التفاصيل البيبلوغرافية
| العنوان: |
Production of α‐Ketoisocaproate and α‐Keto‐β‐Methylvalerate by Engineered L‐Amino Acid Deaminase. |
| المؤلفون: |
Yuan, Yuxiang1,2, Song, Wei1,2, Liu, Jia1,2, Chen, Xiulai1,2, Luo, Qiuling1,2, Liu, Liming1,2 |
| المصدر: |
ChemCatChem. 5/20/2019, Vol. 11 Issue 10, p2464-2472. 9p. |
| مصطلحات موضوعية: |
*VALERATES, *CAPROATES, *AMINO acids, *DEAMINASES, *STRUCTURAL analysis (Engineering), *MOLECULAR dynamics |
| مستخلص: |
This study aimed to develop an efficient enzymatic strategy for industrial production of α‐ketoisocaproate (α‐KIC) and α‐keto‐β‐methylvalerate (α‐KMV) from L‐leucine and L‐isoleucine, respectively. L‐amino acid deaminase from Proteus mirabilis (PmLAAD) was heterologously expressed in E. coli BL21(DE3) and modified to increase its catalytic efficiency by engineering the PmLAAD substrate‐binding cavity and entrance tunnel. Four essential residues (Q92, M440, T436, and W438) were identified from structural analysis and molecular dynamics simulations. Residue Q92 was mutated to alanine, and the volume of the binding cavity, enzyme activity, and the kcat/Km value of mutant PmLAAD Q92A increased to 994.2 Å3, 191.36 U mg−1, and 1.23 mM−1 min−1, respectively; consequently, the titer and conversion rate of α‐KIC from L‐leucine were 107.1 g L−1 and 98.1 %, respectively. For mutant PmLAADT436/W438A, the entrance tunnel, enzyme activity, and the kcat/Km value increased to 1.71 Å, 170.12 U mg−1, and 0.70 mM−1 min−1, respectively; consequently, the titer and conversion rate of α‐KMV from L‐isoleucine were 98.9 g L−1 and 99.7 %, respectively. Therefore, augmentation of the substrate‐binding cavity and entrance tunnel of PmLAAD can facilitate efficient industrial synthesis of α‐KIC and α‐KMV. [ABSTRACT FROM AUTHOR] |
| قاعدة البيانات: |
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