2022年 第19卷 第12期
《工程（英文）》 >> 2022年 第19卷 第12期 doi: 10.1016/j.eng.2022.03.020
理性设计大幅提高一种丝孢堆黑粉菌来源脂肪酶催化活性——用于合成莫西沙星手性中间体
a The National and Local Joint Engineering Research Center for Biomanufacturing of Chiral Chemicals, Zhejiang University of Technology, Hangzhou 310014, China
b Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China
下一篇 上一篇
摘要
通过脂肪酶不对称拆分外消旋N-乙酰基-哌啶-2,3-二甲酸二甲酯[cis-(±)-1]，从而获得手性中间体 (S,S)-2,8-二氮杂双环壬烷，是合成氟喹诺酮类抗生素莫西沙星重要手性中间体的一条具有吸引力的工艺路线。在前期研究中，筛选得到了一株丝孢堆黑粉菌（SRL）来源的脂肪酶，该脂肪酶具有高热稳定性和pH值稳定性。但SRL来源的脂肪酶活性较低，无法满足工业化应用的需求。因此本研究依据前期南极假丝酵母来源脂肪酶B（CALB）改造策略，采用理性设计方法对SRL进行定点突变改造，获得一个双突变体SRLI194N/V195L。随后，确定了位于活性口袋外的环6 上两个关键氨基酸残基L145 和L154；获得四位点突变体SRL-I194N/V195L/L145V/L154G（V13），活性显著提高，达到87.8 U·mg−1，是野生型SRL的2195 倍（E > 200）。该突变体在50 ℃下的半衰期达到92.5 h。突变体V13（100 mg·L−1）能够高效拆分1 mol·L−1cis-(±)-1，2 h 内底物转化率达到49.9%，实现严格立体选择性（E > 200）。总体而言，研究发现了一株对cis-(±)-1 (S,S)-2,8-二氮杂双环壬烷具有高催化活性、严格立体选择性的脂肪酶，可应用于工业化生产，并为其他具有相似结构的脂肪酶和其他种类酶的活性提高提供了一种通用策略。
图片
图1
图2
图3
图4
图5
图6
图7
图8
图9
参考文献
[ 1 ] Hamad B. The antibiotics market. Nat Rev Drug Discov 2010;9(9):675‒6. 链接1
[ 2 ] Ramesh P, Harini T, Fadnavis NW. Efficient resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate with soluble Candida antarctica lipase B (CAL B). Org Process Res Dev 2015;19(1):296‒301. . 链接1
[ 3 ] Borrelli GM, Trono D. Recombinant lipases and phospholipases and their use as biocatalysts for industrial applications. Int J Mol Sci 2015;16(9):20774‒840. 链接1
[ 4 ] Filho DG, Silva AG, Guidini CZ. Lipases: sources, immobilization methods, and industrial applications. Appl Microbiol Biotechnol 2019;103(18):7399‒423. 链接1
[ 5 ] Xu Y, Wang X, Liu X, Li X, Zhang C, Li W, et al. Discovery and development of a novel short-chain fatty acid ester synthetic biocatalyst under aqueous phase from Monascus purpureus isolated from Baijiu. Food Chem 2021;338:128025. 链接1
[ 6 ] Sarmah N, Revathi D, Sheelu G, Yamuna Rani K, Sridhar S, Mehtab V, et al. Recent advances on sources and industrial applications of lipases. Biotechnol Prog 2018;34(1):5‒28. 链接1
[ 7 ] Li Y, Wang A, Shen Y, Zhang P. Convenient enzymatic resolution of cis-6-benzyltetrahydro-1H-pyrrolo[3,4-b]pyridine-5,7(6H,7aH)-dione using lipase to prepare the intermediate of moxifloxacin. J Mol Catal B Enzym 2014;110:178‒83. . 链接1
[ 8 ] Shah A, Yameen MA, Fatima N, Murtaza G. Chemical synthesis of chitosan/silver nanocomposites films loaded with moxifloxacin: their characterization and potential antibacterial activity. Int J Pharm 2019;561:19‒34. 链接1
[ 9 ] Shen JW, Qi JM, Zhang XJ, Liu ZQ, Zheng YG. Significantly increased catalytic activity of Candida antarctica lipase B for the resolution of cis-(±)-dimethyl 1-acetylpiperidine-2,3-dicarboxylate. Catal Sci Technol 2018;8(18):4718‒25. . 链接1
[10] Reetz MT, Jaeger KE. Enantioselective enzymes for organic synthesis created by directed evolution. Chemistry 2000;6(3):407‒12. 链接1
[11] Böttcher D, Bornscheuer UT. Protein engineering of microbial enzymes. Curr Opin Microbiol 2010;13(3):274‒82. 链接1
[12] Cai X, Jiang H, Zhang T, Jiang B, Mu W, Miao M. Thermostability and specific-activity enhancement of an arginine deiminase from Enterococcus faecalis SK23.001 via semirational design for L-citrulline production. J Agric Food Chem 2018;66(33):8841‒50. 链接1
[13] Krohl PJ, Ludwig SD, Spangler JB. Emerging technologies in protein interface engineering for biomedical applications. Curr Opin Biotechnol 2019;60:82‒8. 链接1
[14] Qu G, Li A, Acevedo-Rocha CG, Sun Z, Reetz MT. The crucial role of methodology development in directed evolution of selective enzymes. Angew Chem Int Ed Engl 2020;59(32):13204‒31. 链接1
[15] Shen JW, Cai X, Dou BJ, Qi FY, Zhang XJ, Liu ZQ, et al. Expression and characterization of a CALB-type lipase from Sporisorium reilianum SRZ2 and its potential in short-chain flavor ester synthesis. Front Chem Sci Eng 2020;14(5):868‒79. . 链接1
[16] Xu J, Cen Y, Singh W, Fan J, Wu L, Lin X, et al. Stereodivergent protein engineering of a lipase to access all possible stereoisomers of chiral esters with two stereocenters. J Am Chem Soc 2019;141(19):7934‒45. 链接1
[17] Verma S, Choudhary RN, Kanadje AP, Banerjee UC. Diversifying arena of drug synthesis: in the realm of lipase mediated waves of biocatalysis. Catalysts 2021;11(11):1328. . 链接1
[18] Li D, Chen X, Chen Z, Lin X, Xu J, Wu Q. Directed evolution of lipase A from Bacillus subtilis for the preparation of enantiocomplementary sec-alcohols. Green Syn Catal 2021;2(3):290‒4. . 链接1
[19] Zhang Y, Zhu Q, Fei Z, Lin X, Xia B, Wu Q. Stereoselectivity-tailored chemo-enzymatic synthesis of enantiocomplementary poly (ω-substituted-δ-valerolactone) enabled by engineered lipase. Eur Polym J 2019;119:52‒60. . 链接1
[20] Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res 2018;46(W1):W296‒303. 链接1
[21] Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 2009;30(16):2785‒91. 链接1
[22] Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph 1996;14(1):33‒8. 链接1
[23] Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010;31(2):455‒61. PMID:19499576.
[24] Kutzner C, Páll S, Fechner M, Esztermann A, de Groot BL, Grubmüller H. More bang for your buck: improved use of GPU nodes for GROMACS 2018. J Comput Chem 2019;40(27):2418‒31doi:10.1002/jcc.26011. PMID:31260119. 链接1
[25] Lindorff-Larsen K, Piana S, Palmo K, Maragakis P, Klepeis JL, Dror RO, et al. Improved side-chain torsion potentials for the Amber ff99SB protein force field. Proteins 2010;78(8):1950‒8. 链接1
[26] ConsortiumUniProt. UniProt: a hub for protein information. Nucleic Acids Res 2015;43(D1):D204‒12. PMID:25348405. 链接1
[27] Uppenberg J, Hansen MT, Patkar S, Jones TA. The sequence, crystal structure determination and refinement of two crystal forms of lipase B from Candida antarctica. Structure 1994;2(4):293‒308. 链接1
[28] Cen Y, Singh W, Arkin M, Moody TS, Huang M, Zhou J, et al. Artificial cysteine-lipases with high activity and altered catalytic mechanism created by laboratory evolution. Nat Commun 2019;10(1):3198. 链接1
[29] Stauch B, Fisher SJ, Cianci M. Open and closed states of Candida antarctica lipase B: protonation and the mechanism of interfacial activation. J Lipid Res 2015;56(12):2348‒58. 链接1
[30] Sumbalova L, Stourac J, Martinek T, Bednar D, Damborsky J. HotSpot Wizard 3.0: web server for automated design of mutations and smart libraries based on sequence input information. Nucleic Acids Res 2018;46(W1):W356‒62. 链接1
[31] Verma S, Meghwanshi GK, Kumar R. Current perspectives for microbial lipases from extremophiles and metagenomics. Biochimie 2021;182:23‒36. 链接1
[32] Stephens PJ, Devlin FJ, Chabalowski CF, Frisch MJ. Ab initio calculation of vibrational absorption and circular dichroism spectra using density functional force fields. J Phys Chem 1994;98(45):11623‒7. . 链接1
[33] Błaszczyk J, Kiełbasiński P. Quarter of a century after: a glimpse at the conformation and mechanism of Candida antarctica Lipase B. Crystals 2020;10(5):404. . 链接1
[34] Ding X, Tang XL, Zheng RC, Zheng YG. Identification and engineering of the key residues at the crevice-like binding site of lipases responsible for activity and substrate specificity. Biotechnol Lett 2019;41(1):137‒46. 链接1
[35] Sánchez DA, Tonetto GM, Ferreira ML. Burkholderia cepacia lipase: a versatile catalyst in synthesis reactions. Biotechnol Bioeng 2018;115(1):6‒24. 链接1
[36] Luan B, Zhou R. A novel self-activation mechanism of Candida antarctica lipase B. Phys Chem Chem Phys 2017;19(24):15709‒14. 链接1
[37] Zisis T, Freddolino PL, Turunen P, van Teeseling MC, Rowan AE, Blank KG. Interfacial activation of Candida antarctica lipase B: combined evidence from experiment and simulation. Biochemistry 2015;54(38):5969‒79. 链接1
京公网安备 11010502051620号
