Cross-linked enzyme-polymer conjugates with excellent stability and detergent-enhanced activity for efficient organophosphate degradation

Abstract

Background

Enzymatic biodegradation of organophosphate pesticides (OPs) is a promising technology to remove these toxic compounds. However, its application in industrial washing was restricted by the lack of efficient immobilized enzymes that can work at high temperatures and high pHs in the presence of various detergents. Therefore, it is necessary to develop a simple method to prepare a robust immobilized enzyme for efficient degradation of OPs.

Results

An organophosphate hydrolase (OPH), PoOPH_M9, was conjugated and immobilized with a commercially available polymer, Pluronic F127. The prepared cross-linked enzyme-polymer conjugate (CLEPC) displayed higher pH stability in the range from 7.0 to 11.0 and a higher optimal temperature (50 °C) than that of free PoOPH_M9 (30 °C). Its half-life and apparent k_cat/K_M reached 12.8 h at 50 °C and 390.3 ± 7.8 mM⁻¹ s⁻¹, respectively, which were even better than that of the traditional cross-linked enzyme aggregates (CLEA, 7.2 h and 10.9 ± 1.7 mM⁻¹ s⁻¹). The activity of PoOPH_M9 CLEPC was further enhanced up to 2.5-fold by the anionic, nonionic and biocompatible detergents, which was first observed. 0.15 mM Malathion was degraded completely by PoOPH_M9 CLEPC after activation within 10 min in the presence of 0.1% (w/w) detergents of all types at pH 9.0 and 25 °C, demonstrating its capability in degrading OPs at practically relevant conditions.

Conclusion

The conjugation of Pluronic F127 in enzyme immobilization could effectively reduce the activity loss of immobilized enzymes and enhance their stability and activity at high temperatures and high pHs. In addition, the activity of CLEPC can be even enhanced in the presence of various detergents. This technology can be extended easily to produce other immobilized polymer-enzyme conjugates due to its simplicity.

Keywords

Organophosphate / Organophosphate hydrolase / Immobilization / Biodegradation / Enzyme-polymer conjugates / Pluronic F127

References

[1] Abe K, Yoshida S, Suzuki Y, Mori J, Doi Y, Takahashi S, Kera Y. Haloalkylphosphorus hydrolases purified from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1. Appl Environ Microbial, 2014, 80: 5866-5873.

[2] Akbar U, Aschenbrenner CD, Harper MR, Johnson HR, Dordick JS, Clark DS. Direct solubilization of enzyme aggregates with enhanced activity in nonaqueous media. Biotechnol Bioeng, 2007, 96: 1030-1039.

[3] Bai YP, Luo XJ, Zhao YL, Li CX, Xu DS, Xu JH. Efficient degradation of malathion in the presence of detergents using an engineered organophosphorus hydrolase highly expressed by Pichia pastoris without methanol induction. J Agric Food Chem, 2017, 65: 9094-9100.

[4] Bigley AN, Xiang DF, Ren ZJ, Xue HR, Hull KG, Romo D, Raushel FM. Chemical mechanism of the phosphotriesterase from Sphingobium sp. strain TCM1, an enzyme capable of hydrolyzing organophosphate flame retardants. J Am Chem Soc, 2016, 138: 2921-2924.

[5] Breger JC, Ancona MG, Walper SA, Oh E, Susumu K, Stewart MH, Deschamps JR, Medintz IL. Understanding how nanoparticle attachment enhances phosphotriesterase kinetic efficiency. ACS Nano, 2015, 9: 8491-8503.

[6] Chen J, Luo XJ, Chen Q, Pan J, Zhou JH, Xu JH. Marked enhancement of Acinetobacter sp. organophosphorus hydrolase activity by a single residue substitution Ile211Ala. Bioresour Bioprocess, 2015, 2: 39-46.

[7] Cherny I, Greisen PJ, Ashani Y, Khare SD, Oberdorfer G, Leader H, Baker D, Tawfik DS. Engineering V-type nerve agents detoxifying enzymes using computationally focused libraries. ACS Chem Biol, 2013, 8: 2394-2403.

[8] Cycon M, Mrozik A, Piotrowska-Seget Z. Bioaugmentation as a strategy for the remediation of pesticide-polluted soil: a review. Chemosphere, 2017, 172: 52-71.

[9] Gao Y, Truong YB, Cacioli P, Butler P, Kyratzis IL. Bioremediation of pesticide contaminated water using an organophosphate degrading enzyme immobilized on nonwoven polyester textiles. Enzyme Microbial Technol, 2014, 54: 38-44.

[10] Giudice ID, Coppolecchia R, Merone L, Porzio E, Carusone TM, Mandrich L, Worek F, Manco G. An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination. Biotechnol Bioeng, 2016, 113: 724-734.

[11] Hernandez AF, Gil F, Lacasana M. Toxicological interactions of pesticide mixtures: an update. Arch Toxicol, 2017, 91: 3211-3223.

[12] Hondred JA, Breger JC, Garland NT, Oh E, Susumu K, Walper SA, Medintz IL, Claussen JC. Enhanced enzymatic activity from phosphotriesterase trimer gold nanoparticle bioconjugates for pesticide detection. Analyst, 2017, 142: 3261-3271.

[13] Jackson CJ, Weir K, Herlt A, Khurana J, Sutherland TD, Horne I, Easton C, Russell RJ, Scott C, Oakeshott JG. Structure-based rational design of a phosphotriesterase. Appl Environ Microbiol, 2009, 75: 5153-5156.

[14] Khare SD, Kipnis Y, Greisen PJ, Takeuchi R, Ashani Y, Goldsmith M, Song YF, Gallaher JL, Silman I, Leader H, Sussman JL, Stoddard BL, Tawfik DS, Baker D. Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis. Nat Chem Biol, 2012, 8: 294-300.

[15] Kim CS, Seo JH, Kang DG, Cha HJ. Engineered whole-cell biocatalyst-based detoxification and detection of neurotoxic organophosphate compounds. Biotechnol Adv, 2014, 32: 652-662.

[16] Kim M, Gkikas M, Huang A, Kang JW, Suthiwangcharoen N, Nagarajan R, Olsen BD. Enhanced activity and stability of organophosphorus hydrolase via interaction with an amphiphilic polymer. Chem Commun, 2014, 50: 5345-5348.

[17] LeJeune KE, Mesiano AJ, Bower SB, Grimsley JK, Wild JR, Russell AJ. Dramatically stabilized phosphotriesterase—polymers for nerve agent degradation. Biotechnol Bioeng, 1997, 54: 105-114.

[18] Li P, Moon SY, Guelta MA, Lin L, Gomez-Gualdron DA, Snurr RQ, Harvey SP, Hupp JT, Farha OK. Nanosizing a metal-organic framework enzyme carrier for accelerating nerve agent hydrolysis. ACS Nano, 2016, 10: 9174-9182.

[19] Luo XJ, Kong XD, Zhao J, Chen Q, Zhou JH, Xu JH. Switching a newly discovered lactonase into an efficient and thermostable phosphotriesterase by simple double mutations His250Ile/Ile263Trp. Biotechnol Bioeng, 2014, 111: 1920-1930.

[20] Luo XJ, Zhao J, Li CX, Bai YP, Reetz MT, Yu HL, Xu JH. Combinatorial evolution of phosphotriesterase toward a robust malathion degrader by hierarchical iteration mutagenesis. Biotechnol Bioeng, 2016, 113: 2350-2357.

[21] Mostafalou S, Abdollahi M. Pesticides: an update of human exposure and toxicity. Arch Toxicol, 2017, 91: 549-599.

[22] Pan J, Kong XD, Li CX, Ye Q, Xu JH, Imanaka T. Crosslinking of enzyme coaggregate with polyethyleneimine: a simple and promising method for preparing stable biocatalyst of Serratia marcescens lipase. J Mol Catal B Enzym, 2011, 68: 256-261.

[23] Pan J, Dang ND, Zheng GW, Cheng B, Ye Q, Xu JH. Efficient production of l-menthol in a two-phase system with SDS using an immobilized Bacillus subtilis esterase. Bioresour Bioprocessing, 2014, 1: 12.

[24] Perzon A, Dicko C, Cobanoğlu Ö, Yükselen O, Eryilmaz J, Dey ES. Cellulase cross-linked enzyme aggregates (CLEA) activities can be modulated and enhanced by precipitant selection. J Chem Technol Biotechnol, 2017, 92: 1645-1649.

[25] Ramalho TC, de Castro AA, Silva DR, Silva MC, Franca TCC, Bennion BJ, Kuca K. Computational enzymology and organophosphorus degrading enzymes: promising approaches toward remediation technologies of warfare agents and pesticides. Curr Med Chem, 2016, 23: 1041-1061.

[26] Raynes JK, Pearce FG, Meade SJ, Gerrard JA. Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: towards bioremediation and chemical detoxification. Biotechnol Prog, 2011, 27: 360-367.

[27] Schenk G, Mateen I, Ng TK, Pedroso MM, Mitic N, Jafelicci M, Marques RFC, Gahan LR, Olis DL. Organophosphate-degrading metallohydrolases: structure and function of potent catalysts for applications in bioremediation. Coordin Chem Rev, 2016, 317: 122-131.

[28] Singh AK, Flounders AW, Volponi JV, Ashley CS, Wally K, Schoeniger JS. Development of sensors for direct detection of organophosphates. part I: immobilization, characterization and stabilization of acetylcholinesterase and organophosphate hydrolase on silica supports. Biosens Bioelectron, 1999, 14: 703-713.

[29] Suthiwangcharoen N, Nagarajan R. Enhancing enzyme stability by construction of polymer–enzyme conjugate micelles for decontamination of organophosphate agents. Biomacromol, 2014, 15: 1142-1152.

[30] Theriot C, Grunden AM. Hydrolysis of organophosphorus compounds by microbial enzymes. Appl Microbial Biotechnol, 2011, 89: 35-43.

[31] Wang MF, Qi W, Jia CX, Ren YF, Su RX, He ZM. Enhancement of activity of cross-linked enzyme aggregates by a sugar-assisted precipitation strategy: technical development and molecular mechanism. J Biotechnol, 2011, 156: 30-38.

[32] Wei W, Du JJ, Li J, Yan M, Zhu Q, Jin X, Zhu XY, Hu ZM, Tang Y, Lu YF. Construction of robust enzyme nanocapsules for effective organophosphate decontamination, detoxification, and protection. Adv Mater, 2013, 25: 2212-2218.

[33] Wu XL, Wang R, Zhang YF, Ge J, Liu Z. Enantioselective ammonolysis of phenylglycine methyl ester with lipase–Pluronic nanoconjugate in tertiary butanol. Catal Lett, 2014, 144: 1407-1410.

[34] Wu XL, Ge J, Zhu JY, Zhang YF, Yong Y, Liu Z. A general method for synthesizing enzyme-polymer conjugates in reverse emulsions using Pluronic as a reactive surfactant. Chem Commun, 2015, 51: 9674-9677.

[35] Yan XY, Jiang YJ, Zhang SP, Gao J, Zhang YF. Dual-functional OPH-immobilized polyamide nanofibrous membrane for effective organophosphorus toxic agents protection. Biochem Eng J, 2015, 98: 47-55.

[36] Zhang YF, Dai Y, Hou M, Li T, Ge J, Liu Z. Chemo-enzymatic synthesis of valrubicin using Pluronic conjugated lipase with temperature responsiveness in organic media. RSC Adv, 2013, 3: 22963-22966.

[37] Zheng GW, Yu HL, Li CX, Pan J, Xu JH. Immobilization of Bacillus subtilis esterase by simple cross-linking for enzymatic resolution of dl-menthyl acetate. J Mol Catal B Enzym, 2011, 70: 138-143.

Funding

National Key Research and Development Program of China(2016YFA0204300)

National Natural Science Foundation of China(21505044)

Natural Science Foundation of Shanghai(18ZR1409900)