Immobilization of Laccase from Trametes orientalis and Its Application for Decolorization of Multifarious Dyes

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

Abstract

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

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.

Figures/Tables 13

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

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

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

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

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

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

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

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

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

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

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

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

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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