|
|
|
| Semi-rational Modification of Cephalosporin C Acylase and Biosynthesis of 7-ACA |
| MA Chen-lu1, TANG Cun-duo1, SHI Hong-ling1, WANG Rui2, YUE Chao1, XIA Min1, WU Min-chen2, KAN Yun-chao1 |
1. Henan Provincial Engineering Laboratory of Insect Bio-reactor, Nanyang Normal University, Nanyang 473061, China;
2. Wuxi Medical School, Jiangnan University, Wuxi 214122, China |
|
|
|
Abstract CPC acylases play a key role in producing 7-ACA by one-step enzymatic method, so its research is very important. In order to obtain the CPC acylase with higher catalytic activity, a type Ⅲ CPC acylase from Pseudomonas sp SE 83 was taken as parent. Some key amino acid residues of CAⅢ were determined using molecular docking approach, and substituted the residues with lesser side chains for those. Then, the coding gene of CAⅢ was site-directed mutagenesis by using Multi Site-Directed Mutagenesis Kit, it was expressed in E. coli BL21(DE3) by means of pET32a plasmid. The recombinant mutant reCAⅢM with higher catalytic activity towards CPC, its specific activity is 26.7 IU/mg, which improved by 3.44 folds to the parent. Moreover, its application in producing 7-ACA by one-step enzymatic method were preliminarily studied. With 40 IU/g CPC of enzyme dosage at 25℃ catalyzed for 12 h, the transformation rate of CPC and yield of 7-ACA were up to 96.3% and 63.4% respectively, which demonstated that this enzyme had a promising prospect in application. The ideal results on the CPC acylase molecular modification were obtained, and it laid a solid foundation for further molecular modification and application of this enzyme. In addition, this also provided referential experience for molecular modification of other enzymes.
|
|
Received: 06 July 2015
Published: 22 December 2015
|
|
|
|
|
| [1] Biocatalytic production of semi-synthetic cephalosporins:process technology and integration. Tramper J, Beeftink HH, Janssen AEM, Ooijkaas LP, van Roon JL, Strubel M, Schron CGPH Synthesis of β-Lactam Antibiotics. Springer Netherlands, 2001:206-249.
[2] Pollegioni L, Rosini E, Molla G. Cephalosporin C acylase:dream and(/or) reality. Appl Microbiol Biot, 2013, 97(6):2341-2355.
[3] Sonawane V C. Enzymatic modifications of cephalosporins by cephalosporin ccylase and other enzymes. Crit Rev Biotechnol, 2006, 26(2):95-120.
[4] Pollegioni L, Molla G, Sacchi S, et al. Properties and applications of microbial D-amino acid oxidases:current state and perspectives. Appl Microbiol Biot, 2008, 78(1):1-16.
[5] 姚舜,罗晖,常雁红,等.一步酶法生产7-氨基头孢烷酸的研究进展.现代化工, 2013, 33(2):11-14. Yao S, Luo H, Chang Y H, et al. Research progress in one-step enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid. Modern Chemical Industry, 2013, 33(2):11-14.
[6] Wang Y, Yu H, Song W, et al. Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel. J Biosci Bioeng, 2012, 113(1):36-41.
[7] Zhu X, Luo H, Chang Y, et al. Characteristic of immobilized cephalosporin C acylase and its application in one-step enzymatic conversion of cephalosporin C to 7-aminocephalosporanic acid. World J Microb Biot, 2011, 27(4):823-829.
[8] Li Y, Chen J, Jiang W, et al. In vivo post-translational processing and subunit reconstitution of cephalosporin acylase from Pseudomonas sp. 130. Eur J Biochem, 1999, 262(3):713-719.
[9] Aramori I, Fukagawa M, Tsumura M, et al. Cloning and nucleotide sequencing of new glutaryl 7-ACA and cephalosporin C acylase genes from Pseudomonas strains. J Ferment Bioeng, 1991, 72(4):232-243.
[10] Deshpande B S, Ambedkar S S, Shewale J G. Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95. World J Microb Biot, 1996, 12(4):373-378.
[11] Conti G, Pollegioni L, Molla G, et al. Strategic manipulation of an industrial biocatalyst-evolution of a cephalosporin C acylase. FEBS J, 2014, 281(10):2443-2455.
[12] Golden E, Paterson R, Tie W J, et al. Structure of a class Ⅲ engineered cephalosporin acylase:comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity. Biochem J, 2013, 451(2):217-226.
[13] Pollegioni L, Lorenzi S, Rosini E, et al. Evolution of an acylase active on cephalosporin C. Protein Sci, 2005, 14(12):3064-3076.
[14] Shin Y C, Jeon J Y, Jung K H, et al. Cephalosporin C acylase mutant and method for preparing 7-ACA using same. US Patent, 0207519A1, 2007.
[15] Li Q, Huang X, Zhu Y. Evaluation of active designs of cephalosporin C acylase by molecular dynamics simulation and molecular docking. J Mol Model, 2014, 20(7):2314.
[16] Xiao Y, Huo X, Qian Y, et al. Engineering of a CPC acylase using a facile pH indicator assay. J Ind Microbiol Biot, 2014, 41(11):1617-1625.
[17] 王颖秋,郑林冲,谢丽萍,等.基于易错PCR的头孢菌素C酰化酶的定向进化.中国医药工业杂志, 2013,4:344-347. Wang Y Q, Zhen L C, Xie L P, et al. Directed evolution of cephalosporin C acylase activity by error-prone PCR. Chinese Journal of Pharmaceuticals, 2013,4:344-347.
[18] 徐雪丽,张伟,刘艳,等.头孢菌素C酰化酶突变位点的研究.中国生物工程杂志, 2015,2:59-65. Xu X L, Zhang W, Liu Y, et al. Study on mutaions of cephalosporin C acylase. China Biotechnology, 2015,2:59-65.
[19] Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 1976, 72:248-254.
[20] Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 1970, 227:680-685.
[21] Perez C, Pastor M, Ortiz A R, et al. Comparative binding energy analysis of HIV-1 protease inhibitors:incorporation of solvent effects and validation as a powerful tool in receptor-based drug design. J Med Chem, 1998, 41:836-852.
[22] Timmers L F, Ducati R G, Sanchez-Quitian Z A, et al. Combining molecular dynamics and docking simulations of the cytidine deaminase from Mycobacterium tuberculosis H37Rv. J Mol Model, 2012, 18(2):467-479.
[23] Liu Y, Gong G, Zhu C, et al. Environmentally safe production of 7-ACA by recombinant Acremonium chrysogenum. Curr Microbiol, 2010, 61(6):609-614. |
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
