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

doi:10.13560/j.cnki.biotech.bull.1985.2021-1173

摘要/Abstract

摘要：

漆酶是一种天然的绿色催化剂,由于具有催化效率高、底物特异性广、对辅因子和特定环境条件要求少、无毒等优点,因而在制浆造纸、生物合成、改善纤维性能、食品加工、生物传感器制造、农林废弃物的生物转化和炼制,特别是环境污染物的生物降解和生物修复等领域具有巨大的应用潜力。但游离漆酶稳定性差且成本较高,固定化方法成为解决该问题的有效手段,其能够有效增强酶的热稳定性及对极端环境的耐受力、使酶易与产物分离以提高酶回收率。本研究基于前期筛选获得的一株高产漆酶白腐真菌菌株东方栓孔菌（Trametes orientalis）所分泌的漆酶（Tolacc-T）,对其纯化酶蛋白以壳聚糖作为载体、戊二醛作为交联剂进行了固定化处理,命名为Tolacc-T@Chit@GA,并优化了固定化条件为戊二醛浓度0.7%（V/V）、交联时间4 h、给酶量6.0 mL、固定化时间6 h。与Tolacc-T相比,Tolacc-T@Chit@GA的pH适应性、耐热变性能力及贮存稳定性均显著增强。循环使用时的稳定性和耐久性也十分明显,循环使用7次后,Tolacc-T@Chit@GA的相对活性仍可保持在80%以上。此外,Tolacc-T@Chit@GA还可使不同类型的染料脱色,尤其对金属络合染料萘酚绿B的脱色效果最佳,气相色谱-质谱联用（gas chromatography-mass spectroscopy,GC-MS）检测确定该染料部分代谢产物为1-萘胺、2-萘酚、1-氨基-2-萘酚、1-亚硝基-2-萘酚、1-亚硝基-2-萘酚-6-磺酸。上述结果证明,东方栓孔菌固定化漆酶Tolacc-T@Chit@GA具有较好的稳定性和可重复利用性,存在广阔的应用前景。

关键词: 漆酶, 白腐真菌, 壳聚糖, 固定化, 生物降解

Abstract:

Laccase as a natural green catalyst has high catalytic efficiency,broad substrate specificity,fewer requirements for cofactors and specific environmental conditions,nontoxicity,etc.,and thus plays a powerful role in a vast of industrial areas:biopulping and papermaking,biosynthesis,enhancement of fiber properties,food processing,biosensor manufacturing,and bioconversion and refinement of agricultural and forestry wastes,especially biodegradation and bioremediation of environmental contaminants. However,the use of free laccase for practical applications is still limited due to its low stability and high production costs. Immobilization can overcome some of the aforementioned limitations by improving the thermostability of the enzyme and its resistance to extreme conditions and chemical agents. Additionally,the immobilized laccase may be easily separated from its reaction products,allowing the enzymes to be employed in continuous bioreactor operations. In this study,a laccase Tolacc-T secreted by a white rot fungal strain Trametes orientalis with high laccase-producing capacity screened previously,was purified and immobilized onto chitosan carrier with crosslinker glutaraldehyde,named Tolacc-T@Chit@GA. The optimal conditions for acquiring Tolacc-T@Chit@GA were 0.7%（V/V）glutaraldehyde concentration,4 h crosslinking time,6.0 mL enzyme amount,and 6 h immobilized time. The pH adaptability,resistance to thermal denaturation,and storage stability of Tolacc-T@Chit@GA were apparently enhanced compared with Tolacc-T. The operational stability and durability during multiple reuses also demonstrated superiority;the relative activity of Tolacc-T@Chit@GA still remained over 80% after 7 cycles of continuous use. Additionally,Tolacc-T@Chit@GA was capable of decolorizing multifarious dyes,in particularly metal-complex dye naphthol green B,whose partially metabolic by-products were identified as 1-naphthylamine,2-naphthol,1-amino-2-naphthol,1-nitroso-2-naphthol,and 1-nitroso-2-naphthol-6-sulfonate through GC-MS detection. These findings suggest that the immobilized laccase Tolacc-T@Chit@GA from T. orientalis presents better stability and reusability and therefore could be used in many applications.

Key words: laccase, white rot fungi, chitosan, immobilization, biodegradation

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.

WANG Hao, TANG Lu-xin, MA Hong-fei, QIAN Kun, SI Jing, CUI Bao-kai. Immobilization of Laccase from Trametes orientalis and Its Application for Decolorization of Multifarious Dyes[J]. Biotechnology Bulletin, 2021, 37(11): 142-157.

图/表 13

表1 供试染料

Table 1 Dyes used in this study

染料 Dye	颜色索引编码 Color index No.	颜色索引名称 Color index name	化学分类 Chemical class	波长 Wave length /nm
活性亮蓝X-BR Reactive brilliant blue X-BR	61205	活性蓝4 Reactive blue 4	蒽醌类 Anthraquinone	603
雷玛唑亮蓝R Remazol brilliant blue R	61200	活性蓝19 Reactive blue 19	蒽醌类 Anthraquinone	592
酸性黑172 Acid black 172	15711	酸性黑172 Acid black 172	偶氮类（含金属） Azo（Metal-containing）	597
刚果红 Congo red	22120	直接红28 Direct red 28	偶氮类 Azo	497
亚甲基蓝 Methylene blue	52015	碱性蓝9 Basic blue 9	菁类 Cyanine	664
中性红 Neutral red	50040	碱性红5 Basic red 5	杂环类 Heterocycle	553
靛蓝 Indigo blue	73000	还原蓝1 Vat blue 1	靛蓝类 Indigo	610
萘酚绿B Naphthol green B	10020	酸性绿1 Acid green 1	亚硝基类（含金属） Nitroso（Metal-containing）	714
结晶紫 Crystal violet	42555	碱性紫3 Basic violet 3	三苯甲烷类 Triphenylmethane	595

图1 戊二醛交联剂浓度对东方栓孔菌固定化漆酶Tolacc-T@Chit@GA活性的影响不同小写字母代表差异显著（P<0.05）,下同

Fig. 1 Effects of concentrations of glutaraldehyde crosslinker on the activity of the immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis Different lowercase letters refer to significant difference（P<0.05）,the same below

图2 交联时间对东方栓孔菌固定化漆酶Tolacc-T@Chit@GA活性的影响

Fig. 2 Effects of crosslinking time on the activity of the immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

图3 给酶量对东方栓孔菌固定化漆酶Tolacc-T@Chit@GA活性的影响

Fig. 3 Effects of enzyme amounts on the activity of the immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

图4 固定化时间对东方栓孔菌固定化漆酶Tolacc-T@Chit@GA活性的影响

Fig. 4 Effects of immobilized time on the activity of the immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

图5 pH对东方栓孔菌游离漆酶Tolacc-T和固定化漆酶Tolacc-T@Chit@GA活性的影响

Fig. 5 Effects of pH on the activity of the free laccase Tolacc-T and immobilized laccase Tolacc-T@Chit@GA from the white rot fungus Trametes orientalis

图6 pH对东方栓孔菌游离漆酶Tolacc-T和固定化漆酶Tolacc-T@Chit@GA稳定性的影响

Fig. 6 Effects of pH on the stability of the free laccase Tolacc-T and immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

图7 温度对东方栓孔菌游离漆酶Tolacc-T和固定化漆酶Tolacc-T@Chit@GA活性的影响

Fig. 7 Effects of temperature on the activity of the free laccase Tolacc-T and immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

图8 温度对东方栓孔菌游离漆酶Tolacc-T和固定化漆酶Tolacc-T@Chit@GA稳定性的影响

Fig. 8 Effects of temperature on the stability of the free laccase Tolacc-T and immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

图9 东方栓孔菌游离漆酶Tolacc-T和固定化漆酶Tolacc-T@Chit@GA的贮存稳定性

Fig. 9 Storage stability of the free laccase Tolacc-T and immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

图10 东方栓孔菌固定化漆酶Tolacc-T@Chit@GA的重复利用性

Fig. 10 Repeatability of the immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

图11 东方栓孔菌游离漆酶Tolacc-T和固定化漆酶Tolacc-T@Chit@GA对不同类型染料的脱色作用

Fig. 11 Decolorization of multifarious dyes by the free lacc-ase Tolacc-T and immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

表2 萘酚绿B经东方栓孔菌固定化漆酶Tolacc-T@Chit@GA降解后的部分代谢产物

Table 2 Partially metabolic by-products of naphthol green B after degradation by the immobilized laccase Tolacc-T@Chit@GA from the white rot fungus T. orientalis

编号 No.	主要片段m/z Main fragment	化合物 Compound	CAS编码 CAS No.
1	143	1-萘胺 1-naphthylamine	134-32-7
2	144	2-萘酚 2-naphthol	135-19-3
3	159	1-氨基-2-萘酚 1-amino-2-naphthol	2834-92-6
4	173	1-亚硝基-2-萘酚 1-nitroso-2-naphthol	131-91-9
5	253	1-亚硝基-2-萘酚-6-磺酸 1-nitroso-2-naphthol-6-sulfonate	—

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