来源于原核生物成团泛菌(Pantoea agglomerans)的苯丙氨酸氨基变位酶(PaPAM)可催化α-苯丙氨酸转变为药用价值更高的(S)-β-苯丙氨酸,然而其酶活较低,热稳定性较差,限制其工业应用。本研究突变苯丙氨酸氨基变位酶酶活中心上方loop环上的氨基酸及与其相互作用位点的氨基酸,降低loop环的柔性,以期稳定酶活中心的结构,提高酶的热稳定性。筛选得到热稳定性显著提高的突变体I91M,50 ℃保温1 h后,剩余酶活达到83%(野生型酶酶活仅剩余30%左右)。该结果有助于苯丙氨酸氨基变位酶的理论研究和工业应用。
Pantoea agglomerans phenylalanine aminomutase (PaPAM) catalyzes the conversion of α-phenylalanine to more valuable (S)-β-phenylalanine. However, its low enzyme activity and poor thermal stability limit its industrial application. In order to improve its thermostability and enzyme activity, this study reduced the flexibility of loops above the active center of PaPAM by mutating amino acids on the loops and amino acids at the interaction site to stabilize the active center structure. A mutant I91M with significantly improved thermostability was screened, which had 83% residual enzyme activity after incubating at 50 ℃ for 1 h, while the activity of wild-type enzyme only had 30% remained. These results are helpful for further studies and industrial applications of PaPAM.
[1] SUSAN S U W, NISHANKA D R, KEVIN D W,et al.Insights into the mechanistic pathway of the Pantoea agglomerans phenylalanine aminomutase[J], 2012, 124: 2 952-2 956.
[2] RATNAYAKE N D, WANNINAYAKE U, GEIGER J H, et al.Stereochemistry and mechanism of a microbial phenylalanine aminomutase[J].Journal of the American Chemical Society,2011, 133(22):8 531-8 533.
[3] SEBASTIAN B, UWE T B.Einfluss einer einzelnen Aminosäure auf den Reaktionsmechanismus von ammonium-lyasen und -mutasen[J].Angewandte Chemie International Edition,2009,121(18):3 412-3 415.
[4] WALKER K D,KLEKKTE K,AKIYAM T,et al. Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in taxol biosynthesis[J]. Journal of Biological Chemistry, 2004, 279(52):53 947-53 954.
[5] HUANG S X,LOHMAN J R,HUANG T,et al.A new member of the 4-methylideneimidazole-5-one-containing aminomutase family from the enediyne kedarcidin biosynthetic pathway[J].Proceedings of the National Academy of Sciences,2013,110(20):8 069-8 074.
[6] RETEY J. Discovery and role of methylidene imidazolone, a highly electrophilic prosthetic group[J].Biochim Biophys Acta,2003,1647(1-2):179-184.
[7] SCHUSTER B, RETEY J.The mechanism of action of phenylalanine ammonia-lyase: the role of prosthetic dehydroalanine[J].Passedover by Nature and Science,1995,92(18):8 433-8 437.
[8] CHRISTIANSON C V,MONTAVON T J,FESTIN G M, et al.The mechanism of MIO-based aminomutases in beta-amino acid biosynthesis[J].Journal of the American Chemical Society,2007,129(51):15 744-15 745.
[9] WANK K,HOU Q,LIU Y.Insight into the mechanism of aminomutase reaction:A case study of phenylalanine aminomutase by computational approach[J].Journal of Molecular Graphics & Modelling,2013,46(46C):65-73.
[10] COOKE H A, CHRISTIANSON C V, BRUNER S D.Structure and chemistry of 4-methylideneimidazole-5-one containing enzymes [J].Current Opinion in Chemical Biology,2009,13(4):460-468.
[11] BARTSCH S, WYBENGA G G, JANSEN M, et al.Redesign of a phenylalanine aminomutase into a phenylalanine ammonia lyase[J].Chem Cat Chem,2013,5:1 797-1 802.
[12] DOURADO D F A R, POHLE S, CARVAIHO A T P,et al.Rational design of a (S)-selective-transaminase for asymmetric synthesis of (1S)-1-(1,1′-biphenyl-2-yl)ethanamine[J].ACS Catalysis,2016,6:7 749-7 759.
[13] KREIJ A D,BURG B V D,VENEMA G,et al.The effects of modifying the surface charge on the catalytic activity of a thermolysin-like protease[J]. Journal of Biological Chemistry, 2002, 277(18):15 432.
[14] JAOUADI B,AGHAJARI N,HASER R,et al.Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis[J].Biochimie,2010,92(4):360.
[15] WU B,SZYMANSKI W,WYBENGA G G, et al.Mechanism-Inspired engineering of phenylalanine aminomutase for enhanced β-regioselective asymmetric amination of cinnamates[J].Angewandte Chemie International Edition,2012,51(2):482-486.
[16] NAIK R R,KIRKPATRICK S M,STONE M O.The thermostability of an α-helical coiled-coil protein and its potential use in sensor applications[J].Biosensors and Bioelectronics,2001,16:1 051.
[17] KHURANA J,SINGH R,KAUR J.Engineering of Bacillus lipase by directed evolution for enhanced thermal stability:effect of isoleucine to threonine mutation at protein surface[J].Molecular Biology Reports,2011,38(5):2 919.
[18] 曹佐武. 有效分离1 kDa小肽的Tricine-SDS-PAGE方法[J].中国生物工程杂志,2004, 24(1):74-76.
[19] BRADFORD MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[J]. Anal Biochem, 1976, 72(1/2): 248-254.
[20] ZHANG F, HUANG N, ZHOU L, et al.Modulating the pH-activity profiles of phenylalanine ammonia-lyase from Anabaena variabilis by modification of center-near surface residues [J].Applied Biochemistry and Biotechnology,2017,183:699-711
