New Strategies For High-efficient And Oriented Immobilization Of Fungal Laccase

Laccase is a Cu-containing polyphenol oxidase, which catalyzes the oxidation of various aromatic amines and phenols with concomitant reduction of oxygen to water. The catalytic characteristics of laccase make it very useful not only in lignin degradation, pulp bleaching, dye decolorization/detoxication, and environmental protection but also in biosensor and biofuel cell development.Similar to other enzymes, the immobilization of laccase is an important strategy for its real application. Traditionally, an enzyme is immobilized via the interaction between a support and a functional group of amino acid residue on the enzyme surface. In this way, it is possible that multiple sites of enzyme are attached on the support, resulting in a disturbed enzyme structure, decreased enzyme activity and inappropriate orientation. It may also hinder the interaction between the active site of an enzyme and its substrate, resulting in decreased activity. Therefore, the oriented and high efficient immobilization becomes the focus of recent research.So far, some oriented immobilization strategies have been developed. Some are based on the affinity interaction between an enzyme and a support. Some are based on the covalent interaction between amino acid residue and coupling group on the support. Currently, some development on the oriented immobilization of enzymes of single active site has been achieved.It is known that laccase is a single-chain macromolecule with multiple redox active sites. The catalytie efficiency of the immobilized laccase therefore depends largely on the strategies used for the immobilization, especially when laccase is immobilized on an electrode for the direct electron transfer and bioelectrocatalysis. It follows that high efficient and oriented immobilization of laccase is significant for the development of laccase-based functional devices. However, few works have been carried out on the oriented immobilization of laccase. This thesis presents several new strategies for high-efficient and oriented immobilization of laccase from the perspective of the bio-modification of enzyme itself and/or chemical modification of the carrier.1. Bio-modification of laccaseRelatively speaking, the biological modification of functional proteins is more suitable than its chemical modification for the orientation assembly of proteins. So far, great progress has been made in the cloning and sequence analysis of the gene of laccase and in the high efficient heterologous expression of laccase. In present study, the molecular biology-based bio-modification of laccase is used to realize the oriented immobilization of laccase.The self-assembly of the modified laccase on Au electrode is based on the Au-S band formed between side chain thiol of cysteine and gold substrate. Therefore, to facilitate the immobilization of laccase, the N-terminal or C-terminal is modified with cysteine. The coordination between the imidazole group of histidine and Ni2+ions has also frequently been used for purification and immobilization of protein.In the present study, a DNA sequence of Cys-(His×6)-was first designed and then fused to the3’/5’DNA terminus of laccase from Trameters sp. AH28-2. After that an expression vector was built and electroporated into Pichia pastoris for expression. Finally, the N-terminus and C-terminus of laccase was successfully modified with Cys-(His×6)-oligopeptide.The enzymatic property of the recombinant laccase was analyzed. The results show that the two recombinant laccases exhibit similar enzymatic properties, indicating that the biological modification has no big effect on enzyme properties and the modified laccase can be used for further immobilization.2. Oriented immobilization of recombinant laccase on gold electrodeThe direct electron transfer of laccase on common electrodes is hardly observed. It was also found that for non-oriented immobilized laccase electrode, the electrocatalytic reduction of oxygen is accompanied by the production of the intermediate H2O2, which decreases the electron transfer efficiency. Thus, the oriented immobilization of laccase on electrode is a key step for enhancing the electron transfer efficiency, and constructing high-performance biosensors and biofuel cells.Based on the Au-S band between the cysteine residue of the recombinant laccase and gold electrode, the recombinant laccase is assembled orientationally on the gold electrode.Enzyme activity test shows that the activity of the immobilized laccase is well-retained. Cyclic voltammetry test shows that the direct electron transfer and the bioelectroreduction of oxygen are achieved on both the electrodes modified the recombinant laccases. However, the two electrodes show different direct electrochemical behaviors. Analysis shows that laccase with different orientation on gold electrode have different electron transfer pathways and different direct electron transfer efficiency. Bioelectrocatalytic reduction of oxygen is easier for laccase modified electrode where the T1site is more close to the electrode surface. The above research is helpful to the in-depth understanding of the direct electrochemistry of laccase and also to the development of laccase-based biofuel cells.3. Oriented immobilization of recombinant laccase on agaroseThe recombinant laccase with Cysteine-6×Histidine tag at N-terminus and C-terminus is immobilized on the surface of NTA-Ni2+-modified agarose via the chelation between the histidine residue and Ni2+ion. In this system, the immobilization site is close to laccase surface but distant away from laccase active site, which is beneficial for the structure retention and high catalytic activity. The multiple immobilization sites also enhance the stability. More importantly, the bio-modified amino acid chain realizes the oriented immobilization with adjustable orientation.The enzyme activity test shows that the laccases with different orientations show different activities under the same testing conditions. Circular dichroism spectrum shows that the secondary structure changes little for the two recombinants prior to or after the immobilization. Analysis shows that it is the orientation that makes the exposure of T1site of laccase to the outer solution different, which results in different accessibility of the same substrate to the T1site and therefore different activity. It is also found after immobilization, thermal stability enhances and reusableility is high.4. Efficient immobilization of non-recombinant laccase on single wall carbon nanotubeLaccase is one of the glucoproteinases. The glycosyl on the surface can be used as sites for immobilization. In the present study, the single wall carbon nanotube is first modified with surfactant (alkyl polyglucoside) to disperse the nano tubes and increase the specific surface area. Based on the affinity between concanavalin A and glycosyl, laccase is then immobilized on carbon nanotubes, forming a new conjugate of single wall carbon nanotube-(n-dodecyl β-D-maltoside)-concanavalin A-glucoproteinases with little structure change and high stability.The experimental results show that the new immobilization strategy has some advantages over the direct immobilization in terms of enzyme loading, specific activity and stability. Circular dichroism and fluorescence spectra show that single wall carbon nanotube-(n-dodecyl β-D-maltoside)-concanavalin A hybrid support has a smaller effect on the enzyme structure compared with bare carbon nanotube, demonstrating that the present self-assembly strategy is effective for laccase immobilization.