D-苯基乳酸(D-phenyllactic acid,D-PLA)是一种具有重要生理活性和广泛应用前景的有机酸,可作为天然防腐剂、医药及精细化工中间体以及高分子材料的手性单体,其绿色高效制备已成为研究热点。该研究旨在通过蛋白质工程手段提升D-PLA合成过程中关键限速酶L-氨基酸脱氨酶(PmLAAD)的催化性能。基于理性设计策略,利用同源建模与分子对接分析解析PmLAAD的结构特征与序列保守性,筛选关键催化位点并实施定点饱和突变。结果获得突变酶PmLAAD (L279 W/P315D)重组菌,其比酶活力为野生型酶重组菌的2.66倍。进一步将表达突变酶的E.coli/pET28a-pmlaad (L279 W/P315D)与表达D-乳酸脱氢酶的E.coli/pET28a-ldh进行细胞偶联,构建以两步催化为核心的级联反应体系,实现L-苯丙氨酸经苯丙酮酸高效转化为D-PLA,产物质量浓度达13.09 g/L。研究表明,通过结构改造可显著提升脱氨酶的催化效率。该研究为通过结构改造提升脱氨酶催化性能、实现手性化合物的绿色合成提供了新的思路与技术支撑。
D-Phenyllactic acid (D-PLA) is an organic acid with significant physiological activity and broad application potential.It serves as a natural preservative, a pharmaceutical and fine chemical intermediate, and a chiral monomer for polymer materials.The green and efficient synthesis of D-PLA has become a major research focus.This study aimed to enhance the catalytic efficiency of the key rate-limiting enzyme, L-amino acid deaminase (PmLAAD), in D-PLA biosynthesis using protein engineering.Homology modeling and molecular docking were applied to analyze the structural features and sequence conservation of PmLAAD, identify key catalytic residues, and guide site-saturation mutagenesis.A recombinant E.coli expressing the mutant enzyme PmLAAD (L279W/P315D) was obtained, whose specific activity was 2.66 times that of the wild-type enzyme strain.The mutant strain was coupled with E.coli expressing D-lactate dehydrogenase to construct a two-step cascade reaction system.This system efficiently converted L-phenylalanine to D-PLA via phenylpyruvate, achieving a product concentration of 13.09 g/L.The results demonstrate that structural modification can significantly improve deaminase activity.This approach provides a promising strategy for the green synthesis of chiral compounds and offers technical support for enhancing enzyme performance through rational protein design.
[1] SONG Y, WANG R, ZHANG Z X, et al.Semi-rational engineering membrane binding domain of L-amino acid deaminase from Proteus vulgaris for enhanced α-ketoisocaproate[J].Frontiers in Microbiology, 2022, 13:1025845.
[2] CHANG J Z, ZHANG Y X, LI Z W, et al.Biosynthesis of α-keto acids and resolution of chiral amino acids by L-amino acid deaminases from Proteus mirabilis[J].Protein Expression and Purification, 2024, 221:106518.
[3] LIU K, YU H R, SUN G Y, et al.Semi-rational design of L-amino acid deaminase for production of pyruvate and d-alanine by Escherichia coli whole-cell biocatalyst[J].Amino Acids, 2021, 53(9):1361-1371.
[4] 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[J].Journal of Biological Chemistry, 2016, 291(20):10457-10475.
[5] XIONG T Z, GAO Q Y, ZHANG J T, et al.Engineering Escherichia coli with a symbiotic plasmid for the production of phenylpyruvic acid[J].RSC Advances, 2024, 14(36):26580-26584.
[6] XIONG T Z, BAI Y J, FAN T P, et al.Biosynthesis of phenylpyruvic acid from L-phenylalanine using chromosomally engineered Escherichia coli[J].Biotechnology and Applied Biochemistry, 2022, 69(5):1909-1916.
[7] HOU Y, HOSSAIN G S, LI J H, et al.Combination of phenylpyruvic acid (PPA) pathway engineering and molecular engineering of L-amino acid deaminase improves PPA production with an Escherichia coli whole-cell biocatalyst[J].Applied Microbiology and Biotechnology, 2016, 100(5):2183-2191.
[8] LI T L, QIN Z, WANG D, et al.Coenzyme self-sufficiency system-recent advances in microbial production of high-value chemical phenyllactic acid[J].World Journal of Microbiology and Biotechnology, 2022, 39(1):36.
[9] RAJANIKAR R V, NATARAJ B H, NAITHANI H, et al.Phenyllactic acid:A green compound for food biopreservation[J].Food Control, 2021, 128:108184.
[10] ZHENG Z J, XIA M J, FANG X C, et al.Enhanced biosynthesis of chiral phenyllactic acid from L-phenylalanine through a new whole-cell biocatalyst[J].Bioprocess and Biosystems Engineering, 2018, 41(8):1205-1212.
[11] HOSSAIN G S, SHIN H D, LI J H, et al.Integrating error-prone PCR and DNA shuffling as an effective molecular evolution strategy for the production of α-ketoglutaric acid by L-amino acid deaminase[J].RSC Advances, 2016, 6(52):46149-46158.
[12] 石选平, 王玥, 乔郅钠, 等.蛋白质工程改造L-氨基酸脱氨酶高效合成苯丙酮酸[J].生物工程学报, 2025, 41(9):3521-3536.
SHI X P, WANG Y, QIAO Z N, et al.Protein engineering for the modification of a L-amino acid deaminase for efficient synthesis of phenylpyruvic acid[J].Chinese Journal of Biotechnology, 2025, 41(9):3521-3536.
[13] LIU J, LIU J M, YANG B, et al.Production of phenylpyruvic acid by engineered L-amino acid deaminase from Proteus mirabilis[J].Biotechnology Letters, 2022, 44(5):635-642.
[14] WEI B, KANG Y L, ZHAO Y X, et al.Rational design of spontaneous self-cyclization enzymes in vivo and in vitro with improved thermal tolerance and activity[J].ACS Catalysis, 2024, 14(7):5469-5480.
[15] LI Z L, PEI S X, CHEN Z Y, et al.Machine learning-assisted amidase-catalytic enantioselectivity prediction and rational design of variants for improving enantioselectivity[J].Nature Communications, 2024, 15:8778.
[16] WANG Z Q, LI C H, LIU J, et al.Random mutagenesis and semi-rational design enhance the tolerance of Metabacillus litoralis C44 α-L-rhamnosidase[J].International Journal of Biological Macromolecules, 2025, 309:142971.
[17] HU M, SONG J X, MIAO S T, et al.Rational design of soluble expressed human aldehyde dehydrogenase 2 with high stability and activity in pepsin and trypsin[J].International Journal of Biological Macromolecules, 2024, 265:131091.
[18] FAN A W, WANG Z Y, QU H J, et al.Semi-rational design of Proteus mirabilis l-amino acid deaminase for expanding its substrate specificity in α-keto acid synthesis from L-amino acids[J].Catalysts, 2022, 12(2):175.
[19] ZHOU J H, HUANG M L.Navigating the landscape of enzyme design:From molecular simulations to machine learning[J].Chemical Society Reviews, 2024, 53(16):8202-8239.
[20] LUTZ S.Beyond directed evolution:Semi-rational protein engineering and design[J].Current Opinion in Biotechnology, 2010, 21(6):734-743.
[21] ZABALA A O, CACHO R A, TANG Y.Protein engineering towards natural product synthesis and diversification[J].Journal of Industrial Microbiology and Biotechnology, 2012, 39(2):227-241.
[22] ZHU Y M, CHEN P, DONG Q Z, et al.Protein engineering of transaminase facilitating enzyme cascade reaction for the biosynthesis of azasugars[J].iScience, 2024, 27(3):109034.
[23] HOSSACK E J, HARDY F J, GREEN A P.Building enzymes through design and evolution[J].ACS Catalysis, 2023, 13(19):12436-12444.
[24] SUN B Y, SUI H L, LIU Z W, et al.Structure-guided engineering of a flavin-containing monooxygenase for the efficient production of indirubin[J].Bioresources and Bioprocessing, 2022, 9(1):70.
[25] MACARTHUR M W, THORNTON J M.Influence of proline residues on protein conformation[J].Journal of Molecular Biology, 1991, 218(2):397-412.
[26] NESTL B M, HAUER B.Engineering of flexible loops in enzymes[J].ACS Catalysis, 2014, 4(9):3201-3211.
[27] ZHANG J Z, LI X.Novel strategy for phenyllactic acid biosynthesis from phenylalanine by whole cell recombinant Escherichia coli coexpressing L-phenylalanine oxidase and L-lactate dehydrogenase[J].Biotechnology Letters, 2018, 40(1):165-171.
