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 WANG Fang,et al..Molecular modification of L-aspartate β-decarboxylase to improve its catalytic activity under acidic condition[J].Chinese Journal of Applied & Environmental Biology,2018,24(06):1411-1417.[doi:10.19675/j.cnki.1006-687x.2018.01022]





Molecular modification of L-aspartate β-decarboxylase to improve its catalytic activity under acidic condition
汪芳 杨套伟 周俊平 徐美娟 张显 饶志明
江南大学生物工程学院,工业微生物教育部重点实验室 无锡 214122
WANG Fang et al.
Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China
Pseudomonas dacunhae L-aspartate β-decarboxylase enzymatic property substrate entrance tunnel site mutation
克隆了德阿昆哈假单胞菌(Pseudomonas dacunhae)来源的L-天冬氨酸β-脱羧酶基因(Asd),实现其在Escherichia coli 中的异源表达,但该酶在酸性环境中活性较低,不利于工业生产. 为通过定点突变技术提高该酶在酸性环境中的活力,选择底物通道区域内的5个氨基酸残基作为突变位点,构建6个突变体,随后分析突变体的酶学特性. 结果表明,相比于野生型Asd,大多数突变体的比酶活显著下降,只有突变体N34D比酶活(71.67 U/mg)比野生型(65.95 U/mg)略高;另外,突变体N34D在3.5 < pH < 5.5酸性环境中的酶活力与野生型Asd相比均得到了提高,其中pH 5.0条件下突变体N34D相对酶活达到82%,而野生型Asd相对酶活只有50%左右;而在pH 5.5-8.5范围内突变体N34D的酶活力与野生型相当. 最后将突变体N34D应用于催化合成L-丙氨酸,在最适催化条件(pH 5.5)下,突变体N34D催化合成L-丙氨酸的产量是野生型Asd的1.5倍. 本研究通过对德阿昆哈假单胞菌来源的N34位点进行突变,成功提高了天冬氨酸β-脱羧酶在酸性环境中的酶活力,并提高了其合成L-丙氨酸的能力,这对酶法生产L-丙氨酸具有重要的指导意义. (图4 表4 参24)
L-Aspartate beta-decarboxylase gene (Asd) from Pseudomonas dacunhae was heterologously expressed in Escherichia coli. However, it showed low activity under acidic condition, thereby limiting its wide application in industrial process. In this study, we aimed to improve the activity of Asd under acidic condition by using site mutation. The non-conservative residues located in the substrate entrance tunnel were selected, and the enzymatic properties of variants were investigated. Finally, the whole-cell transformation was performed with variants. The activities of most variants were significantly lower than those of wild type (WT, 65.95 U/mg), except N34D, which showed higher activity of 71.67 U/mg. The mutant N34D showed higher activity at the pH range from 3.5 to 5.5, and the relative activity was over 82% at pH 5.0, which was considerably higher than that of the wild-type (50%). The activity of mutant N34D at pH range of 5.0-8.5 was the same with the wild-type. Under the optimal catalytic conditions, the production of L-alanine by N34D was 1.5 times higher than that of the wild-type. The activity of variant N34D was successfully increased under acidic condition, which shows a promising application for the production of L-alanine at an industrial scale.


1 王雪根, 朱建良. L-丙氨酸的生产及应用[J]. 南京工业大学学报(自科版), 1998, 20 (1): 88-92 [Wang XG, Zhu JL. The production and applications of L-alanin [J]. J Nanjing Technol Univ (Nat Sci Ed), 1998, 20 (1): 88-92]
2 Donowitz M, Hendler R, Spiro HM, Binder HJ, Felig P. Glucagon secretion in acute and chronic pancreatitis [J]. Ann Int Med, 1975, 83 (6): 778-781
3 严振寰, 吴自慎, 桂自其, 王成刚. 4-羟基水杨醛丙氨酸合锌(Ⅱ)的合成表征及其抗癌活性的初步研究[J]. 华中师范大学学报(自科版), 1989 (4): 57-61 [Yan ZH, Wu ZS, Ghi ZQ, Wang CG. Synthesis and characterization of 4-hydroxysali cylaldehyde-alanine to zinc (II) and its preliminary study for anticancer activity [J]. J Centr Chin Nor Univ(Nat Sci Ed), 1989 (4): 57-61]
4 Chibata I, Kakimoto T, Kato J. Enzymatic production of L-alanine by Pseudomonas dacunhae [J]. Appl Microbiol, 1965, 13 (5): 638-645
5 Chibata I, Tosa T, Takamatsu S. Continuous l-alanine production using two different immobilized microbial cell preparations on an industrial scale [J]. Methods Enzymol, 1987, 136 (136C): 472-479
6 Koichi S, Masaharu M, Takaya H. A process for producing l-aspartic acid and converting maleic acid to fumaric acid [D]. EP, 1998 (179): 85
7 Szymańska G, Sobierajski B, Chmiel A. Immobilized cells of recombinant Escherichia coli strain for continuous production of L-aspartic acid [J]. Pol J Microbiol, 2011, 60 (2): 105-112
8 Takamatsu S, Tosa T. Production of L-alanine and D-aspartic acid [J]. Bioprocess Technol, 1993, 16 (8): 25-35
9 Novogrodsky A, Nishimura JS, Meister A. Transamination and beta-decarboxylation of aspartate catalyzed by the same pyridoxal phosphate-enzyme [J]. J Biol Chem, 1963, 238 (238): 1903-1905
10 Wang NC, Lee CY. Enhanced transaminase activity of a bifunctional L-aspartate 4-decarboxylase [J]. Biochem Biophys Res Commun, 2007, 356 (2): 368-373
11 Lima S, Sundararaju B, Huang C, Khristoforov R, Momany C, Phillips RS. The crystal structure of the pseudomonas dacunhae aspartate-β-decarboxylase dodecamer reveals an unknown oligomeric assembly for a pyridoxal-5′-phosphate-dependent enzyme [J]. J Mol Biol, 2009, 388 (1): 98-108
12 Chen HJ, Ko TP, Lee CY, Wang NC, Wang HJ. Structure, assembly, and mechanism of a PLP-dependent dodecameric -aspartate β-decarboxylase [J]. Structure, 2009, 17 (4): 517-529
13 Phillips RS, Lima S, Khristoforov R, Sudararaju B. Insights into the mechanism of Pseudomonas dacunhae aspartate beta-decarboxylase from rapid-scanning stopped-flow kinetics [J]. Biochemistry, 2010, 49 (24): 5066-5073
14 Wang NC, Ko TP, Lee CY. Inactive S298R disassembles the dodecameric L-aspartate 4-decarboxylase into dimers [J]. Biochem Biophys Res Commun, 2008, 374 (1): 134-137
15 Urban P, Truan G, Pompon D. Access channels to the buried active site control substrate specificity in CYP1A P450 enzymes [J]. Biochim Biophys Acta, 2015, 1850 (4): 696-707
16 Kingsley LJ, Lill MA. Substrate tunnels in enzymes: structure-function relationships and computational methodology [J]. Proteins-struct Funct Bioinformatics, 2015, 83 (4): 599-611
17 Yan X, Wang J, Yu S, Zhu J, Sheng W. Facilitating the evolution of esterase activity from a promiscuous enzyme (mhg) with catalytic functions of amide hydrolysis and carboxylic acid perhydrolysis by engineering the substrate entrance tunnel [J]. Appl Environ Microbiol, 2016, 82 (22): 6748-6756
18 Wang Y, Yu HM, Song WS, An M, Zhang J, Luo H, Shen ZY. Overexpression of synthesized cephalosporin C acylase containing mutations in the substrate transport tunnel [J]. J Biosci Bioeng, 2012, 113 (1): 36-41
19 Studer RA, Dessailly BH, Orengo CA. Residue mutations and their impact on protein structure and function: detecting beneficial and pathogenic changes [J]. Biochem J, 2013, 449 (3): 581-594
20 Fan F, You Z, Li Z. A butterfly effect: highly insecticidal resistance caused by only a conservative residue mutated of drosophila melanogaster acetylcholinesterase [J]. J Mol, 2009, 15 (10): 1229-1236
21 Sanchis J, Fernandez L, Carballeira JD, Drone J, Gumulya Y, Hobenreich H, Kahakeaw D, Kille S, Lohmer R, Peyralans JJ, Podtetenieff J, Prasad S, Soni P, Taglieber A, Zilly FE, Reetz MT. Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates [J]. Appl microbiol biotechnol, 2008, 81 (2): 387-397
22 Novogrodsky A, Nishimura JS, Meister A. Transamination and beta-decarboxylation of aspartate catalyzed by the same pyridoxal phosphate-enzyme [J]. J Biol Chem, 1963, 238 (238): 1903-1905
23 白东亭, 许丽锋. 重组大肠杆菌的包涵体[J]. 中国生物制品学杂志, 1996 (4): 188-191 [Bai DT, Xu LF. Inclusion bodies of recombinant Escherichia coli [J]. Chin J Biol, 1996 (4): 188-191]
24 吕梦圆, 吕永玲, 夏祥, 林建国, 王常高, 蔡俊, 胡瑛. 大肠杆菌重组几丁质酶的表达、包涵体复性及酶学性质研究[J]. 食品工业科技, 2015, 36 (22): 168-172, 178 [ Lü MY, Lü YL, Xia X, Lin JG, Wang CG, Cai J, Hu Y. Expression and inclusion body renaturation of recombinant chitinase in Escherichia coli and its enzymology properties [J]. Sci Technol Food Ind, 2015, 36 (22): 168-172, 178]

更新日期/Last Update: 2018-12-25