漆酶是一种单电子氧化还原酶,在日常生活中是一种优良的生态替代酶。木霉作为生长迅速的生防菌,在漆酶生产中具有明显优势。该研究从江西省南昌市土壤中筛选获得一株产漆酶的棘孢木霉菌株Tasjk26,在最优培养条件(碳源为麦芽糖,氮源为胰蛋白胨,pH 6.0,28 ℃,200 r/min)时酶活力达到2.56 U/mL。粗酶液60%(质量分数)(NH4)2SO4沉淀透析后,DEAE-纤维素阴离子层析纯化,酶被纯化了13.2倍,酶的回收率为23.9%,SDS-PAGE检测结果表明该酶分子质量为70 kDa。纯化后的酶以ABTS为底物时酶促反应米氏常数Km为0.175 mol/L。酶最适反应pH值为3,最适反应温度为40 ℃。该酶对有机染料孔雀绿、结晶紫、刚果红、中性红和甲基橙均有降解能力,降解率分别为69.1%、60.9%、54.6%、40.1%、22.2%。研究表明该菌株在有机污染物治理方面有重要价值。
Laccase is a multifunctional single electron redox enzyme, which is used as an excellent ecological substitute enzyme in various applications in daily life.As a fungal biocontrol agents, Trichoderma exhibited rapid growth and significant potential for laccase production.In this study, a Trichoderma asperellum strain Tasjk26 with high laccase production was isolated from soil samples collected in Nanchang, Jiangxi, China.Under the optimized culture conditions, which maltose as carbon source, tryptone as nitrogen source, pH 6.0, temperature 28 ℃ and rotation speed 200 r/min, the strain showed laccase activity of 2.56 U/mL.Following a 60% saturation ammonium sulfate precipitation and dialysis, the crude enzyme solution was purified using DEAE-cellulose anion exchange chromatography.The purification rate of laccase reached 13.2 times, and the recovery rate was 23.9%.SDS-PAGE electrophoresis analysis confirmed that the molecular weight of the purified enzyme was 70 kDa.When ABTS was used as substrate, the Michaelis constant (Km) of the purified laccase was 0.175 mol/L.The laccase showed the best activity at pH 3 and temperature 40 ℃.In addition, it also can degrade organic dyes such as malachite green, crystal violet, Congo red, neutral red and methyl orange, which the degradation efficiency is 69.1%, 60.9%, 54.6%, 40.1% and 22.2% respectively.These findings emphasize the important value of Tasjk26 strain in the remediation of organic pollutants and provide a promising biocatalytic tool for environmental purification.
[1] YANG J, LIN Y H, YANG X D, et al.Degradation of tetracycline by immobilized laccase and the proposed transformation pathway[J].Journal of Hazardous Materials, 2017, 322:525-531.
[2] 郭金英, 赵翠敏, 张云龙, 等.血红栓菌液体发酵产漆酶培养条件的研究[J].中国食用菌, 2019, 38(11):52-56.
GUO J Y, ZHAO C M, ZHANG Y L, et al.Study on optimization of liquid fermentation cultivation conditions for laccase production by Trametes sanguinea[J].Edible Fungi of China, 2019, 38(11):52-56.
[3] MORENO A D, IBARRA D, EUGENIO M E, et al.Laccases as versatile enzymes:From industrial uses to novel applications[J].Journal of Chemical Technology & Biotechnology, 2020, 95(3):481-494.
[4] 闫莉, 刘海波, 唐德羲, 等.生物催化引发合成高分子化木质素[J].中国造纸学报, 2024, 39(2):35-43.
YAN L, LIU H B, TANG D X, et al.Polymerization of lignin by biocatalytic initiation method[J].Transactions of China Pulp and Paper, 2024, 39(2):35-43.
[5] JANUSZ G, PAWLIK A, ŚWIDERSKA-BUREK U, et al.Laccase properties, physiological functions, and evolution[J].International Journal of Molecular Sciences, 2020, 21(3):966.
[6] VASILIADOU I A, SÁNCHEZ-VÁZQUEZ R, MOLINA R, et al.Biological removal of pharmaceutical compounds using white-rot fungi with concomitant FAME production of the residual biomass[J].Journal of Environmental Management, 2016, 180:228-237.
[7] 肖旭倩, 胡正龙, 邵广晴.细菌降解三苯甲烷类染料废水的研究进展[J].中国资源综合利用, 2019, 37(7):83-85.
XIAO X Q, HU Z L, SHAO G Q.Research progress in degradation of triphenylmethane dye wastewater by bacteria[J].China Resources Comprehensive Utilization, 2019, 37(7):83-85.
[8] ZENG X K, CAI Y J, LIAO X R, et al.Decolorization of synthetic dyes by crude laccase from a newly isolated Trametes trogii strain cultivated on solid agro-industrial residue[J].Journal of Hazardous Materials, 2011, 187(1-3):517-525.
[9] 杨珍福, 陈惠, 郭相, 等.两株田头菇属野生菌株的鉴定及生物学特性[J].北方园艺, 2021(6):116-122.
YANG Z F, CHEN H, GUO X, et al.Identification and biological characteristics of two strains from wild Agrocybe[J].Northern Horticulture, 2021(6):116-122.
[10] SHI Y W, NIU X X, YANG H M, et al.Optimization of the fermentation media and growth conditions of Bacillus velezensis BHZ-29 using a Plackett-Burman design experiment combined with response surface methodology[J].Frontiers in Microbiology, 2024, 15:1355369.
[11] 李润根, 于鑫, 孙琴琴, 等.龙牙百合褐斑病病原的鉴定、生物学特性及室内药剂筛选[J].核农学报, 2024, 38(8):1487-1495.
LI R G, YU X, SUN Q Q, et al.Identification, characterization the leaf brown spot pathogen in Lilium brownii var.viridulum and screening fungicides against Alternaria arborescens[J].Journal of Nuclear Agricultural Sciences, 2024, 38(8):1487-1495.
[12] ABD EL-LATIF A S, ZOHRI A A, EL-AREF H M, et al.Kinetic studies on optimized extracellular laccase from Trichoderma harzianum PP389612 and its capabilities for azo dye removal[J].Microbial Cell Factories, 2024, 23(1):150.
[13] 彭微, 丁威, 吴宜, 等.基于酿酒废水发酵生产棘孢木霉工艺条件优化及应用研究[J].食品与发酵科技, 2023, 59(4):78-84;91.
PENG W, DING W, WU Y, et al.Optimization of fermentation conditions for Trichoderma echinospora production from liquor-making wastewater and its application[J].Food and Fermentation Science & Technology, 2023, 59(4):78-84;91.
[14] 刘芹, 崔筱, 孔维威, 等.白灵侧耳菌渣中漆酶的分离纯化与酶学性质[J].食用菌学报, 2019, 26(4):90-99.
LIU Q, CUI X, KONG W W, et al.Purification and characterization of a laccase from Pleurotus tuoliensis cultivation residue[J].Acta Edulis Fungi, 2019, 26(4):90-99.
[15] 田嘉慧, 封佳丽, 卢俊桦, 等.一色齿毛菌漆酶LacT-1的分离纯化与性质研究[J].生物技术通报, 2021, 37(8):186-194.
TIAN J H, FENG J L, LU J H, et al.Isolation, purification and characterization of laccase LacT-1 from Cerrena unicolor[J].Biotechnology Bulletin, 2021, 37(8):186-194.
[16] 唐禄鑫, 王雅娴, 彭明意,等.真菌漆酶及其生产、固定化与应用[J].菌物学报, 2023, 42(9):1821-1837.
TANG L X, WANG Y X, PENG M Y, et al. Fungal laccase and its production, immobilization, and application:A review[J].Mycosystema, 2023, 42(9):1821-1837.
[17] 黄翠欣, 张桂容, 刘军, 等.一株产纤维素酶菌株的分离纯化及其产酶性质研究[J].食品与发酵工业, 2023, 49(17):37-43.
HUANG C X, ZHANG G R, LIU J, et al.Isolation and purification of a cellulose-producing strain and its enzyme-producing properties[J].Food and Fermentation Industries, 2023, 49(17):37-43.
[18] 张亚楠, 王亚勋, 宋立立.枯草芽孢杆菌发酵蟹壳粉产蛋白酶的研究[J].中国饲料, 2024(13):42-47.
ZHANG Y N, WANG Y X, SONG L L.Study on protease production from crab shell meal fermented by Bacillus subtilis[J].China Feed, 2024(13):42-47.
[19] 李丽, 张学成, 肖亚中.栓菌4281漆酶催化几种不同结构染料脱色[J].生物学杂志, 2023, 40(2):33-37.
LI L, ZHANG X C, XIAO Y Z.Decolorization of several different dyes by Trametes sp.4281 laccase[J].Journal of Biology, 2023, 40(2):33-37.
[20] 吴丽雅. 深海海单胞菌来源漆酶LacMp1在大肠杆菌中的高效表达及其在染料脱色中的应用[D].合肥:合肥学院, 2023.
WU L Y.Expression of Marinomonas profundimaris sp-derived laccase LacMp1 in Escherichia coli and its application in dye decolorization[D].Hefei:Hefei university, 2023.
