一色齿毛菌漆酶LacT-1的分离纯化与性质研究

doi:10.13560/j.cnki.biotech.bull.1985.2020-1408

摘要/Abstract

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K_m值为0.075 mmol/L,V_max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。

关键词: 漆酶, 一色齿毛菌, 分离纯化, 酶学性质, 降解染料

Abstract:

Laccase may react with many types of substrates and has a broad application prospect,but the laccase properties from different strains differ. In this study,the laccase in the fermentation supernatant of a newly isolated fungus Cerrena unicolor was separated and purified sequentially by（NH₄）₂SO₄ precipitation,hydrophobic chromatography（phenyl sepharose）,ion exchange chromatography（DEAE sepharose）and gel chromatography（TSK gel G2000SWxl）,and named as LacT-1. The molecular weight of purified LacT-1 was approximately 65 kD with specific activity of 41.31 U/mg. The recovery rate was 6.57% after 21.86 times of purification. The optimal temperature of LacT-1 was 60℃ and the optimal pH was 4.8. The LacT-1 activity was relatively stable with the temperature at 0-30℃ or pH 5.4-8. TCA promoted laccase activity. The Michaelis constant K_m of LacT-1 with ABTS as substrate was 0.075 mmol/L,while the corresponding V_max was 250 000 mmol/（L·min）. The degradation rates of methyl orange,acid red 1,reactive black 5,Coomassie brilliant blue R250,malachite green,crystal violet and bromophenol blue ranged in 71.64% to 95.17%. The above results indicate that the LacT-1 has practical application potential because of excellent pH stability,strong binding force with ABTS and it can effectively degrade some common dyes.

Key words: laccase, Cerrena unicolor, isolation and purification, enzymatic properties, degradation of dyes

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.

图/表 11

参考文献 33

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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

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/%
化合物 Compound	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

化合物 Compound	相对酶活Relative enzyme activity/%
化合物 Compound	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	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