrile hydratase from Rhodococcus erythropolis A4[J]. J Mol Catal B-Enzym,2008,50(2):107-113
90Rodrigues R C, Ortiz C, Berenguer-Murcia á, et al. Modifying enzyme activity and selectivity byimmobilization[J]. Chem Soc Rev,2013,42(15):6290-6307
91Perezgasga L, Sánchez-Sánchez L, Aguila S, et al. Substitution of the Catalytic Metal and ProteinPEGylation Enhances Activity and Stability of Bacterial Phosphotriesterase[J]. Appl Biochem Biotech,2012,166(5):1236-1247
92Yu H, Li J, Zhang D, et al. Improving the thermostability of N-carbamyl-D-amino acidamidohydrolase by error-prone PCR[J]. Appl Microbiol Biot,2009,82(2):279-285
93Park Y-M, Phi Q T, Song B-H, et al. Thermostability of chimeric cytidine deaminase variantsproduced by DNA shuffling[J]. J Microbiol Biotechn,2009,19(12):1536-1541
94Buettner K, Hertel T C, Pietzsch M. Increased thermostability of microbial transglutaminase bycombination of several hot spots evolved by random and saturation mutagenesis[J]. Amino acids,2012,42(2-3):987-996
95Komeda H, Ishikawa N, Asano Y. Enhancement of the thermostability and catalytic activity ofd-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3by directed evolution[J]. J MolCatal B-Enzym,2003,21(4):283-290
96Acharya P, Rajakumara E, Sankaranarayanan R, et al. Structural Basis of Selection andThermostability of Laboratory Evolved Bacillus subtilis Lipase[J]. J Mol Biol,2004,341(5):1271-1281
97Oh K-H, Nam S-H, Kim H-S. Improvement of oxidative and thermostability of N-carbamyl-D-aminoacid amidohydrolase by directed evolution[J]. Protein Eng,2002,15(8):689-695
98Malakauskas S M, Mayo S L. Design, structure and stability of a hyperthermophilic protein variant[J].Nat Struct Mol Biol,1998,5(6):470-475
99Dombkowski A A. Disulfide by Design: a computational method for the rational design of disulfidebonds in proteins[J]. Bioinformatics,2003,19(14):1852-1853
100Huang P-S, Ban Y-E A, Richter F, et al. RosettaRemodel: a generalized framework for flexiblebackbone protein design[J]. PloS one,2011,6(8): e24109
101Zhang W, Mullaney E J, Lei X G. Adopting selected hydrogen bonding and ionic interactions fromAspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase[J].Appl Environ Microb,2007,73(9):3069-3076
102Mabrouk S B, Aghajari N, Ali M B, et al. Enhancement of the thermostability of the maltogenicamylase MAUS149by Gly312Ala and Lys436Arg substitutions[J]. Bioresource Technol,2011,102(2):1740-1746
103Schwehm J M, Kristyanne E S, Biggers C C, et al. Stability effects of increasing the hydrophobicity ofsolvent-exposed side chains in staphylococcal nuclease[J]. Biochemistry,1998,37(19):6939-6948
104Nicholson H, Anderson D, Dao Pin S, et al. Analysis of the interaction between charged side chainsand the alpha-helix dipole using designed thermostable mutants of phage T4lysozyme[J]. Biochemistry,1991,30(41):9816-9828
105Vieille C, Zeikus G J. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms forthermostability[J]. Microbiol Mol Biol R,2001,65(1):1-43
106Yoshida H, Kojima K, Witarto A B, et al. Engineering a chimeric pyrroloquinoline quinone glucosedehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity[J]. Protein Eng,1999,12(1):63-70
107周晓丽.蛋白质结构域重组构建新功能酶[D]:[博士学位论文].吉林:吉林大学,2012
108Takano K, Okamoto T, Okada J, et al. Stabilization by fusion to the C-terminus of hyperthermophileSulfolobus tokodaii RNase HI: a possibility of protein stabilization tag[J]. PLoS One,2011,6(1): e16226
109García-Fruitós E, Vázquez E, Díez-Gil C, et al. Bacterial inclusion bodies: Making gold from waste[J].Trends Biotechnol,2011,30(2):65-70
110De Groot N S, Sabate R, Ventura S. Amyloids in bacterial inclusion bodies[J]. Trends Biochem Sci,2009,34(8):408-416
111Wang L, Maji S K, Sawaya M R, et al. Bacterial inclusion bodies contain amyloid-like structure[J].Plos Biol,2008,6(8): e195
112Morell M, Bravo R, Espargaró A, et al. Inclusion bodies: specificity in their aggregation process andamyloid-like structure[J]. Bba-Mol Cell Res,2008,1783(10):1815-1825
113De Groot N S, Ventura S. Protein activity in bacterial inclusion bodies correlates with predictedaggregation rates[J]. J Biotechnol,2006,125(1):110-113
114García-Fruitós E, González-Montalbán N, Morell M, et al. Aggregation as bacterial inclusion bodiesdoes not imply inactivation of enzymes and fluorescent proteins[J]. Microb Cell Fact,2005,4(1):27-35
115Dellus-Gur E, Toth-Petroczy A, Elias M, et al. What makes a protein fold amenable to functionalinnovation? Fold polarity and stability trade-offs[J]. J Mol Biol,2013,425(14):2609-2621
116Wu W, Xing L, Zhou B H, et al. Active protein aggregates induced by terminally attachedself-assembling peptide ELK16in Escherichia coli[J]. Microb Cell Fact,2011,10(1):9-13
117Zhang S, Lockshin C, Herbert A, et al. Zuotin, a putative Z-DNA binding protein in Saccharomycescerevisiae[J]. EMBO J,1992,11(10):3787-3794
118Tokuriki N, Tawfik D S. Stability effects of mutations and protein evolvability[J]. Curr Opin StrucBiol,2009,19(5):596-604
119Goldsmith M, Tawfik D S. Enzyme engineering by targeted libraries[J]. Method Enzymol,2013,523:257-283
120Yu H, Huang H. Engineering Proteins for Thermostability through Rigidifying Flexible Sites (RFS)[J].Biotechnol Adv,2013,32(2):308–315
121王帅.基于同源建模的蛋白质结构预测方法的研究[D]:[硕士学位论文].哈尔滨:哈尔滨工业大学,2006
122王志珍.蛋白质折叠和分子伴侣[J].生物学通报,2004,39(5):1-6
123Yu H, Liu J, Shen Z. Modeling catalytic mechanism of nitrile hydratase by semi-empirical quantummechanical calculation[J]. J Mol Graph Model,2008,27(4):522-528
124Miyanaga A, Fushinobu S, Ito K, et al. Mutational and structural analysis of cobalt-containing nitrilehydratase on substrate and metal binding[J]. Eur J Biochem,2004,271(2):429-438
125Peplowski L, Kubiak K, Nowak W. Insights into catalytic activity of industrial enzyme Co-nitrilehydratase[J]. J Mol Model,2007,13(6-7):725-730
126Wu R, Gong W, Liu T, et al. A QM/MM Molecular Dynamics Study of Purine-Specific NucleosideHydrolase[J]. J Phys Chem B,2012,116(6):1984-1991
127Lonsdale R, Hoyle S, Grey D T, et al. Determinants of Reactivity and Selectivity in Soluble EpoxideHydrolase from QM/MM Modeling[J]. Biochemistry,2012,51(8):1774-1786
128Hou Q, Wang J, Gao J, et al. QM/MM studies on the catalytic mechanism of PhenylethanolamineN-methyltransferase[J]. Bba-Proteins Proteom,2012,1824(4):533-541
129De Marco A. Strategies for successful recombinant expression of disulfide bond-dependent proteins inEscherichia coli[J]. Microb Cell Fact,2009,8(1):26-35
130Khatib F, Cooper S, Tyka M D, et al. Algorithm discovery by protein folding game players[J]. ProcNatl Acad Sci U S A,2011,108(47):18949-18953
131R thlisberger D, Khersonsky O, Wollacott A M, et al. Kemp elimination catalysts by computationalenzyme design[J]. Nature,2008,453(7192):190-195
132Eiben C B, Siegel J B, Bale J B, et al. Increased Diels-Alderase activity through backbone remodelingguided by Foldit players[J]. Nat Biotechnol,2012,30(2):190-192
133Okamoto S, Eltis L D. The biological occurrence and trafficking of cobalt[J]. Metallomics,2011,3(10):963-970
134Kim S-H, Oriel P. Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp.BR449into Escherichia coli[J]. Enzyme Microb Tech,2000,27(7):492-501
135Petrillo K L, Wu S, Hann E C, et al. Over-expression in Escherichia coli of a thermally stable andregio-selective nitrile hydratase from Comamonas testosteroni5-MGAM-4D[J]. Appl Microbiol Biot,2005,67(5):664-670
136Salis H M, Mirsky E A, Voigt C A. Automated design of synthetic ribosome binding sites to controlprotein expression[J]. Nat Biotechnol,2009,27(10):946-950
137Icev A, Ruiz C, Ryder E F. Distance-enhanced association rules for gene expression[J]. Gene,2003,10(2):140-144
138Sahdev S, Khattar S K, Saini K S. Production of active eukaryotic proteins through bacterialexpression systems: a review of the existing biotechnology strategies[J]. Mol Cell Biochem,2008,307(1):249-264
139Tasaki T, Sriram S M, Park K S, et al. Regulation of Protein Function by N-Terminal DegradationSignal[J]. Biochemistry,2012,81(2):62-69
140Varshavsky A. The N-end rule pathway and regulation by proteolysis[J]. Protein Sci,2011,20(8):1298-1345
141Sriram S M, Kim B Y, Kwon Y T. The N-end rule pathway: emerging functions and molecularprinciples of substrate recognition[J]. Nat Rev Mol Cell Bio,2011,12(11):735-747
142Pival S L, Birner-Gruenberger R, Krump C, et al. D-Xylulose kinase from Saccharomyces cerevisiae:Isolation and characterization of the highly unstable enzyme, recombinantly produced in Escherichiacoli[J]. Protein Expres Purif,2011,79(2):223-230
143Licausi F, Kosmacz M, Weits D A, et al. Oxygen sensing in plants is mediated by an N-end rulepathway for protein destabilization[J]. Nature,2011,479(7373):419-422
144Lee P C W, Sowa M E, Gygi S P, et al. Alternative Ubiquitin Activation/Conjugation Cascades Interactwith N-End Rule Ubiquitin Ligases to Control Degradation of RGS Proteins[J]. Mol Cell,2011,43(3):392-405
145Gibbs D J, Lee S C, Isa N M, et al. Homeostatic response to hypoxia is regulated by the N-end rulepathway in plants[J]. Nature,2011,479(7373):415-418
146Sriram S M, Kwon Y T. The molecular principles of N-end rule recognition[J]. Nat Struct Mol Biol,2010,17(10):1164-1170
147Fang N, Zhong C Q, Liang X L, et al. Improvement of extracellular production of a thermophilicsubtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide[J]. ApplMicrobiol Biotechnol,2010,85(5):1473-1481
148Dougan D, Truscott K, Zeth K. The bacterial N-end rule pathway: expect the unexpected[J]. MolMicrobiol,2010,76(3):545-558
149Choi W S, Jeong B C, Joo Y J, et al. Structural basis for the recognition of N-end rule substrates by theUBR box of ubiquitin ligases[J]. Nat Struct Mol Biol,2010,17(10):1175-1181
150Chen J, Yu H, Liu C, et al. Improving stability of nitrile hydratase by bridging the salt-bridges inspecific thermal-sensitive regions[J]. J Biotechnol,2013,164(2):354-362
151Peplowski L, Kubiak K, Nowak W. Mechanical aspects of nitrile hydratase enzymatic activity. Steeredmolecular dynamics simulations of Pseudonocardia thermophila JCM3095[J]. Chem Phys Lett,2008,467(1):144-149
152Kubiak K, Nowak W. Molecular dynamics simulations of the photoactive protein nitrile hydratase[J].Biophys J,2008,94(10):3824-3838
153Zhang Y, Zeng Z, Zeng G, et al. Enzyme–Substrate Binding Landscapes in the Process of NitrileBiodegradation Mediated by Nitrile Hydratase and Amidase[J]. Appl Biochem Biotech,2013,170(7):1-10
154Kwon W S, Da Silva N A, Kellis Jr J T. Relationship between thermal stability, degradation rate andexpression yield of barnase variants in the periplasm of Escherichia coli[J]. Protein Eng,1996,9(12):1197-1202
155Choi J, Lee S. Secretory and extracellular production of recombinant proteins using Escherichiacoli[J]. Appl Microbiol Biotechnol,2004,64(5):625-635
156Johnston J A, Johnson E S, Waller P R H, et al. Methotrexate inhibits proteolysis of dihydrofolatereductase by the N-end rule pathway[J]. J Biol Chem,1995,270(14):8172-8178
157Schmidt R, Bukau B, Mogk A. Principles of general and regulatory proteolysis by AAA+proteases inEscherichia coli[J]. Res Microbiol,2009,160(9):629-636
158Wu S, Fallon R, Payne M. Over-production of stereoselective nitrile hydratase from Pseudomonasputida5B in Escherichia coli: activity requires a novel downstream protein[J]. Appl Microbiol Biot,1997,48(6):704-708
159Ba L A, Doering M, Burkholz T, et al. Metal trafficking: from maintaining the metal homeostasis tofuture drug design[J]. Metallomics,2009,1(4):292-311
160Teilum K, Olsen J G, Kragelund B B. Protein stability, flexibility and function[J]. Biochim BiophysActa,2011,1814(8):969-976
161Bahar I, Lezon T R, Bakan A, et al. Normal mode analysis of biomolecular structures: functionalmechanisms of membrane proteins[J]. Chem Rev,2010,110(3):1463-1468
162Hamelberg D, McCammon J A. Fast peptidyl cis-trans isomerization within the flexible Gly-rich flapsof HIV-1protease[J]. J Am Chem Soc,2005,127(40):13778-13779
163Liu Y, Cui W, Xia Y, et al. Self-Subunit Swapping Occurs in Another Gene Type of Cobalt NitrileHydratase[J]. PLoS One,2012,7(11): e50829
164Ma Y, Yu H, Pan W, et al. Identification of nitrile hydratase-producing Rhodococcus ruber TH andcharacterization of an amiE-negative mutant[J]. Bioresource Technol,2010,101(1):285-291
165Chen J, Yu H, Liu C, et al. Improving stability of nitrile hydratase by bridging the salt-bridges inspecific thermal-sensitive regions[J]. J Biotechnol,2012,164(2):354-362
166Zhang S, Holmes T, Lockshin C, et al. Spontaneous assembly of a self-complementary oligopeptide toform a stable macroscopic membrane[J]. Proc Natl Acad Sci U S A,1993,90(8):3334-3338
167Lazar K L, Miller-Auer H, Getz G S, et al. Helix-turn-helix peptides that form α-helical fibrils: Turnsequences drive fibril structure[J]. Biochemistry,2005,44(38):12681-12689
168Zhou B, Xing L, Wu W, et al. Small surfactant-like peptides can drive soluble proteins into active
aggregates[J]. Microb Cell Fact,2012,11(10):26-34
2Wu J, Kinoshita K, Khosla C, et al. Biochemical analysis of the substrate specificity of theβ-ketoacyl-acyl carrier protein synthase domain of module2of the erythromycin polyketide synthase[J].Biochemistry,2004,43(51):16301-16310
3Kobayashi M, Shimizu S. Cobalt proteins[J]. Eur J Biochem,1999,261(1):1-9
4Wu J, Bell A F, Luo L, et al. Probing hydrogen-bonding interactions in the active site of medium-chainacyl-CoA dehydrogenase using Raman spectroscopy[J]. Biochemistry,2003,42(40):11846-11856
5Wang K, Wang B, Yang H-L, et al. New strategy for specific activation of recombinant microbialpro-transglutaminase by introducing an enterokinase cleavage site[J]. Biotechnol Lett,2013,35(3):383-388
6Asano Y, Tani Y, Yamada H. A new enzyme "Nitrile hydratase" which degrades acetonitrile incombination with amidase[J]. Agr Biol Chem,1980,44(9):2251-2252
7Kato Y, Nakamura K, Sakiyama H, et al. Novel heme-containing lyase, phenylacetaldoximedehydratase from Bacillus sp. strain OxB-1: purification, characterization, and molecular cloning of thegene[J]. Biochemistry,2000,39(4):800-809
8Foerstner K U, Doerks T, Muller J, et al. A nitrile hydratase in the eukaryote Monosiga brevicollis[J].PLoS One,2008,3(12): e3976
9Marron A O, Akam M, Walker G. Nitrile hydratase genes are present in multiple eukaryoticsupergroups[J]. PloS one,2012,7(4): e32867
10King N, Westbrook M J, Young S L, et al. The genome of the choanoflagellate Monosiga brevicollisand the origin of metazoans[J]. Nature,2008,451(7180):783-788
11Mascharak P K. Structural and functional models of nitrile hydratase[J]. Coordin Chem Rev,2002,225(1):201-214
12董礼,陈敏,李梅,等.柴胡红景天中一个新氰苷类化合物[J].药学学报,2009,44(12):1383-1386
13Komeda H, Kobayashi M, Shimizu S. Characterization of the gene cluster of high-molecular-massnitrile hydratase (H-NHase) induced by its reaction product in Rhodococcus rhodochrous J1[J]. Proc NatlAcad Sci U S A,1996,93(9):4267-4272
14Battistel E, Bernardi A, Maestri P. Enzymatic decontamination of aqueous polymer emulsionscontaining acrylonitrile[J]. Biotechnol Lett,1997,19(2):131-134
15Schmid A, Dordick J, Hauer B, et al. Industrial biocatalysis today and tomorrow[J]. Nature,2001,409(6817):258-268
16Vejvoda V, Martínková L, Veselá A B, et al. Biotransformation of nitriles to hydroxamic acids via anitrile hydratase amidase cascade reaction[J]. J Mol Catal B-Enzym,2011,71(1):51-55
17Babu V, Choudhury B. Competitive adsorptions of nitrile hydratase and amidase on polyacrylonitrileand its effect on surface modification[J]. Colloid Surface B,2012,89(2):277-282
18Nagasawa T, Shimizu H, Yamada H. The superiority of the third-generation catalyst, Rhodococcusrhodochrou J1nitrile hydratase, for industrial production of acrylamide[J]. Appl Microbiol Biot,1993,40(2-3):189-195
19Yamada H, Kobayashi M. Nitrile hydratase and its application to industrial production ofacrylamide[J]. Biosci Biotech Bioch,1996,60(9):1391-1400
20Chen A C, Damian D L. Nicotinamide and the skin[J]. Australas J Dermatol,2014,7(4):41-45
21Mu G, Luan F, Liu H, et al. Use of experimental design and artificial neural network in optimization ofcapillary electrophoresis for the determination of nicotinic acid and nicotinamide in food compared withhigh-performance liquid chromatography[J]. Food Anal Method,2013,6(1):191-200
22肖志斌,洪挺,陆豫.二氧化锰催化水解3-氰基吡啶生成烟酰胺[J].南昌大学学报:理科版,2011,35(4):365-369
23Prasad S, Bhalla T C. Nitrile hydratases (NHases): At the interface of academia and industry[J].Biotechnol Adv,2010,28(6):725-741
24Yamaki T, Oikawa T, Ito K, et al. Cloning and sequencing of a nitrile hydratase gene fromPseudonocardia thermophila JCM3095[J]. J Ferment Bioeng,1997,83(5):474-477
25Cameron R A, Sayed M, Cowan D A. Molecular analysis of the nitrile catabolism operon of thethermophile Bacillus pallidus RAPc8[J]. Bba-Gen Subjects,2005,1725(1):35-46
26Hourai S, Miki M, Takashima Y, et al. Crystal structure of nitrile hydratase from a thermophilicBacillus smithii[J]. Biochem Bioph Res Co,2003,312(2):340-345
27Chiyanzu I, Cowan D A, Burton S G. Immobilization of Geobacillus pallidus RAPc8nitrile hydratase(NHase) reduces substrate inhibition and enhances thermostability[J]. J Mol Catal B-Enzym,2010,63(3):109-115
28Nagasawa T, Takeuchi K, Nardi-Dei V, et al. Optimum culture conditions for the production ofcobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1[J]. Appl Microbiol Biot,1991,34(6):783-788
29Nagasawa T, Takeuchi K, Yamada H. Characterization of a new cobalt-containing nitrile hydratasepurified from urea-induced cells of Rhodococcus rhodochrous J1[J]. Eur J Biochem,1991,196(3):581-589
30李建军,李纪生.二腈的区域和对映选择性生物水解反应-腈水合酶与二腈分子作用模式初探[J].化学学报,2001,59(10):1827-1830
31Moreau J, Azza S, Bigey F, et al. Application of high-performance liquid chromatography to the studyof the biological transformation of adiponitrile[J]. J Chromatogr B,1994,656(1):197-202
32Shen Y, Wang M, Li X, et al. Highly efficient synthesis of5-cyanovaleramide byRhodococcusruberCGMCC3090resting cells[J]. J Chem Technol Biot,2012,87(10):1396-1400
33Petrillo K L, Wu S, Hann E C, et al. Over-expression in Escherichia coli of a thermally stable andregio-selective nitrile hydratase from Comamonas testosteroni5-MGAM-4D[J]. Appl Microbiol Biot,2005,67(5):664-670
34Shapiro R, DiCosimo R, Hennessey S M, et al. Discovery and development of a commercial synthesisof azafenidin[J]. Org Process Res Dev,2001,5(6):593-598
35Wu J, Zaleski T J, Valenzano C, et al. Polyketide double bond biosynthesis. Mechanistic analysis ofthe dehydratase-containing module2of the picromycin/methymycin polyketide synthase[J]. J Am ChemSoc,2005,127(49):17393-17404
36Wu J, Bell A F, Jaye A A, et al. Ring current effects in the active site of medium-chain Acyl-CoAdehydrogenase revealed by NMR spectroscopy[J]. J Am Chem Soc,2005,127(23):8424-8432
37Wu J, Cheung T, Grande C, et al. Biochemical characterization of human SET and MYNDdomain-containing protein2methyltransferase[J]. Biochemistry,2011,50(29):6488-6497
38Wu J, He W, Khosla C, et al. Chain elongation, macrolactonization, and hydrolysis of natural andreduced hexaketide substrates by the picromycin/methymycin polyketide synthase[J]. Angew Chem-GerEdit,2005,117(46):7729-7732
39Zhou Z, Hashimoto Y, Kobayashi M. Nitrile degradation by Rhodococcus: Useful microbialmetabolism for industrial productions[J].日本放线菌学会志,2005,19(1):18-26
40Zhou Z, Hashimoto Y, Kobayashi M. Self-subunit swapping chaperone needed for the maturation ofmultimeric metalloenzyme nitrile hydratase by a subunit exchange mechanism also carries out theoxidation of the metal ligand cysteine residues and insertion of cobalt[J]. J Biol Chem,2009,284(22):14930-14938
41Zhou Z, Hashimoto Y, Shiraki K, et al. Discovery of posttranslational maturation by self-subunitswapping[J]. Proc Natl Acad Sci U S A,2008,105(39):14849-14854
42Nagashima S, Nakasako M, Dohmae N, et al. Novel non-heme iron center of nitrile hydratase with aclaw setting of oxygen atoms[J]. Nat Struct Mol Biol,1998,5(5):347-351
43Miyanaga A, Fushinobu S, Ito K, et al. Mutational and structural analysis of cobalt‐containing nitrilehydratase on substrate and metal binding[J]. Eur J Biochem,2004,271(2):429-438
44Arakawa T, Kawano Y, Kataoka S, et al. Structure of thiocyanate hydrolase: a new nitrile hydratasefamily protein with a novel five-coordinate cobalt (III) center[J]. J Mol Biol,2007,366(5):1497-1509
45Brodkin H R, Novak W R, Milne A C, et al. Evidence of the participation of remote residues in thecatalytic activity of co-type nitrile hydratase from Pseudomonas putida[J]. Biochemistry,2011,50(22):4923-4935
46Swartz R D, Coggins M K, Kaminsky W, et al. Nitrile Hydration by Thiolate-and Alkoxide-LigatedCo-NHase Analogues. Isolation of Co (III)-Amidate and Co (III)-Iminol Intermediates[J]. J Am Chem Soc,2011,133(11):3954-3963
47Song L, Wang M, Shi J, et al. High resolution X-ray molecular structure of the nitrile hydratase fromRhodococcus erythropolis AJ270reveals posttranslational oxidation of two cysteines into sulfinic acids anda novel biocatalytic nitrile hydration mechanism[J]. Biochem Biophys Res Commun,2007,362(2):319-324
48Mitra S, Holz R C. Unraveling the catalytic mechanism of nitrile hydratases[J]. J Biol Chem,2007,282(10):7397-7404
49Yamanaka Y, Hashimoto K, Ohtaki A, et al. Kinetic and structural studies on roles of the serine ligandand a strictly conserved tyrosine residue in nitrile hydratase[J]. J Biol Inorg Chem,2010,15(5):655-665
50Yu H, Liu J, Shen Z. Modeling catalytic mechanism of nitrile hydratase by semi-empirical quantummechanical calculation[J]. J Mol Graph Model,2008,27(4):522-528
51Kuhn M L, Martinez S, Gumataotao N, et al. The Fe-Type Nitrile Hydratase from Comamonastestosteroni Ni1Does Not Require an Activator Accessory Protein for Expression in Escherichia coli[J].Biochem Biophys Res Commun,2012,424(3):365-370
52Okamoto S, Van Petegem F, Patrauchan M A, et al. AnhE, a metallochaperone involved in thematuration of a cobalt-dependent nitrile hydratase[J]. J Biol Chem,2010,285(33):25126-25133
53Nojiri M, Yohda M, Odaka M, et al. Functional expression of nitrile hydratase in Escherichia coli:requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine[J]. JBiochem,1999,125(4):696-704
54Nishiyama M, Horinouchi S, Kobayashi M, et al. Cloning and characterization of genes responsiblefor metabolism of nitrile compounds from Pseudomonas chlororaphis B23[J]. J Bacteriol,1991,173(8):2465-2472
55Hashimoto Y, Nishiyama M, Horinouchi S, et al. Nitrile hydratase gene from Rhodococcus sp. N-774requirement for its downstream region for efficient expression[J]. Biosci Biotechnol Biochem,1994,58(10):1859-1864
56Wu S, Fallon R D, Payne M S. Over-production of stereoselective nitrile hydratase from Pseudomonasputida5B in Escherichia coli: activity requires a novel downstream protein[J]. Appl Microbiol Biot,1997,48(6):704-708
57Lu J, Zheng Y, Yamagishi H, et al. Motif CXCC in nitrile hydratase activator is critical for NHasebiogenesis in vivo[J]. FEBS Lett,2003,553(3):391-396
58Mitra S, Job K M, Meng L, et al. Analyzing the catalytic role of Asp97in the methionineaminopeptidase from Escherichia coli[J]. FEBS J,2008,275(24):6248-6259
59Deng H, Callender R, Zhu J, et al. Determination of the ionization state and catalytic function ofGlu-133in Peptide deformylase by difference FTIR spectroscopy[J]. Biochemistry,2002,41(33):10563-10569
60Cai J, Han C, Hu T, et al. Peptide deformylase is a potential target for anti-Helicobacter pylori drugs:Reverse docking, enzymatic assay, and X-ray crystallography validation[J]. Protein Sci,2006,15(9):2071-2081
61Chae P S, Kim M, Jeung C S, et al. Peptide-cleaving catalyst selective for peptide deformylase[J]. JAm Chem Soc,2005,127(8):2396-2397
62Waldron K J, Rutherford J C, Ford D, et al. Metalloproteins and metal sensing[J]. Nature,2009,460(7257):823-830
63Schwarz G, Mendel R R, Ribbe M W. Molybdenum cofactors, enzymes and pathways[J]. Nature,2009,460(7257):839-847
64Tottey S, Waldron K J, Firbank S J, et al. Protein-folding location can regulate manganese-bindingversus copper-or zinc-binding[J]. Nature,2008,455(7216):1138-1142
65Hu Y, Fay A W, Ribbe M W. Molecular insights into nitrogenase FeMo cofactor insertion: the role ofHis362of the MoFe protein α subunit in FeMo cofactor incorporation[J]. J Biol Inorg Chem,2007,12(4):449-460
66Fay A W, Hu Y, Schmid B, et al. Molecular insights into nitrogenase FeMoco insertion-The role of His274and His451of MoFe protein [alpha] subunit[J]. J Inorg Biochem,2007,101(11-12):1630-1641
67Hu Y, Fay A W, Schmid B, et al. Molecular Insights into Nitrogenase FeMoco Insertion[J]. J BiolChem,2006,281(41):30534-30541
68Changela A, Chen K, Xue Y, et al. Molecular basis of metal-ion selectivity and zeptomolar sensitivityby CueR[J]. Science,2003,301(5638):1383-1387
69Ribbe M W, Burgess B K. The chaperone GroEL is required for the final assembly of themolybdenum-iron protein of nitrogenase[J]. Proc Natl Acad Sci U S A,2001,98(10):5521-5526
70Lei M, Lu W, Meng W, et al. Structure of PAK1in an autoinhibited conformation reveals a multistageactivation switch[J]. Cell,2000,102(3):387-397
71Meng W, Swenson L L, Fitzgibbon M J, et al. Structure of mitogen-activated protein kinase-activatedprotein (MAPKAP) kinase2suggests a bifunctional switch that couples kinase activation with nuclearexport[J]. J Biol Chem,2002,277(40):37401-37405
72Kataoka S, Arakawa T, Hori S, et al. Functional expression of thiocyanate hydrolase is promoted by itsactivator protein, P15K[J]. FEBS Lett,2006,580(19):4667-4672
73Murakami T, Nojiri M, Nakayama H, et al. Post-translational modification is essential for catalyticactivity of nitrile hydratase[J]. Protein Sci,2000,9(5):1024-1030
74Stevens J M, Belghazi M, Jaouen M, et al. Post-translational modification of Rhodococcus R312andComamonas NI1nitrile hydratases[J]. J Mass Spectrom,2003,38(9):955-961
75Shearer J, Callan P E, Amie J. Use of Metallopeptide Based Mimics Demonstrates That theMetalloprotein Nitrile Hydratase Requires Two Oxidized Cysteinates for Catalytic Activity[J]. Inorg Chem,2010,49(19):9064-9077
76Arakawa T, Kawano Y, Katayama Y, et al. Structural Basis for Catalytic Activation of ThiocyanateHydrolase Involving Metal-Ligated Cysteine Modification[J]. J Am Chem Soc,2009,131(41):14838-14843
77Reddie K G, Carroll K S. Expanding the functional diversity of proteins through cysteine oxidation[J].Curr Opin Chem Biol,2008,12(6):746-754
78Zhou Z, Hashimoto Y, Cui T, et al. Unique biogenesis of high-molecular mass multimericmetalloenzyme nitrile hydratase: intermediates and a proposed mechanism for self-subunit swappingmaturation[J]. Biochemistry,2010,49(44):9638-9648
79高毅,刘铭,曹竹安.生物法生产丙烯酰胺过程中腈水合酶的抑制和失活[J].过程工程学报,2005,5(2):193-196
80罗积杏,薛建萍,沈寅初.我国生物法生产丙烯酰胺的现状及研发概况[J].上海化工,2007,32(2):17-21
81刘铭,李春,高毅,等. Nocardia sp. RS合成腈水合酶过程及其高酶活的表达工艺[J].过程工程学报,2003,3(6):555-559
82Cantarella L, Gallifuoco A, Spera A, et al. Nitrile, amide and temperature effects on amidase-kineticsduring acrylonitrile bioconversion by nitrile-hydratase/amidase in-situ cascade system[J]. BioresourceTechnol,2013,142(3):320-328
83Cantarella L, Gallifuoco A, Malandra A, et al. Application of continuous stirred membrane reactor to3-cyanopyridine bioconversion using the nitrile hydratase–amidase cascade system of Microbacteriumimperiale CBS498-74[J]. Enzyme Microb Tech,2010,47(3):64-70
84孙云鹏,于慧敏,孙旭东,等.游离细胞腈水合酶催化丙烯腈水合反应的双稳态反应动力学[J].化工学报,2010(7):1783-1789
85张蕾.腈水合酶固定化细胞的载体选择及复合固定化的研究[D]:[硕士学位论文].哈尔滨:哈尔滨理工大学,2009
86张云天.利用膜生物反应器微生物转化丙烯酰胺的研究[D]:[硕士学位论文].哈尔滨:哈尔滨理工大学,2008
87黄绣川,曾亮,胡常伟,等.腈水合酶催化水合制备丙烯酰胺反应的研究[J].石油化工,2005,33(10):978-982
88马武生.诺卡氏菌(Nocardia sp.) HD9611发酵优化控制及其游离细胞法转化丙烯酰胺的研究[D]:[硕士学位论文].河南:河南大学,2004
89Kubá D, Kaplan O, Eli áková V, et al. Biotransformation of nitriles to amides using soluble andimmobilized nitrile hydratase from Rhodococcus erythropolis A4[J]. J Mol Catal B-Enzym,2008,50(2):107-113
90Rodrigues R C, Ortiz C, Berenguer-Murcia á, et al. Modifying enzyme activity and selectivity byimmobilization[J]. Chem Soc Rev,2013,42(15):6290-6307
91Perezgasga L, Sánchez-Sánchez L, Aguila S, et al. Substitution of the Catalytic Metal and ProteinPEGylation Enhances Activity and Stability of Bacterial Phosphotriesterase[J]. Appl Biochem Biotech,2012,166(5):1236-1247
92Yu H, Li J, Zhang D, et al. Improving the thermostability of N-carbamyl-D-amino acidamidohydrolase by error-prone PCR[J]. Appl Microbiol Biot,2009,82(2):279-285
93Park Y-M, Phi Q T, Song B-H, et al. Thermostability of chimeric cytidine deaminase variantsproduced by DNA shuffling[J]. J Microbiol Biotechn,2009,19(12):1536-1541
94Buettner K, Hertel T C, Pietzsch M. Increased thermostability of microbial transglutaminase bycombination of several hot spots evolved by random and saturation mutagenesis[J]. Amino acids,2012,42(2-3):987-996
95Komeda H, Ishikawa N, Asano Y. Enhancement of the thermostability and catalytic activity ofd-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3by directed evolution[J]. J MolCatal B-Enzym,2003,21(4):283-290
96Acharya P, Rajakumara E, Sankaranarayanan R, et al. Structural Basis of Selection andThermostability of Laboratory Evolved Bacillus subtilis Lipase[J]. J Mol Biol,2004,341(5):1271-1281
97Oh K-H, Nam S-H, Kim H-S. Improvement of oxidative and thermostability of N-carbamyl-D-aminoacid amidohydrolase by directed evolution[J]. Protein Eng,2002,15(8):689-695
98Malakauskas S M, Mayo S L. Design, structure and stability of a hyperthermophilic protein variant[J].Nat Struct Mol Biol,1998,5(6):470-475
99Dombkowski A A. Disulfide by Design: a computational method for the rational design of disulfidebonds in proteins[J]. Bioinformatics,2003,19(14):1852-1853
100Huang P-S, Ban Y-E A, Richter F, et al. RosettaRemodel: a generalized framework for flexiblebackbone protein design[J]. PloS one,2011,6(8): e24109
101Zhang W, Mullaney E J, Lei X G. Adopting selected hydrogen bonding and ionic interactions fromAspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase[J].Appl Environ Microb,2007,73(9):3069-3076
102Mabrouk S B, Aghajari N, Ali M B, et al. Enhancement of the thermostability of the maltogenicamylase MAUS149by Gly312Ala and Lys436Arg substitutions[J]. Bioresource Technol,2011,102(2):1740-1746
103Schwehm J M, Kristyanne E S, Biggers C C, et al. Stability effects of increasing the hydrophobicity ofsolvent-exposed side chains in staphylococcal nuclease[J]. Biochemistry,1998,37(19):6939-6948
104Nicholson H, Anderson D, Dao Pin S, et al. Analysis of the interaction between charged side chainsand the alpha-helix dipole using designed thermostable mutants of phage T4lysozyme[J]. Biochemistry,1991,30(41):9816-9828
105Vieille C, Zeikus G J. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms forthermostability[J]. Microbiol Mol Biol R,2001,65(1):1-43
106Yoshida H, Kojima K, Witarto A B, et al. Engineering a chimeric pyrroloquinoline quinone glucosedehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity[J]. Protein Eng,1999,12(1):63-70
107周晓丽.蛋白质结构域重组构建新功能酶[D]:[博士学位论文].吉林:吉林大学,2012
108Takano K, Okamoto T, Okada J, et al. Stabilization by fusion to the C-terminus of hyperthermophileSulfolobus tokodaii RNase HI: a possibility of protein stabilization tag[J]. PLoS One,2011,6(1): e16226
109García-Fruitós E, Vázquez E, Díez-Gil C, et al. Bacterial inclusion bodies: Making gold from waste[J].Trends Biotechnol,2011,30(2):65-70
110De Groot N S, Sabate R, Ventura S. Amyloids in bacterial inclusion bodies[J]. Trends Biochem Sci,2009,34(8):408-416
111Wang L, Maji S K, Sawaya M R, et al. Bacterial inclusion bodies contain amyloid-like structure[J].Plos Biol,2008,6(8): e195
112Morell M, Bravo R, Espargaró A, et al. Inclusion bodies: specificity in their aggregation process andamyloid-like structure[J]. Bba-Mol Cell Res,2008,1783(10):1815-1825
113De Groot N S, Ventura S. Protein activity in bacterial inclusion bodies correlates with predictedaggregation rates[J]. J Biotechnol,2006,125(1):110-113
114García-Fruitós E, González-Montalbán N, Morell M, et al. Aggregation as bacterial inclusion bodiesdoes not imply inactivation of enzymes and fluorescent proteins[J]. Microb Cell Fact,2005,4(1):27-35
115Dellus-Gur E, Toth-Petroczy A, Elias M, et al. What makes a protein fold amenable to functionalinnovation? Fold polarity and stability trade-offs[J]. J Mol Biol,2013,425(14):2609-2621
116Wu W, Xing L, Zhou B H, et al. Active protein aggregates induced by terminally attachedself-assembling peptide ELK16in Escherichia coli[J]. Microb Cell Fact,2011,10(1):9-13
117Zhang S, Lockshin C, Herbert A, et al. Zuotin, a putative Z-DNA binding protein in Saccharomycescerevisiae[J]. EMBO J,1992,11(10):3787-3794
118Tokuriki N, Tawfik D S. Stability effects of mutations and protein evolvability[J]. Curr Opin StrucBiol,2009,19(5):596-604
119Goldsmith M, Tawfik D S. Enzyme engineering by targeted libraries[J]. Method Enzymol,2013,523:257-283
120Yu H, Huang H. Engineering Proteins for Thermostability through Rigidifying Flexible Sites (RFS)[J].Biotechnol Adv,2013,32(2):308–315
121王帅.基于同源建模的蛋白质结构预测方法的研究[D]:[硕士学位论文].哈尔滨:哈尔滨工业大学,2006
122王志珍.蛋白质折叠和分子伴侣[J].生物学通报,2004,39(5):1-6
123Yu H, Liu J, Shen Z. Modeling catalytic mechanism of nitrile hydratase by semi-empirical quantummechanical calculation[J]. J Mol Graph Model,2008,27(4):522-528
124Miyanaga A, Fushinobu S, Ito K, et al. Mutational and structural analysis of cobalt-containing nitrilehydratase on substrate and metal binding[J]. Eur J Biochem,2004,271(2):429-438
125Peplowski L, Kubiak K, Nowak W. Insights into catalytic activity of industrial enzyme Co-nitrilehydratase[J]. J Mol Model,2007,13(6-7):725-730
126Wu R, Gong W, Liu T, et al. A QM/MM Molecular Dynamics Study of Purine-Specific NucleosideHydrolase[J]. J Phys Chem B,2012,116(6):1984-1991
127Lonsdale R, Hoyle S, Grey D T, et al. Determinants of Reactivity and Selectivity in Soluble EpoxideHydrolase from QM/MM Modeling[J]. Biochemistry,2012,51(8):1774-1786
128Hou Q, Wang J, Gao J, et al. QM/MM studies on the catalytic mechanism of PhenylethanolamineN-methyltransferase[J]. Bba-Proteins Proteom,2012,1824(4):533-541
129De Marco A. Strategies for successful recombinant expression of disulfide bond-dependent proteins inEscherichia coli[J]. Microb Cell Fact,2009,8(1):26-35
130Khatib F, Cooper S, Tyka M D, et al. Algorithm discovery by protein folding game players[J]. ProcNatl Acad Sci U S A,2011,108(47):18949-18953
131R thlisberger D, Khersonsky O, Wollacott A M, et al. Kemp elimination catalysts by computationalenzyme design[J]. Nature,2008,453(7192):190-195
132Eiben C B, Siegel J B, Bale J B, et al. Increased Diels-Alderase activity through backbone remodelingguided by Foldit players[J]. Nat Biotechnol,2012,30(2):190-192
133Okamoto S, Eltis L D. The biological occurrence and trafficking of cobalt[J]. Metallomics,2011,3(10):963-970
134Kim S-H, Oriel P. Cloning and expression of the nitrile hydratase and amidase genes from Bacillus sp.BR449into Escherichia coli[J]. Enzyme Microb Tech,2000,27(7):492-501
135Petrillo K L, Wu S, Hann E C, et al. Over-expression in Escherichia coli of a thermally stable andregio-selective nitrile hydratase from Comamonas testosteroni5-MGAM-4D[J]. Appl Microbiol Biot,2005,67(5):664-670
136Salis H M, Mirsky E A, Voigt C A. Automated design of synthetic ribosome binding sites to controlprotein expression[J]. Nat Biotechnol,2009,27(10):946-950
137Icev A, Ruiz C, Ryder E F. Distance-enhanced association rules for gene expression[J]. Gene,2003,10(2):140-144
138Sahdev S, Khattar S K, Saini K S. Production of active eukaryotic proteins through bacterialexpression systems: a review of the existing biotechnology strategies[J]. Mol Cell Biochem,2008,307(1):249-264
139Tasaki T, Sriram S M, Park K S, et al. Regulation of Protein Function by N-Terminal DegradationSignal[J]. Biochemistry,2012,81(2):62-69
140Varshavsky A. The N-end rule pathway and regulation by proteolysis[J]. Protein Sci,2011,20(8):1298-1345
141Sriram S M, Kim B Y, Kwon Y T. The N-end rule pathway: emerging functions and molecularprinciples of substrate recognition[J]. Nat Rev Mol Cell Bio,2011,12(11):735-747
142Pival S L, Birner-Gruenberger R, Krump C, et al. D-Xylulose kinase from Saccharomyces cerevisiae:Isolation and characterization of the highly unstable enzyme, recombinantly produced in Escherichiacoli[J]. Protein Expres Purif,2011,79(2):223-230
143Licausi F, Kosmacz M, Weits D A, et al. Oxygen sensing in plants is mediated by an N-end rulepathway for protein destabilization[J]. Nature,2011,479(7373):419-422
144Lee P C W, Sowa M E, Gygi S P, et al. Alternative Ubiquitin Activation/Conjugation Cascades Interactwith N-End Rule Ubiquitin Ligases to Control Degradation of RGS Proteins[J]. Mol Cell,2011,43(3):392-405
145Gibbs D J, Lee S C, Isa N M, et al. Homeostatic response to hypoxia is regulated by the N-end rulepathway in plants[J]. Nature,2011,479(7373):415-418
146Sriram S M, Kwon Y T. The molecular principles of N-end rule recognition[J]. Nat Struct Mol Biol,2010,17(10):1164-1170
147Fang N, Zhong C Q, Liang X L, et al. Improvement of extracellular production of a thermophilicsubtilase expressed in Escherichia coli by random mutagenesis of its N-terminal propeptide[J]. ApplMicrobiol Biotechnol,2010,85(5):1473-1481
148Dougan D, Truscott K, Zeth K. The bacterial N-end rule pathway: expect the unexpected[J]. MolMicrobiol,2010,76(3):545-558
149Choi W S, Jeong B C, Joo Y J, et al. Structural basis for the recognition of N-end rule substrates by theUBR box of ubiquitin ligases[J]. Nat Struct Mol Biol,2010,17(10):1175-1181
150Chen J, Yu H, Liu C, et al. Improving stability of nitrile hydratase by bridging the salt-bridges inspecific thermal-sensitive regions[J]. J Biotechnol,2013,164(2):354-362
151Peplowski L, Kubiak K, Nowak W. Mechanical aspects of nitrile hydratase enzymatic activity. Steeredmolecular dynamics simulations of Pseudonocardia thermophila JCM3095[J]. Chem Phys Lett,2008,467(1):144-149
152Kubiak K, Nowak W. Molecular dynamics simulations of the photoactive protein nitrile hydratase[J].Biophys J,2008,94(10):3824-3838
153Zhang Y, Zeng Z, Zeng G, et al. Enzyme–Substrate Binding Landscapes in the Process of NitrileBiodegradation Mediated by Nitrile Hydratase and Amidase[J]. Appl Biochem Biotech,2013,170(7):1-10
154Kwon W S, Da Silva N A, Kellis Jr J T. Relationship between thermal stability, degradation rate andexpression yield of barnase variants in the periplasm of Escherichia coli[J]. Protein Eng,1996,9(12):1197-1202
155Choi J, Lee S. Secretory and extracellular production of recombinant proteins using Escherichiacoli[J]. Appl Microbiol Biotechnol,2004,64(5):625-635
156Johnston J A, Johnson E S, Waller P R H, et al. Methotrexate inhibits proteolysis of dihydrofolatereductase by the N-end rule pathway[J]. J Biol Chem,1995,270(14):8172-8178
157Schmidt R, Bukau B, Mogk A. Principles of general and regulatory proteolysis by AAA+proteases inEscherichia coli[J]. Res Microbiol,2009,160(9):629-636
158Wu S, Fallon R, Payne M. Over-production of stereoselective nitrile hydratase from Pseudomonasputida5B in Escherichia coli: activity requires a novel downstream protein[J]. Appl Microbiol Biot,1997,48(6):704-708
159Ba L A, Doering M, Burkholz T, et al. Metal trafficking: from maintaining the metal homeostasis tofuture drug design[J]. Metallomics,2009,1(4):292-311
160Teilum K, Olsen J G, Kragelund B B. Protein stability, flexibility and function[J]. Biochim BiophysActa,2011,1814(8):969-976
161Bahar I, Lezon T R, Bakan A, et al. Normal mode analysis of biomolecular structures: functionalmechanisms of membrane proteins[J]. Chem Rev,2010,110(3):1463-1468
162Hamelberg D, McCammon J A. Fast peptidyl cis-trans isomerization within the flexible Gly-rich flapsof HIV-1protease[J]. J Am Chem Soc,2005,127(40):13778-13779
163Liu Y, Cui W, Xia Y, et al. Self-Subunit Swapping Occurs in Another Gene Type of Cobalt NitrileHydratase[J]. PLoS One,2012,7(11): e50829
164Ma Y, Yu H, Pan W, et al. Identification of nitrile hydratase-producing Rhodococcus ruber TH andcharacterization of an amiE-negative mutant[J]. Bioresource Technol,2010,101(1):285-291
165Chen J, Yu H, Liu C, et al. Improving stability of nitrile hydratase by bridging the salt-bridges inspecific thermal-sensitive regions[J]. J Biotechnol,2012,164(2):354-362
166Zhang S, Holmes T, Lockshin C, et al. Spontaneous assembly of a self-complementary oligopeptide toform a stable macroscopic membrane[J]. Proc Natl Acad Sci U S A,1993,90(8):3334-3338
167Lazar K L, Miller-Auer H, Getz G S, et al. Helix-turn-helix peptides that form α-helical fibrils: Turnsequences drive fibril structure[J]. Biochemistry,2005,44(38):12681-12689
168Zhou B, Xing L, Wu W, et al. Small surfactant-like peptides can drive soluble proteins into active
aggregates[J]. Microb Cell Fact,2012,11(10):26-34
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