巴尔喀什蘑菇子实体漆酶纯化与理化特性及对酚类化合物的降解能力

doi:10.6048/j.issn.1001-4330.2023.02.021

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (2): 432-439.DOI: 10.6048/j.issn.1001-4330.2023.02.021

• 园艺特产·植物保护·微生物·土壤肥料·节水灌溉 • 上一篇下一篇

巴尔喀什蘑菇子实体漆酶纯化与理化特性及对酚类化合物的降解能力

努尔孜亚·亚力买买提(), 郝敬喆(), 贾文捷, 陈浩宇, 罗影, 贾培松()

新疆农业科学院植物保护研究所/农业农村部西北荒漠绿洲作物有害生物综合治理重点实验室,乌鲁木齐 830091

收稿日期:2022-05-27 出版日期:2023-02-20 发布日期:2023-03-31
通信作者: 郝敬喆(1969-),男,江苏人,研究员,研究方向为微生物病理学,(E-mail)urmqhjz@qq.com; 贾培松(1984-),男,河北人,副研究员,研究方向为食用菌驯化与栽培,(E-mail)jps-fly@163.com
作者简介:努尔孜亚·亚力买买提(1977-),女,新疆伊犁人,副研究员,研究方向为食用菌驯化与栽培, (E-mail)nrzy729@163.com
基金资助:
国家自然科学基金地区基金(32060709);国家自然科学基金地区基金(31560577);现代农业产业技术体系建设专项资金(CARS-20)

Purification and Physicochemical Properties of Laccase from Fruiting Bodies of Agaricus balchaschensis

Nurziya Yarmamat(), HAO Jingzhe(), JIA Wenjie, CHEN Haoyu, LUO Ying, JIA Peisong()

Key Laboratory of Intergraded Management of Harmful Crop Vermin in Northwestern Oasis of China, Ministry of Agriculture and Rural Affairs of the P.R.C. /Institute of Plant Protection,Xinjiang Academy of Agricultural Sciences,Urumqi 830091,China

Received:2022-05-27 Online:2023-02-20 Published:2023-03-31
Correspondence author: HAO Jingzhe (1970-), researcher, research area: micropathology,(E-mail) urmqhjz@qq.com; JIA Peisong (1984-), associate researcher, research area: edible fungi research, (E-mail)jps-fly@163.com
Supported by:
National Natural Science Foundation Project(32060709);National Natural Science Foundation Project(31560577);Earmarked Fund for CARS(CARS-20)

摘要/Abstract

摘要：

【目的】 研究巴尔喀什蘑菇子实体的生物学特性、漆酶理化性质及对酚类化合物的降解能力,为野生巴尔喀什蘑菇的栽培驯化以及栽培料高效利用提供理论依据。【方法】 利用纤维素CM-cellulose阳离子交换和Q-Sepharose阴离子交换以及Superdex 75凝胶层析过滤纯化技术,纯化分析该漆酶的降解代谢底物与抗金属离子抑制能力,确定该漆酶活性最适温度与pH值。【结果】 获得本漆酶为65 kDa的单亚基蛋白,总纯化倍数为354.58,比活力17.02 U/mg,该酶N端10个氨基酸的序列为SGGPEQNTTA,经过NBCI-BLAST后发现该漆酶与糙皮侧耳、杂色云芝等的漆酶具有同源性。本漆酶底物差异性较大,从强到弱主要为ABTS>二甲氧基酚>甲苯胺。最适pH值为2.2,属中低温活性;最适反应温度为40℃,为中低温活性漆酶。Cu²⁺对该漆酶催化ABTS有轻微促进作用,Hg²⁺对其催化ABTS有很强的抑制作用。【结论】 巴尔喀什蘑菇子实体漆酶为65 kDa的单亚基蛋白,是一种中低温活性耐酸性漆酶,底物特异性为ABTS>二甲氧基酚>甲苯胺。

关键词: 巴尔喀什蘑菇; 漆酶; 纯化; 天然底物

Abstract:

【Objective】 To determine the physiochemical properties and biological functions of the laccase from the fruiting bodies of wild Agaricus balchaschensis, this study to explore the biological characteristics of the fruiting bodies, the physiochemical properties of the laccase, and the degradation ability of phenolic compoundstheoretical basis for the cultivation domestication of wild A. balchaschensis, as well as the efficient use of cultivating materials. 【Methods】 Cellulose CM-cellulose cation exchange column, Q-Sepharose anion exchange column, and Superdex 75 gel chromatography filtration and purification technique were used to analyze the degradative metabolic substrate of the laccase and its inhibition ability against metal ions. The optimum temperature and pH value for the activity laccase were determined. 【Results】 The laccase obtained from the fruiting bodies of A. balchaschensis was a 65 kDa single subunit protein. The total purification multiple was 354.58 and the specific activity was17.02 U/mg. The sequence of the 10 N-terminal amino acids of the enzyme was SGGPEQNTTA. Through NBCI-BLAST, it was found that the laccase was homologous to those of Pleurotus ostreatus and Versicolor Versicolor. The laccase was substrate-specific and varied greatly based on different metabolic substrates. The major metabolic substrates ranked from strong to weak were ABTS > dimethoxyphenol > toluidine. The optimum pH value was 2.2, so the laccase was acid-resistant. The optimum reaction temperature was 40℃, so the laccase was active at medium-low temperatures. Cu²⁺ can slightly promote the laccase to catalyze ABTS, while Hg²⁺ can strongly inhibit its catalysis of ABTS. 【Conclusion】 The results showed that the laccase from the fruiting bodies of A. balchaschensis was a 65 kDa single subunit protein.

Key words: Agaricus balchaschensis; laccase; purif; substrates

中图分类号:

S646

努尔孜亚·亚力买买提, 郝敬喆, 贾文捷, 陈浩宇, 罗影, 贾培松. 巴尔喀什蘑菇子实体漆酶纯化与理化特性及对酚类化合物的降解能力[J]. 新疆农业科学, 2023, 60(2): 432-439.

Nurziya Yarmamat, HAO Jingzhe, JIA Wenjie, CHEN Haoyu, LUO Ying, JIA Peisong. Purification and Physicochemical Properties of Laccase from Fruiting Bodies of Agaricus balchaschensis[J]. Xinjiang Agricultural Sciences, 2023, 60(2): 432-439.

图/表 8

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分离方式 Separation mode	收集组分 Collect components	收集量 Collection volume(mg)	总活性 Total activity(U)	比活力 Specific activity (U/mg)	纯化倍数 Purification factor
DEAE-cellulose	Crude extract	4142.75	198.85	0.048	1
CM-cellulose cation exchange	C₁	19.89	1.01	0.051	1.06
	C₂	125.86	9.82	0.078	1.63
	C₃	363.31	68.30	0.188	3.92
Q-Sepharose anion exchange	Q₁	83.82	31.26	0.373	7.77
Superdex 75	SU1	6.4	108.93	17.02	354.58

分离方式 Separation mode	收集组分 Collect components	收集量 Collection volume(mg)	总活性 Total activity(U)	比活力 Specific activity (U/mg)	纯化倍数 Purification factor
DEAE-cellulose	Crude extract	4142.75	198.85	0.048	1
CM-cellulose cation exchange	C₁	19.89	1.01	0.051	1.06
	C₂	125.86	9.82	0.078	1.63
	C₃	363.31	68.30	0.188	3.92
Q-Sepharose anion exchange	Q₁	83.82	31.26	0.373	7.77
Superdex 75	SU1	6.4	108.93	17.02	354.58

Mushroom laccase	N-terminal sequence
BHBClaccase	SGGPEQNTTA
Agaricus bisporus laccaseI	KTRTFDFDL
Agaricus bisporus laccaseII	DTKTFNFDL
Basidiomycete PM1laccase	SIGPVADLT
Ceriporiopsis subvermispora laccaseI	AIGPVTDLE
Lepista nuda laccase	AIGPAADLHIVNKDISPDGF
Pleurotus ostreatus laccase	SIGPRGTLN
Pleurotus eryngii laccaseI	AXKKLDFHI
Pleurotus eryngii laccaseII	ATKKLDFHI
Phlebia radiata laccase	SIGPVTDFH
Pycnoporus cinnabarius laccase	AIGPVADLT
Russula virescens laccase	AIGPTAELVV
Trametes sp F1635 laccase	AIGPVADLTIINNAV
Trametes versicolor laccaseI	AIGPVASLV
Trametes versicolor laccaseII	GIGPVADLT
Trametes versicolor laccaseIII	GIGPVADLT
Tricholoma matsutake laccase	ADPVGIVGD
Tricholoma giganteum laccase	DDPQQAVIDD
Tricholoma mongolicum laccase	GIGPVADLYVGNRIL

Mushroom laccase	N-terminal sequence
BHBClaccase	SGGPEQNTTA
Agaricus bisporus laccaseI	KTRTFDFDL
Agaricus bisporus laccaseII	DTKTFNFDL
Basidiomycete PM1laccase	SIGPVADLT
Ceriporiopsis subvermispora laccaseI	AIGPVTDLE
Lepista nuda laccase	AIGPAADLHIVNKDISPDGF
Pleurotus ostreatus laccase	SIGPRGTLN
Pleurotus eryngii laccaseI	AXKKLDFHI
Pleurotus eryngii laccaseII	ATKKLDFHI
Phlebia radiata laccase	SIGPVTDFH
Pycnoporus cinnabarius laccase	AIGPVADLT
Russula virescens laccase	AIGPTAELVV
Trametes sp F1635 laccase	AIGPVADLTIINNAV
Trametes versicolor laccaseI	AIGPVASLV
Trametes versicolor laccaseII	GIGPVADLT
Trametes versicolor laccaseIII	GIGPVADLT
Tricholoma matsutake laccase	ADPVGIVGD
Tricholoma giganteum laccase	DDPQQAVIDD
Tricholoma mongolicum laccase	GIGPVADLYVGNRIL

阳离子盐 Cationic salt	相对酶活Relative enzyme activity(%)
阳离子盐 Cationic salt	5 mmol/L	2.5 mmol/L	1.25 mmol/L
Li⁺	62.12± 0.55	97.97 ± 0.41	98.42± 0.11
Na⁺	63.38 ± 0.07	97.75 ± 0.49	98.33 ± 0.1
Mg²⁺	60.63 ± 0.52	98.74 ± 0.49	97.21 ± 0.01
Al³⁺	58.96 ± 0.16	96.51 ± 0.77	97.57 ± 0.25
K⁺	50.52 ± 0.07	89.68 ± 0.67	91.37 ± 0.17
Ca²⁺	58.96 ± 0.78	96.71± 0.79	97.72± 0.13
Cr²⁺	62.22 ± 0.29	98.27 ± 0.08	99.53 ± 0.07
Mn²⁺	47.39 ± 0.42	92.54± 0.01	99.41 ± 0.18
Fe³⁺	63.58 ± 0.15	95.95 ± 0.23	97.82± 0.11
Cu²⁺	105.35 ± 0.15	101.77 ± 2.61	98.88 ± 2.07
Zn²⁺	60.11± 0.27	84.98 ± 0.41	98.72 ± 0.07
Cd²⁺	71.71 ± 0.17	76.42 ± 0.85	96.78 ± 0.01
Ba²⁺	61.28 ± 0.47	93.58 ± 0.01	99.41 ± 1.2
Hg²⁺	6.94 ± 0.05	9.55 ± 0.04	12.21 ± 0.84
Pb²⁺	85.32 ± 0.64	95.7 ± 0.69	94.66 ± 0.25

巴尔喀什蘑菇子实体漆酶纯化与理化特性及对酚类化合物的降解能力

Purification and Physicochemical Properties of Laccase from Fruiting Bodies of Agaricus balchaschensis

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参考文献 20

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Metrics

底物 Substrate	波长 Wavel ength	相对酶活 Relative enzyme activity
咖啡酸 Caffeic acid	420	0.930±0.03
没食子酸 Gallic acid	450	0.60±0.04
甲苯胺 Toluidine	366	15.77±0.01
联苯胺 Benzidine	366	8.10±0.03
邻苯二酚 Catechol	392	1.00±0.06
2,6二甲氧基酚 2,6 dimethoxyhenool	460	1.10±0.05
愈创木酚 Guaiacol	470	0.90±0.03
对苯二酚 Hydroquinone	248	34±0.01
ABTS	405	100±0.25

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