Molecular Modification of L-amino Acid Deaminase and Optimization of α-ketoglutaric Acid Production by Whole-cell Biocatalysis

doi:10.13523/j.cb.20190308

China Biotechnology

2019, Vol. 39

Issue (3): 56-64 DOI: 10.13523/j.cb.20190308

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.

Key words： L-amino acid deaminase Condition optimization Site-saturation mutagenesis Whole-cell biocatalyst

Received: 06 September 2018 Published: 12 April 2019

ZTFLH:

Q819

Corresponding Authors: Long LIU E-mail: longliu@jiangnan.edu.cn

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Cite this article:

Yue WANG,Jiang-hua LI,Guo-cheng DU,Long LIU. Molecular Modification of L-amino Acid Deaminase and Optimization of α-ketoglutaric Acid Production by Whole-cell Biocatalysis. China Biotechnology, 2019, 39(3): 56-64.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190308 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I3/56

Table 1 Primer for site-saturation mutagenesis

Fig.1 The optimization of induction temperature

Fig. 2 The optimization of expression cell age and the OD₆₀₀ value at the end of culture

Fig.3 The optimization of induction time

Fig.4 The optimization of IPTG concentration and the OD₆₀₀ value at the end of culture

Fig.5 Effects of seed culture time on alpha-ketoglutarate production

Fig. 6 Effects of temperature on alpha-ketoglutarate production

Fig.7 Effects of pH on alpha-ketoglutarate production

Fig.8 Effects of biocatalytic time on alpha-ketoglutarate production

Fig.9 Effects of DCW on alpha-ketoglutarate production

Fig.10 The docking result of L-amino acid deaminase and L-glutamic acid (a) The docking position between L-glutamic acid and L-amino acid deaminase (b) FAD active site (c) Mutation site selection (d) L-amino acid desminase and L-glutamic acid action position

Fig.11 Positive result of site-directed mutation

Fig.12 Positive result of multi-site compound mutation

Fig.13 Effects of catalytic conditions on alpha-ketoglutarate production after mutation (a) Effects of temperature on alpha-ketoglutarate production (b) Effects of pH on alpha-ketoglutarate production (c) Effects of DCW on alpha-ketoglutarate production


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