|Table of Contents|

Construction and application of immobilized laccase reactor(PDF)

Chinese Journal of Applied & Environmental Biology[ISSN:1006-687X/CN:51-1482/Q]

2021 05
Research Field:
Publishing date:


Construction and application of immobilized laccase reactor
LIN Zhiwei YE Xiuyun LIN Juan & YANG Jie?
Fujian Key Laboratory of Marine Enzyme Engineering, Fuzhou University, Fuzhou 350116, China
laccase enzyme immobilization bioreactor malachite green

Laccases are valuable for a variety of industrial applications. Nonetheless, large-scale applications of laccases are limited by the economy and efficiency of the enzymes. Enzyme stability can be improved by immobilization. In the present study, the laccase from Cerrena sp. HYB07 was covalently immobilized on chitosan, and continuous decolorization of dyes was achieved with a lab-scale bioreactor, with guaiacol as the substrate; the immobilized enzyme activity was 43.5 U/g after optimization of immobilization conditions. Immobilized laccase displayed higher thermostability, with a half-life at 65 ℃, 5.3 times that of the free enzyme. The reactor was composed of an injection cell, reaction vessel, and collector, set to batch and cycle modes. The immobilized enzyme was used in an enzyme reactor to decolorize Remazol Brilliant Blue Reactive (RBBR) and malachite green (MG). After ten decolorization cycles, the activity of immobilized laccase and the decolorization rates of the dyes remained at 60% . Compared with the batch mode, the decolorization efficiency of the immobilized enzyme was higher in the circulating mode. Decolorization of MG by the immobilized enzyme was more thorough than that of the free enzyme. After laccase treatment, the microbial toxicity, phytotoxicity, and cytotoxicity of MG were significantly reduced. The stability and decolorization efficiency of laccase improved after immobilization. Furthermore, immobilized laccase has better operational stability in a bioreactor. The results of this study can prove the application value of laccase in dye decolorization and detoxification.


1 Yang J, Li WJ, Tzi Bun Ng, Deng XZ, Lin J, Ye XY. Laccases: production, expression regulation, and applications in pharmaceutical biodegradation [J]. Frontiers Microbiol, 2017, 8: 832
2 Tadesse MA, D’Annibale A, Galli C, Gentili P, Sergi F. An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates [J]. Org Biomol Chem, 2008, 6 (5): 868-878
3 Dwivedi UN, Singh P, Pandey VP, Kumar A. Structure-function relationship among bacterial, fungal and plant laccases [J]. J Mol Catal B Enzym, 2011, 68 (2): 117-128
4 Wen X, Du C, Wan J, Zeng G, Huang D, Yin L, Deng R, Tan S, Zhang J. Immobilizing laccase on Kaolinite and its application in treatment of Malachite Green effluent with the coexistence of Cd (II) [J]. Chemosphere, 2018, 217: 843-850
5 Yang J, Xu XQ, Yang XD, Lin J. Cross-linked enzyme aggregates of Cerrena laccase: preparation, enhanced NaCl tolerance and decolorization of Remazol Brilliant Blue Reactive [J]. J Taiwan Inst Chem Eng, 2016, 65: 1-7
6 Fernández-Fernández M, Sanromán María A, Moldes D. Recent developments and applications of immobilized laccase [J]. Biotechnol Adv, 2013, 31 (8): 1808-1825
7 Nguyen TA, Fu CC, Juang RS. Effective removal of sulfur dyes from water by biosorption and subsequent immobilized laccase degradation on crosslinked chitosan beads [J]. Chem Eng J, 2016, 304: 313-324
8 Davis S, Burns R. Covalent iImmobilization of laccase on activated carbon for phenolic effluent treatment [J]. Appl Microbiol Biotechnol, 1992, 37 (4): 474-479
9 Tavares APM, Rodríguez O, Fernández-Fernández M, Domínguez A, Moldes D, Sanromán María A, Macedo Eugénia A. Immobilization of laccase on modified silica: stabilization, thermal inactivation and kinetic behaviour in 1-ethyl-3-methylimidazolium ethylsulfate ionic liquid [J]. Bioresour Technol, 2013, 131: 405-412
10 Wang F, Hu Y, Guo C, Liu CZ. Enhanced phenol degradation in coking wastewater by immobilized laccase on magnetic mesoporous silica nanoparticles in a magnetically stabilized fluidized bed [J]. Bioresour Technol, 2012, 110: 120-124
11 Bilal M, Jing Z, Zhao YP, Iqbal Hafiz MN. Immobilization of fungal laccase on glutaraldehyde cross-linked chitosan beads and its bio-catalytic potential to degrade bisphenol A [J]. Biocatal Agric Biotechnol, 2019, 19: UNSP 101174
12 Naghdi M, Taheran M, Brar SK, Kermanshahi-Pour A, Verma M, Surampalli RY. Fabrication of nanobiocatalyst using encapsulated laccase onto chitosan-nanobiochar composite [J]. Int J Biol Macromol, 2019, 124: 530-536
13 Nadaroglu H, Mosher G. Biodegradation of some azo dyes from wastewater with laccase from Weissella viridescens LB37 immobilized on magnetic chitosan nanoparticles [J]. J Water Process Eng, 2019, 31: UNSP 100866
14 Zheng F, Cui BK, Wu XJ, Meng G, Liu HX, Si J. Immobilization of laccase onto chitosan beads to enhance its capability to degrade synthetic dyes [J]. Int Biodeterior Biodegrad, 2016, 110: 69-78
15 Nguyen LN, Hai FI, Dosseto A, Richardson C, Price WE, Nghiem LD. Continuous adsorption and biotransformation of micropollutants by granular activated carbon-bound laccase in a packed-bed enzyme reactor [J]. Bioresour Technol, 2016, 210: 108-16
16 HajKacem S, Galai S, Hernández Fernandez FJ, Pérez de Los Ríos A, Smaali I, Quesada Medina J. Bioreactor Membranes for laccase immobilization optimized by ionic liquids and cross-linking agents [J]. Appl Biochem Biotechnol, 2020, 190 (1): 1-17
17 Nguyen LN, Hai FI, Price WE, Leusch FD, Roddick F, Ngo HH, Guo W, Magram SF, Nghiem LD. The effects of mediator and granular activated carbon addition on degradation of trace organic contaminants by an enzymatic membrane reactor [J]. Bioresour Technol, 167: 169-177
18 Yang J, Yang XD, Ye XY, Lin J. Destaining of coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase [J]. Anal Biochem, 2016, 493: 27-29
19 江聪, 尹大强, 许鑫琦, 林娟, 叶秀云, 杨捷. 齿毛菌漆酶对活性亮蓝的高效脱色 [J]. 厦门大学学报(自然科学版), 2018, 57 (3): 337-345 [Jiang C, Yin DQ, Xu XQ, Lin J, Ye XY, Yang J. Efficient decolorization of Remazol Brilliant Blue R by Cerrena unicolor laccase [J]. J Xiamen Univ (Nat Sci Ed), 2018, 57 (3): 337-345]
20 Yang J, Lin Q, Ng TB, Ye XY, Lin J. Purification and characterization of a novel laccase from Cerrena sp. HYB07 with dye decolorizing ability [J]. PLoS ONE, 2014, 9 (10): e110834
21 Yang J, Lin Q, Ng TB, Ye XY, Lin J. Degradation of tetracycline by immobilized laccase and the proposed transformation pathway [J]. J Hazard Mater, 2016, 322 (Pt B): 525-531
22 马晓瑞, 秦家亮, 吴海珍, 许毅, 曹瑜, 乔代蓉, 曹毅. Fe3O4@SiO2-CHO共价固定脂肪酶条件的响应面优化及固定化酶催化生产生物柴油[J]. 应用与环境生物学报, 2017, 23 (2): 256-263 [Ma XR, Qin JL, Wu HZ, Xu Y, Cao Y, Qiao DR, Cao Y. Preparation of a Fe3O4@SiO2-CHO carrier and immobilized lipase as a recyclable catalyst for biodiesel production [J]. Chin J Appl Environ Biol, 2017, 23 (2): 256-263]
23 Bilal M, Asgher M, Iqbal M, Hu H, Zhang X. Chitosan beads immobilized manganese peroxidase catalytic potential for detoxification and decolorization of textile effluent [J]. Int J Biol Macromol, 2016, 89: 181-189.
24 Srivastava PK, Anand A. Immobilization of acid phosphatase from Vigna aconitifolia seeds on chitosan beads and its characterization [J]. Int J Biol Macromol, 2014, 64: 150-154.
25 Kadam AA, Jang J, Jee SC, Sung JS, Lee DS. Chitosan-functionalized supermagnetic Halloysite Nanotubes for covalent laccase immobilization [J]. Carbohydr Polym, 2018, 194: 208-216
26 Verma ML, Kumar S, Das A, Randhawa JS. Chamundeeswari Munusamy Chitin and chitosan-based support materials for enzyme immobilization and biotechnological applications [J]. Environ Chem Let, 2020, 18: 315-323
27 Bilala M, Iqbal Hafiz MN. Lignin peroxidase immobilization on Ca-alginate beads and its dye degradation performance in a packed bed reactor system [J]. Biocatal Agric Biotechnol, 2019, 20: 101205
28 Zhu J, Sun G. Lipase immobilization on glutaraldehyde-activated nanofibrous membranes for improved enzyme stabilities and activities [J]. Relat Fount Player, 2012, 72: 839-845
29 Yang J, Wang Z, Lin Y, Ng TB, Ye XY, Lin J. Immobilized Cerrena sp. laccase: preparation, thermal inactivation, and operational stability in malachite green decolorization [J]. Sci Rep, 2017, 7 (1): 16429.
30 Berrio J, Plou FJ, Ballesteros A, Martínez ?ngel T, Martínez MJ. Immobilization of Pycnoporous coccineus laccase on Eupergit C: stability increase and treatment of oil mill wastewaters [J]. Biocatal Biotransform, 2007, 25 (2-4): 130-134.
32 Yang J, Yang X, Lin Y, Ng TB, Lin J, Ye X. Laccase-catalyzed decolorization of Malachite Green: performance optimization and degradation mechanism [J]. PLoS ONE, 2015, 10 (5): e0127714
33 Chao C, Guan HJ, Zhang J, Liu Y, Zhao YF, Zhang B. Immobilization of laccase onto porous polyvinyl alcohol/halloysite hybrid beads for dye removal [J]. Water Sci Technol, 2018, 77 (3): 809-818
34 Kadam AA, Jang J, Jee SC, Sung JS, Lee DS. Chitosan-functionalized supermagnetic Halloysite Nanotubes for covalent laccase immobilization [J]. Carbohydr Polym, 2018, 194: 208-216
35 Gennari A, Mobayed FH, Catto André L, Benvenutti EV, Volpato G, de SCFV. Kluyveromyces lactis β-galactosidase immobilized on collagen: catalytic stability on batch and packed-bed reactor hydrolysis [J]. React Kinet Mechanisms Catal, 2019, 127: 583-599
36 Reda FM, El-Mekkawy RM, Hassan NS. Detoxification and bioremediation of Sulfa drugs and synthetic dyes by Streptomyces mutabilis A17 laccase produced in solid state fermentation [J]. J Pure Appl Microbiol, 2019, 13 (1): 85-96
37 Huang J, Yang Y, Wang Y, Zhang M, Liu Y. Immobilization of a laccase 2,2’-azino-bis-(3-ethylbenzothiazoline)-6-sulfonic acid system to layered double hydroxide/alginate biohybrid beads for biodegradation of malachite green dye [J]. Biomed Res Int, 2018, 2018: 5471961
38 El-Fakharany EM, Hassan MA, Taha TH. Production and application of extracellular laccase produced by Fusarium oxysporum EMT [J]. Intl J Agric Biol, 18 (5): 939-947
39 Shanmugam S, Ulaganathan P, Swaminathan K, Sadhasivam S, Wu YR. Enhanced biodegradation and detoxification of malachite green by Trichoderma asperellum laccase: degradation pathway and product analysis [J]. Int Biodeterior Biodegrad, 2017, 125: 258-268


Last Update: 2021-10-25