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Molecular Modification of L-amino Acid Deaminase and Optimization of α-ketoglutaric Acid Production by Whole-cell Biocatalysis |
Yue WANG,Jiang-hua LI,Guo-cheng DU,Long LIU() |
Key Laboratory of Carbohydrate Chemistry and Biotechnology Ministry of Education Jiangnan University, Wuxi 214122, China |
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Abstract Alpha-ketoglutaric acid (α-KG), which is a keto acid product deaminated by glutamic acid, is widely used in food, medicine, fine chemicals and other fields as an important organic acid. To improve the yeild and the efficiency of biotransformation for the synthesis of α-ketoglutaric acid. First, by optimizing the conditions of whole-cell biocatalyst preparation and whole-cell biocatalysis conditions. Optimization conditions include the temperature, pH, inducer concentration, induction time in the whole-cell biocatalyst preparation process and the temperature, pH, biocatalyst concentration, biocatalytic time in the whole-cell biocatalysis process. Determine the optimal conditions of each item by detecting the amount of product α-KG. After the conditions were optimized, the maximum yield was increased by 54.9 % and the molar conversion was 39.6 %. Secondly, the directed evolution of L-amino acid deaminase by site-directed mutagenesis increased its catalytic ability. Through multiple mutations, screening, the yield of α-ketoglutaric acid biocatalytic synthesized by monosodium glutamate with optimal mutant E.coli BL21-pET-20b (+)-pm1152 was 100.9 g/L, and the molar conversion rate was 64.7 %, an increase of 66.3% compared to the control strain. The maximal yield and molar conversion of L-glutamic acid to α-KG was reached under the following optimal conditions: 20 g/L whole-cell biocatalyst, 30 ℃, pH 6.0, and 60-h biocatalysis, strain: E.coli BL21-pET-20b (+)-pm1152. The results showed that the conditional optimization and saturation mutation could effectively increase the whole-cell biocatalyst of recombinant E. coli to synthesize α-ketoglutaric acid.
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Received: 06 September 2018
Published: 12 April 2019
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Corresponding Authors:
Long LIU
E-mail: longliu@jiangnan.edu.cn
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[1] |
Matzi V, Lindenmann J, Muench A , et al. The impact of preoperative micronutrient supplementation in lung surgery. A prospective randomized trial of oral supplementation of combined alpha-ketoglutaric acid and 5-hydroxymethylfurfural. European Journal of Cardio-thoracic Surgery, 2007,32(5):776-782.
doi: 10.1016/j.ejcts.2007.07.016
pmid: 17768058
|
|
|
[2] |
Sauer M, Porro D, Mattanovich D , et al. Microbial production of organic acids: Expanding the markets. Trends in Biotechnology, 2008,26(2):100-108.
doi: 10.1016/j.tibtech.2007.11.006
pmid: 18191255
|
|
|
[3] |
Jinap S, Hajeb P . Glutamate. its applications in food and contribution to health. Appetite, 2010,55(1):1-10.
doi: 10.1016/j.appet.2010.05.002
pmid: 20470841
|
|
|
[4] |
Stottmeister U, Aurich A, Wilde H , et al. White biotechnology for green chemistry: Fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses. Journal of Industrial Microbiology Biotechnology, 2005,32(11-12):651-664.
doi: 10.1007/s10295-005-0254-x
|
|
|
[5] |
牛盼清, 张震宇, 刘立明 . 酶法转化L-谷氨酸生产α-酮戊二酸. 生物工程学报, 2014,30(8):1318-1322.
|
|
|
[5] |
Niu P Q, Zhang Z Y, Liu L M . Enzymatic production of α-ketoglutaric acid by L-glutamate oxidase from L-glutamic acid. Chinese Journal of Biotechnology, 2014,30(8):1318-1322.
|
|
|
[6] |
Liu L, Hossain G S, Shin H D , et al. One-step production of alpha-ketoglutaric acid from glutamic acid with an engineered L-amino acid deaminase from Proteus mirabilis. Journal of Biotechnology, 2013,164(1):97-104.
doi: 10.1016/j.jbiotec.2013.01.005
pmid: 23333917
|
|
|
[7] |
Hossain G S, Li J H, Shin H D , et al. Improved production of alpha-ketoglutaric acid (alpha-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of L-amino acid deaminase and deletion of the alpha-KG utilization pathway. Journal of Biotechnology, 2014,187:71-77.
doi: 10.1016/j.jbiotec.2014.07.431
|
|
|
[8] |
Hou Y, Hossain G S, Li J H , et al. Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: Comparison of enzymatic and whole-cell biotransformation approaches. Applied Microbiology and Biotechnology, 2015,99(20):8391-8402.
doi: 10.1007/s00253-015-6757-0
|
|
|
[9] |
Li R X, Sakir H G, Li J H , et al. Rational molecular engineering ofl-amino acid deaminase for production of α-ketoisovaleric acid froml-valine by Escherichia coli. Rsc Advances, 2017,7(11):6615-6621.
doi: 10.1039/C6RA26972A
|
|
|
[10] |
Song Y, Li J H, Shin H D , et al. One-step biosynthesis of alpha-ketoisocaproate from L-leucine by an Escherichia coli whole-cell biocatalyst expressing an L-amino acid deaminase from Proteus vulgaris. Scientific Reports, 2015,5:12614.
doi: 10.1038/srep12614
|
|
|
[11] |
Carvalho de, CarlaC C R . Enzymatic and whole cell catalysis: Finding new strategies for old processes. Biotechnology Advances, 2011,29(1):75-83.
doi: 10.1016/j.biotechadv.2010.09.001
pmid: 20837129
|
|
|
[12] |
Baek J O, Seo J W, Kwon O , et al. Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli. Journal of Basic Microbiology, 2011,51(2):129-135.
doi: 10.1002/jobm.201000086
pmid: 21298676
|
|
|
[13] |
Faust A, Niefind K, Hummel W . The structure of a bacterial L-amino acid oxidase from Rhodococcus opacus gives new evidence for the hydride mechanism for dehydrogenation. Journal of Molecular Biology, 2007,367(1):234-248.
doi: 10.1016/j.jmb.2006.11.071
pmid: 17234209409300143002282013
|
|
|
[14] |
Pelmont J, Arlaud G, Rossat A M . L-aminoacide oxydases des enveloppes de Proteus mirabilis: Propriétés générales. Biochimie, 1972,54(10):1359-1374.
doi: 10.1016/S0300-9084(72)80076-2
|
|
|
[15] |
Hossain G S, Li J H, Shin H D , et al. Bioconversion of l-glutamic acid to α-ketoglutaric acid by an immobilized whole-cell biocatalyst expressing L-amino acid deaminase from Proteus mirabilis. Journal of Biotechnology, 2014,169(Complete):112-120.
doi: 10.1016/j.jbiotec.2013.10.026
pmid: 24172254
|
|
|
[16] |
Motta P, Molla G, Pollegioni L , et al. Structure-function relationships in L-amino acid deaminase, a flavoprotein belonging to a novel class of biotechnologically relevant enzymes. Journal of Biological Chemistry, 2016,291(20):10457-10475.
doi: 10.1074/jbc.M115.703819
pmid: 27022028
|
|
|
[17] |
宋阳 . L-氨基酸脱氨酶分子改造及生物合成α-酮异己酸. 无锡: 江南大学, 2018.
|
|
|
[17] |
Song Y . Molecular modification of L-amino acid deaminase and biosynthesis of α-ketoisocaproate. Wuxi: Jiangnan University, 2018.
|
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