| Laccase（EC 1.10.3.2）is a blue copper-containing polyphenol oxidase,belonging to the family of blue Multicopper Oxidases（MCOs）,which are generally found in nature as monomeric,dimeric or tetrameric glycoproteins.Over the years,research on laccase has been conducted in many fields,including biology,chemistry and environment.In addition,laccase has a wide range of application in environmental protection,textile,printing and dyeing,food,chemical synthesis,etc.,which has aroused widespread attention and great interest.With the deepening of research,it has been found that some laccases are different from the typical blue laccase,do not contain four copper ions,and are called"white laccase"or"yellow laccase",and show different and unique properties from the blue laccase.They are considered to be better biocatalysts than blue laccase,because of their ability to oxidize non-phenolic substrates without any medium,In this study,a novel bacterial white laccase was successfully cloned from microorganisms and heterologously expressed in Escherichia coli by mining genomic data,and the sequence and structure of the enzyme were bioinformatically analyzed and the physicochemical properties of the recombinant enzyme were investigated by using a variety of bioinformatics tools.Subsequently,the limitations of the enzyme in specific applications were solved by enzyme immobilization and incorporation of deep eutectic solvent system and it was used for the degradation of antibiotics.In addition,homology modeling and molecular simulation of the enzyme and the substrates were used to understand the interaction mode between the enzyme and the substrate,and on the basis of which the range of substrates that can be acted upon was broadened by constructing a highly active mutant.This paper mainly focuses on the three aspects of digging enzyme,changing enzyme and using enzyme.The main research contents and results are as follows:1.The non-blue laccase Melac13220 was successfully cloned and expressed from the bacterium Methylobacterium extorquens conserved in our laboratory through NCBI database as well as bioinformatics analysis.The sequence and structure of the enzyme were analyzed and the enzymatic properties of the recombinant enzyme were characterized by using various bioinformatics tools.Upon homologous sequence analysis,it was found that the sequence similarity between this enzyme and other multicopper oxidases was only 20-32%.It was further demonstrated by sequence evolution tree analysis that the enzyme belongs to a unique branch of unreported non-blue laccase.Based on its metal ion content,it can be categorized as a"white laccase".The enzyme was characterized by its enzymatic properties,which were different from those of typical blue laccase.The purified Melac13220 was colorless,and no absorption peaks were found near 610 nm for T1 copper and 330 nm for T3 binuclear copper,but there was an absorption peak at 260 nm.The optimal temperature of the enzyme is 65°C,and it can maintain good stability at 40°C and 50°C,with half-lives of 50 h and 9h,respectively;the optimal p H of Melac13220 is 1.5,which is an acidophilic enzyme with high activity in acidic environments;the activity of the enzyme can be slightly increased by Cu2+and Co2+,and it can be increased more than 5-fold when Fe2+is present,which also plays a role in stabilizing the enzyme to some extent.To a certain extent,it also plays a role in stabilizing the enzyme.Unlike most laccases,Melac 13220can decolorize Congo red and indigo carmine dyes without any redox mediator.2.Melac13220 has certain limitations in application,such as low stability and susceptibility to external factors.Therefore,a peptide sequence,LCTPSR,was introduced into the amino,carboxy and double ends of Melac13220,which was catalyzed by FGE to generate aldehyde group and then covalently bound to the magnetic nanoparticles with amino group through Schiff base reaction,to realize the specific fixed-point covalent binding between the enzyme and the magnetic nanoparticles.Subsequently,the immobilization conditions of Melac13220 were optimized,and the site-specific covalent immobilization method was found to have the highest immobilization efficiency and viability recovery rate when compared with the Melac13220 immobilized enzyme obtained by the immobilization methods of physical embedding and chemical cross-linking.The optimal temperature of Melac13220 was found to be 80°C,which was 15°C higher than that of the free state,and the immobilized Melac13220 showed more significant tolerance to some organic solvents.DSC results showed that the melting temperature of the enzyme was increased from 57°C in the free state to 79°C after the immobilization process.decolorization of the dye,which resulted in the complete decolorization of Congo red within 10 h.The results showed that Melac13220 could be used for the decolorization of Congo red within 10h.3.Antibiotics play an indispensable role in the medical field.However,the ecological imbalance caused by antibiotic residues has become an urgent problem.Among many antibiotics,theβ-lactam antibiotic ampicillin,which has a wider range of applications due to its excellent cost-effectiveness,has high environmental residues and thus causes more prominent environmental problems.In the absence of a redox mediator,free Melac13220 and immobilized Si O2-Fe3O4-Melac13220-CQ exhibited oxidative degradation of theβ-lactam antibiotic ampicillin,with Si O2-Fe3O4-Melac13220-CQ showing a higher degradation efficiency.It was deduced that the degradation of ampicillin by Melac13220 mainly included hydroxylation,decarboxylation,and ring-opening of theβ-lactam ring.4.In an attempt to improve the stability of Melac13220,a natural deep eutectic solvent consisting of a hydrogen bond acceptor（HBA）and a hydrogen bond donor（HBD）was added to the Melac13220 system as a novel green solvent to try to improve the stability of the enzyme.A total of six natural deep eutectic solvents,namely,glycerol-betaine（GBDES）,glycerol-chlorocholine（GCDES）,lactic acid-betaine（LBDES）,lactic acid-chlorocholine（LCDES）,xylitol-betaine（XBDES）,and xylitol-chlorocholine（XCDES）,were used to investigate the effects on enzyme activity and stability.The results showed that different concentrations of GBDES,LBDES,LCDES,XBDES,and XCDES exhibited the ability to enhance the enzyme activity of Melac13220,while GCDES had a slight inhibitory effect on Melac13220.Deep eutectic solvents（DES）induced conformational changes in the enzyme and promoted protein folding,which in turn affected enzyme activity and thermal stability.The higher activation effect of lactate-and betaine-based DESs could be attributed to the formation of self-hydrogen bonds in addition to hydrogen bonds in the presence of DES,which could significantly increase the enzyme activity of Melac13220.5.In order to further improve the catalytic activity and broaden the substrate applicability of Melac13220,mutants V212L,R248S,and K394M were constructed by computer-assisted homology modeling and docking analysis of the enzyme and substrate molecules.The enzyme activity screening revealed that all three mutants showed different degrees of increase in enzyme activity compared with the wild type,and the substrate profiles of the mutants were also broader.Mutation of the basic amino acid at the enzyme-substrate binding pocket to a non-polar methionine resulted in a significant change in the catalytic ability of the enzyme,indicating that the amino acid structure at the binding pocket plays an important role in the catalytic efficiency and substrate specificity of the enzyme. |
