One-Pot Enzymatic Synthesis of d-Arylalanines Using Phenylalanine Ammonia Lyase and l-Amino Acid Deaminase
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The phenylalanine ammonia-lyase (AvPAL) from Anabaena variabilis catalyzes the amination of substituent trans-cinnamic acid (t-CA) to produce racemic d,l-enantiomer arylalanine mixture owing to its low stereoselectivity. To produce high optically pure d-arylalanine, a modified AvPAL with high d-selectivity is expected. Based on the analyses of catalytic mechanism and structure, the Asn347 residue in the active site was proposed to control stereoselectivity. Therefore, Asn347 was mutated to construct mutant AvPAL-N347A, the stereoselectivity of AvPAL-N347A for d-enantiomer arylalanine was 2.3-fold higher than that of wild-type AvPAL (WtPAL). Furthermore, the residual l-enantiomer product in reaction solution could be converted into the d-enantiomer product through stereoselective oxidation by PmLAAD and nonselective reduction by reducing agent NH3BH3. At optimal conditions, the conversion rate of t-CA and optical purity (enantiomeric excess (eeD)) of d-phenylalanine reached 82% and exceeded 99%, respectively. The two enzymes displayed activity toward a broad range of substrate and could be used to efficiently synthesize d-arylalanine with different groups on the phenyl ring. Among these d-arylalanines, the yield of m-nitro-d-phenylalanine was highest and reached 96%, and the eeD exceeded 99%. This one-pot synthesis using AvPAL and PmLAAD has prospects for industrial application.
KeywordsPhenylalanine ammonia lyase l-Amino acid deaminase d-Arylalanine One-pot synthesis Gene cloning and expression Mutation
The authors gratefully acknowledged Prof. Zheming Zhou from the School of Biotechnology, Jiangnan University for the facilities and infrastructure. The authors also acknowledged the help rendered by Weifeng Sun from Xihua University in proofreading the manuscript.
This work was supported by the National Natural Science Foundation of China (21506172; 31671797), Natural Sciences Foundation supported by Anhui Province universities (KJ2016A801) and Anhui Polytechnic University Youth Talent Support Program (2016BJRC006).
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Conflict of Interest
The authors declare that they have no conflict of interest.
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