生物技术通报 ›› 2021, Vol. 37 ›› Issue (8): 186-194.doi: 10.13560/j.cnki.biotech.bull.1985.2020-1408
田嘉慧1(), 封佳丽1, 卢俊桦1, 毛林静1, 胡著然2, 王莹1(), 楚杰1()
收稿日期:
2020-11-18
出版日期:
2021-08-26
发布日期:
2021-09-10
作者简介:
田嘉慧,女,硕士研究生,研究方向:工业微生物;E-mail: 基金资助:
TIAN Jia-hui1(), FENG Jia-li1, LU Jun-hua1, MAO Lin-jing1, HU Zhu-ran2, WANG Ying1(), CHU Jie1()
Received:
2020-11-18
Published:
2021-08-26
Online:
2021-09-10
摘要:
漆酶可以与多种底物反应,具有广阔的应用前景,但不同漆酶其性质有一定差异。本文研究的漆酶,由一色齿毛菌(Cerrena unicolor)发酵上清经过(NH4)2SO4沉淀、疏水层析(phenyl sepharose)、离子交换层析(DEAE sepharose)和凝胶层析(TSK gel G2000SWxl)纯化得到,命名为LacT-1。该酶分子量约为65 kD,比活力为41.31 U/mg,回收率为6.57%,纯化倍数为21.86。通过对LacT-1进行酶学性质研究发现:该酶最适温度为60℃,最适pH为4.8,酶活在低于30℃和pH为5.4-8时较稳定;三氯乙酸(TCA)对漆酶酶活有促进作用;LacT-1催化底物ABTS的米氏常数Km值为0.075 mmol/L,Vmax为250 000 mmol/(L·min);对甲基橙、酸性红1、活性黑5、考马斯亮蓝、孔雀石绿、结晶紫和溴酚蓝的降解率在71.64%-95.17%之间。研究结果表明该酶具有较强的pH稳定性,并且与ABTS的结合力较强,可以有效降解一些常见的染料,具有一定的实际应用价值。
田嘉慧, 封佳丽, 卢俊桦, 毛林静, 胡著然, 王莹, 楚杰. 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 Jia-hui, FENG Jia-li, LU Jun-hua, MAO Lin-jing, HU Zhu-ran, WANG Ying, CHU Jie. Isolation,Purification and Characterization of Laccase LacT-1 from Cerrena unicolor[J]. Biotechnology Bulletin, 2021, 37(8): 186-194.
纯化步骤 Purification step | 总酶活 Total enzyme activity/U | 总蛋白 Total protein/mg | 比活力 Specific activity/(U·mg-1) | 纯化倍数 Purification fold | 回收率 Recovery rate/% |
---|---|---|---|---|---|
粗酶液Crude enzyme | 8 395.66 | 4 451.7 | 1.89 | 1 | 100 |
硫酸铵沉淀(NH4)2SO4 precipitation | 3 904.04 | 1 112.93 | 3.51 | 1.86 | 46.50 |
疏水层析Phenyl sepharose | 2 520.62 | 445.17 | 5.66 | 2.99 | 30.02 |
DEAE离子层析DEAE sepharose | 1 401.68 | 66.78 | 20.99 | 11.13 | 16.70 |
凝胶层析TSK gel G2000SWxl | 551.68 | 13.36 | 41.31 | 21.86 | 6.57 |
表1 一色齿毛菌漆酶各纯化步骤的纯化效率
Table 1 Purification efficiency of each purification step of laccase from C. unicolor
纯化步骤 Purification step | 总酶活 Total enzyme activity/U | 总蛋白 Total protein/mg | 比活力 Specific activity/(U·mg-1) | 纯化倍数 Purification fold | 回收率 Recovery rate/% |
---|---|---|---|---|---|
粗酶液Crude enzyme | 8 395.66 | 4 451.7 | 1.89 | 1 | 100 |
硫酸铵沉淀(NH4)2SO4 precipitation | 3 904.04 | 1 112.93 | 3.51 | 1.86 | 46.50 |
疏水层析Phenyl sepharose | 2 520.62 | 445.17 | 5.66 | 2.99 | 30.02 |
DEAE离子层析DEAE sepharose | 1 401.68 | 66.78 | 20.99 | 11.13 | 16.70 |
凝胶层析TSK gel G2000SWxl | 551.68 | 13.36 | 41.31 | 21.86 | 6.57 |
化合物 Compound | 相对酶活Relative enzyme activity/% | ||
---|---|---|---|
0 | 1 mmol·L-1 | 10 mmol·L-1 | |
SDS | 100±1.45 | 29.32±1.98 | 0 |
β-ME | 100±0.23 | 0 | 0 |
EDTA-2Na | 100±2.44 | 89.64±2.51 | 61.55±1.27 |
H2O2 | 100±2.01 | 123.66±2.47 | 59.78±1.57 |
草酸Oxalic acid | 100±0.32 | 90.83±4.31 | 103.80±3.72 |
抗坏血酸Ascorbic acid | 100±0.67 | 0 | 0 |
TCA | 100±1.22 | 111.41±6.58 | 145.95±3.14 |
表2 化合物对LacT-1活性的影响
Table 2 Effect of compounds on the activity of LacT-1
化合物 Compound | 相对酶活Relative enzyme activity/% | ||
---|---|---|---|
0 | 1 mmol·L-1 | 10 mmol·L-1 | |
SDS | 100±1.45 | 29.32±1.98 | 0 |
β-ME | 100±0.23 | 0 | 0 |
EDTA-2Na | 100±2.44 | 89.64±2.51 | 61.55±1.27 |
H2O2 | 100±2.01 | 123.66±2.47 | 59.78±1.57 |
草酸Oxalic acid | 100±0.32 | 90.83±4.31 | 103.80±3.72 |
抗坏血酸Ascorbic acid | 100±0.67 | 0 | 0 |
TCA | 100±1.22 | 111.41±6.58 | 145.95±3.14 |
染料 Dye | 染料类型 Dye type | CAS | 分子式 Molecular formula | 结构式 Chemical formula | 最大吸收波长Maximum absorption wavelength | 12 h降解率12 h degradation rate/% |
---|---|---|---|---|---|---|
甲基橙 Methyl orange | 偶氮 Azo dyes | 547-58-0 | C14H14N3SO3Na | 465 | 75.71±1.34 | |
酸性红1 Acid red 1 | 偶氮 Azo dyes | 3734-67-6 | C18H13N3NaO8S2 | 505 | 76.79±2.78 | |
刚果红 Congo red | 偶氮 Azo dyes | 573-58-0 | C32H22N6Na2O6S2 | 350 | 48.59±2.32 | |
活性黑5 Reactive black 5 | 偶氮 Azo dyes | 17095-24-8 | C26H21N5Na4O19S6 | 590 | 79.60±1.23 | |
考马斯亮蓝R250 Coomassie brilliant blue R250 | 苯甲烷 Phenylmethane dyes | 6104-59-2 | C45H44N3NaO7S2 | 555 | 71.64±3.65 | |
孔雀石绿 Malachite Green | 苯甲烷 Phenylmethane dyes | 569-64-2 | C23H25ClN2 | 595 | 95.17±0.44 | |
结晶紫 Crystal violet | 苯甲烷 Phenylmethane dyes | 548-62-9 | C25H30N3Cl | 545 | 90.61±3.73 | |
溴酚蓝 Bromophenol blue | 苯甲烷 Phenylmethane dyes | 115-39-9 | C19H10Br4O5S | 595 | 74.28±3.56 |
表3 LacT-1对染料的降解
Table 3 Degradation of dyes by LacT-1
染料 Dye | 染料类型 Dye type | CAS | 分子式 Molecular formula | 结构式 Chemical formula | 最大吸收波长Maximum absorption wavelength | 12 h降解率12 h degradation rate/% |
---|---|---|---|---|---|---|
甲基橙 Methyl orange | 偶氮 Azo dyes | 547-58-0 | C14H14N3SO3Na | 465 | 75.71±1.34 | |
酸性红1 Acid red 1 | 偶氮 Azo dyes | 3734-67-6 | C18H13N3NaO8S2 | 505 | 76.79±2.78 | |
刚果红 Congo red | 偶氮 Azo dyes | 573-58-0 | C32H22N6Na2O6S2 | 350 | 48.59±2.32 | |
活性黑5 Reactive black 5 | 偶氮 Azo dyes | 17095-24-8 | C26H21N5Na4O19S6 | 590 | 79.60±1.23 | |
考马斯亮蓝R250 Coomassie brilliant blue R250 | 苯甲烷 Phenylmethane dyes | 6104-59-2 | C45H44N3NaO7S2 | 555 | 71.64±3.65 | |
孔雀石绿 Malachite Green | 苯甲烷 Phenylmethane dyes | 569-64-2 | C23H25ClN2 | 595 | 95.17±0.44 | |
结晶紫 Crystal violet | 苯甲烷 Phenylmethane dyes | 548-62-9 | C25H30N3Cl | 545 | 90.61±3.73 | |
溴酚蓝 Bromophenol blue | 苯甲烷 Phenylmethane dyes | 115-39-9 | C19H10Br4O5S | 595 | 74.28±3.56 |
[1] |
Hakulinen N, Kiiskinen LL, Kruus K, et al. Crystal structure of a laccase from Melanocarpus albomyces with an intact trinuclear copper site[J]. Nat Struct Biol, 2002, 9(8):601-605.
pmid: 12118243 |
[2] |
Zheng BG, Quan L, Hao RW, et al. Bacterial laccases:promising biological green tools for industrial applications[J]. Cellular and Molecular Life Sciences, 2018, 75:3569-3592.
doi: 10.1007/s00018-018-2883-z pmid: 30046841 |
[3] |
Mate DM, Alcalde M. Laccase:A multi-purpose biocatalyst at the forefront of biotechnology[J]. Microbial Biotechnology, 2017, 10(6):1457-1467.
doi: 10.1111/mbt2.2017.10.issue-6 URL |
[4] | Petr B. Fungal laccases occurrence and properties[J]. FEMS Microbiology Reviews, 2010(2):215-242. |
[5] | 廖海君, 李蕊伽, 陶敏, 等. 杂优-2平菇漆酶的分离纯化及酶学性质[J]. 食品科学, 2016(37):147-153. |
Liao HJ, Li RJ, Tao M, et al. Isolation, purification and characterization of laccase from Pleurotus ostreatus Heterosis-2[J]. Food Science, 2016(37):147-153. | |
[6] | 杨娟, 邹亚杰, 张瑞颖, 等. 白灵侧耳漆酶分离纯化及其酶学性质研究[J]. 菌物学报, 2015, 34(3):456-464. |
Yang J, Zou YJ, Zhang RY, et al. Purification and property of laccase from Pleurotus eryngii var. tuoliensis[J]. Mycosystema, 2015, 34(3):456-464. | |
[7] | 翁彩红, 冯宏昌, 赖春芬, 等. 松杉灵芝漆酶的分离纯化及酶学性质研究[J]. 药物生物技术, 2017, 24(1):21-26. |
Weng CH, Feng HC, Lai CF, et al. Isolation, purification and characterization of laccase from Ganodermatsugae[J]. Chinese Journal of Pharmaceutial Biotechnology, 2017, 24(1):21-26. | |
[8] | Antecka A, Blatkiewicz M, Boruta T, et al. Comparison of downstream processing methods in purification of highly active laccase[J]. Springer Open Choice, 2019, 42(10):1635-1645. |
[9] |
Zhang J, Ma F, Zhang X, et al. Directed evolution of a homodimeric Laccase from Cerrena unicolor BBP6 by random mutagenesis and in vivo assembly[J]. International Journal of Molecular Sciences, 2018, 19(10):2989-3000.
doi: 10.3390/ijms19102989 URL |
[10] |
Kachlishvili E, Asatiani M, Kobakhidze A, et al. Trinitrotoluene and mandarin peels selectively affect lignin-modifying enzyme production in white-rot basidiomycetes[J]. Springerplus, 2016, 5(1):1-9.
doi: 10.1186/s40064-015-1659-2 URL |
[11] | Janusz G, Sulej J, Jaszek M, et al. Effect of different wavelengths of light on laccase, cellobiose dehydrogenase, and proteases produced by Cerrena unicolor, Pycnoporus sanguineus and Phlebia lindtneri[J]. Acta Biochim Pol, 2016, 63(2):223-228. |
[12] |
İzlen DÇ, Rıza A, Yalçın G, et al. Determination of the color removal efficiency of laccase enzyme depending on dye class and chromophore[J]. Water Sci Technol, 2019, 80(1):134-143.
doi: 10.2166/wst.2019.255 URL |
[13] |
Anna M, Marta K, Magdalena J, et al. Laccase purified from Cerrena unicolor exerts antitumor activity against leukemic cells[J]. Oncol Lett, 2016, 11(3):2009-2018.
doi: 10.3892/ol.2016.4220 URL |
[14] | Szymon M, Herbert P, Anthony F, et al. Laccase enzyme polymerization by soft plasma jet for durable bioactive coatings[J]. Polymers(Basel) 2018, 10(5):532-551. |
[15] | 马倩倩, 赵丽红, 聂飞. 糙皮侧耳C1诱变菌株F-13漆酶催化染料脱色[J]. 科学技术与工程, 2019, 19(6):304-307. |
Ma QQ, Zhao LH, Nie F. Decolorization of dye catalyzed by Pleurotus ostreatus C1 mutagenic strain F-13 laccase[J]. Science Technology and Engineering, 2019, 19(6):304-307. | |
[16] |
Bagewadi ZK, Mulla SI, Ninnekar HZ. Purification and immobilization of laccase from Trichoderma harzianum strain HZN10 and its application in dye decolorization[J]. Genetic Engineering and Biotechnology Journal, 2017, 15(1):139-150.
doi: 10.1016/j.jgeb.2017.01.007 URL |
[17] | 马倩倩. 白腐菌Pleurotus ostreatus漆酶纯化和酶学性质研究[D]. 锦州:辽宁工业大学, 2019. |
Ma QQ. Purification and property of laccase from white rot fungus Pleurotus ostreatus[D]. Jinzhou:Liaoning University of Technology, 2019. | |
[18] |
Wang SS, Ning YJ, Wang SN, et al. Purification, characterization, and cloning of an extracellular laccase with potent dye decolorizing ability from white rot fungus Cerrena unicolor GSM-01[J]. Int J Biol Macromol, 2017, 95:920-927.
doi: 10.1016/j.ijbiomac.2016.10.079 URL |
[19] |
Manavalan A, Manavalan T, Murugesan K, et al. Characterization of a solvent, surfactant and temperature-tolerant laccase from Pleurotus sp. MAK-II and its dye decolorizing property[J]. Biotechnology Letters, 2015, 37(12):2403-2409.
doi: 10.1007/s10529-015-1937-7 pmid: 26315605 |
[20] | 杨清香, 王涛, 王栋, 等. 平菇漆酶的性质和应用研究[J]. 环境科学与技术, 2008, 31(9):39-41. |
Yang QX, Wang T, Wang D, et al. Characteristics and application of laccase from Pleuotus ostreatus[J]. Environmental Science & Technology, 2008, 31(9):39-41. | |
[21] | 董天保. 紫杉木齿菌漆酶的分离纯化及其对木质素的降解研究[D]. 武汉:华中科技大学, 2013. |
Dong TB. Study of purification and lignin deradation of laccase from Echinodontium taxodii[D]. Wuhan:Huazhong University of Science and Technology, 2013. | |
[22] | 姚良同, 魏玮, 张建军, 等. 滑菇Pholiota nameko SW-01菌株产漆酶营养条件及部分酶学性质研究[J]. 安徽农业科学, 2014, 42(4):969-971, 980. |
Yao LT, Wei W, Zhang JJ, et al. Optimization of parameters on laccase production and properties of laccase from Pholiota nameko SW-01[J]. Journal of Anhui Agricultural Sciences, 2014, 42(4):969-971, 980. | |
[23] | 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):94-103. |
Liu Q, Cui X, Kong WW, et al. Purification and property of laccase from Pleurotus eryngii var. tuoliensis[J]. Acta Edulis Fungi, 2019, 26(4):94-103. | |
[24] | 窦隆, 陈莎, 李传仁, 等. 糙皮侧耳P5漆酶酶学性质的初步研究[J]. 长江大学学报:自然科学版, 2015(1):61-64. |
Dou W, Chen S, Li CR, et al. Preliminary study on property of laccase from Pleurotus ostreatus P5[J]. Journal of Yangtze University:Natural Science Edition, 2015(1):61-64. | |
[25] | 刘骊, 单书凯, 等. 杏鲍菇菌糠漆酶分离纯化及部分酶学性质研究[J]. 江西农业大学学报, 2017, 39(5):989-995. |
Liu L, Shan SK, et al. Purification and properties of laccase from spent mushroom compost of Pleurotuseryngii[J]. Acta Agriculturae Universitatis Jiangxiensis, 2017, 39(5):989-995. | |
[26] |
Forootanfar H, Faramarzi MA, Shahverdi AR, et al. Purification and biochemical characterization of extracellular laccase from the ascomycete Paraconiothyrium variabile[J]. Bioresource Technology, 2011, 102(2):1808-1814.
doi: 10.1016/j.biortech.2010.09.043 pmid: 20933400 |
[27] | 陈维新. 过渡金属离子对漆酶催化活性的影响[J]. 分析测试学报, 2004(4):78-80. |
Chen WX. Effect of metal ions on the laccase activity[J]. Journal of Instrumental Analysis. 2004(4):78-80. | |
[28] | 张莉. 白腐菌(Trametes pubescens MB89)漆酶酶学性质及其在酚类化合物的降解特性研究[D]. 杨凌:西北农林科技大学, 2009. |
Zhang Li. Characteristics of Laccase from white rot fungus(T. pubescens MB89)and its use in phenolic compounds degradation[D]. Yangling:Northwest A&F University, 2009. | |
[29] | 骆燕. 一株高产漆酶野生金针菇菌株的鉴定及酶学性质研究[J]. 食用菌, 2018, 40(3):7-11. |
Luo Y. Study on identification of a laccase-producing wild edible fungi and on its enzymatic properties[J]. Edible Fungus, 2018, 40(3):7-11. | |
[30] | 邵小虎. 苏云金芽胞杆菌自溶素蛋白用于构建表面展示系统的研究[D]. 武汉:华中农业大学, 2009. |
Shao XH. Cell surface display system for Bacillus thuringiensis:a novel development using cell well-bond autolysins[D]. Wuhan:Huazhong Agricultural University, 2009. | |
[31] | 傅恺, 付时雨, 张丽, 等. 亚热带木腐真菌产漆酶及其酶学性质[J]. 华南理工大学学报, 2011, 39(9):152-157. |
Fu K, Fu SY, Zhang L, et al. Production and enzymatic properties of Laccase from a subtropical wood-rot fungus[J]. Journal of South China University of Technology, 2011, 39(9):152-157. | |
[32] | 凌庆枝, 董丽辉, 范三微, 等. 亮菌漆酶的初步研究[J]. 食品科学, 2009(19):190-193. |
Ling QZ, Dong LH, Fan SW, et al. Preliminary study on Laccase from Armillariella tabescens[J]. Food Science, 2009(19):190-193. | |
[33] |
Ji Z, Lei S, Hao Z, et al. A novel homodimer laccase from Cerrena unicolor BBP6:Purification, characterization, and potential in dye decolorization and denim bleaching[J]. PLoS One, 2018, 13(8):e0202440.
doi: 10.1371/journal.pone.0202440 URL |
[1] | 赵赛赛, 张小丹, 贾晓妍, 陶大炜, 刘可玉, 宁喜斌. 高产硝酸盐还原酶Staphylococcus simulans ZSJ6的复合诱变选育及其酶学性质研究[J]. 生物技术通报, 2023, 39(4): 103-113. |
[2] | 杨俊钊, 张新蕊, 赵国柱, 郑菲. 新型GH5家族多结构域纤维素酶的结构与功能研究[J]. 生物技术通报, 2023, 39(4): 71-80. |
[3] | 王雨辰, 丁尊丹, 关菲菲, 田健, 刘国安, 伍宁丰. 耐热漆酶ba4基因鉴定与酶学性质分析[J]. 生物技术通报, 2022, 38(8): 252-260. |
[4] | 贾晨波, 苏一黄, 马秀梅, 王春利, 徐春燕. 端梗霉Z45产漆酶培养基的优化及其对染料的脱色[J]. 生物技术通报, 2022, 38(6): 252-260. |
[5] | 毛国涛, 王杰, 王凯, 王方园, 曹乐言, 张宏森, 宋安东. 水生栖热菌漆酶TaLac的性质分析及对孔雀石绿染料的脱除[J]. 生物技术通报, 2022, 38(4): 261-268. |
[6] | 常晴, 束月蓉, 王文韬, 蒋昊, 延泉德, 钱政, 高雪纯, 吴金鸿, 张勇. 来自Yeosuana marina sp. JLT21内切型海藻酸裂解酶的异源表达及酶学表征[J]. 生物技术通报, 2022, 38(2): 123-131. |
[7] | 张丰文, 周丽亚, 董超, 史延茂. 纳豆中抗氧化肽的分离纯化与活性研究[J]. 生物技术通报, 2022, 38(2): 158-165. |
[8] | 王小桃, 邹杭, 吴怡, 向省维, 吕华, 刘超兰, 林家富, 王欣荣, 褚以文, 宋涛. Paraglaciecola hydrolytica中新型β-琼胶酶Aga2的异源表达及酶学性质分析[J]. 生物技术通报, 2022, 38(11): 258-268. |
[9] | 岑潇龙, 雷曦, 马诗云, 陈倩茹, 黄遵锡, 周峻沛, 张蕊. 金黄色葡萄球菌透明质酸裂解酶HylS的异源表达与特性研究[J]. 生物技术通报, 2022, 38(1): 157-167. |
[10] | 陈明雨, 倪烜, 司友斌, 孙凯. 固定化真菌漆酶在环境有机污染修复中的应用研究进展[J]. 生物技术通报, 2021, 37(6): 244-258. |
[11] | 张瑶心, 王亮节, 郑文, 徐汉琴, 郑恋, 钟静. 产几丁质酶的无色杆菌ZWW8的发酵产酶及酶学性质研究[J]. 生物技术通报, 2021, 37(4): 96-106. |
[12] | 王志新, 鲁雷震, 周景波, 封成玲, 贾紫伟, 宁亚维, 贾英民. 抗真菌肽研究进展[J]. 生物技术通报, 2021, 37(3): 206-218. |
[13] | 熊雪, 李鹏, 张贵合, 向准, 陶文广, 周光燕, 和耀威. 不同栽培基质诱导对香菇液体发酵产漆酶活性的影响[J]. 生物技术通报, 2021, 37(12): 50-59. |
[14] | 王豪, 唐禄鑫, 马鸿飞, 钱坤, 司静, 崔宝凯. 东方栓孔菌漆酶的固定化及其对不同类型染料的脱色作用[J]. 生物技术通报, 2021, 37(11): 142-157. |
[15] | 刘珊, 叶伟, 朱牧孜, 李赛妮, 邓张双, 章卫民. 一种新型酰基转移酶GPAT的克隆、表达与酶学性质研究[J]. 生物技术通报, 2021, 37(11): 257-266. |
阅读次数 | ||||||
全文 |
|
|||||
摘要 |
|
|||||