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

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

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。

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 byproducts 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.