Bacillus Licheniformis YlmD And CotA: Heterologous Expression, Physochemical Characterization And Catalytic Application In Transformation Of Lignin Model Compounds

Laccase?EC 1.10.3.2?is a copper-containing oxidase enzymes occuring widely in higher plants,fungi,insects and bacteria with the dual functions of synthesis and degradation.Generally,commercial laccases extracted from fungi and plants cannot adapt to the high temperature and alkaline operation conditions in pulp and paper industry.However,laccases from bacteria especially for Bacillus spore coat protein?CotA?have excellent properties such as heat resistance,alkali resistance and solvent resistance.The study was aimed to identify exogenous expression and high temperature enzyme activity of proteins YlmD and CotA from Bacillus licheniformis as well as explore mechanisms of laccases reaction with ionic liquids and lignin models.Firstly,codons of YlmD gene were optimized,and then genes were synthesized and amplified.Subsequently,recombinant pET-28a?+?-YlmD plasmid was constructed and transformed to Escherichia coli BL21?DE3?in order to produce the YlmD protein which was then used for purification and identification.Same as wild B.licheniformis,the recombinant E.coli-YlmD produced a transparent ring on the medium with 2,6-DMP,showing the resistance to phenol and quinones probably due to the production of oxidoreductase.Besides,it was verified in vitro test that YlmD reduced quinone structure in 2,6-DMP.It may be on account of?1?the reduction of oxidized substrates or?2?the further oxidation and decolorization of the oxidized substrates.Secondly,recombinant pPIC9K-CotA plasmid with histidine tag was constructed.BliCotA protein was expressed in P.pastoris,and the crude enzyme was separated by chromatography,Cu ions absorption,and ultra-filtration desalination to obtain pure CotA?purity>82%?.Thirdly,in order to know catalytic properties of BliCotA in high temperature,we established a method to determine laccase activities and kinetics in high temperature.The operating temperature was 5⁸3°C and the detection resolution can reach 0.0001 Abs.The spectrometer has the function of continuous and rapid scanning of full wavelength spectrum.This system was used to compare the high-temperature enzymatic activity of BliCotA laccase,N 51003 laccase and Pleurotus laccases.BliCotA laccase was thermophilic whose optimum temperature was 80°C at pH 4 and maximum enzyme activity was 32 U/mg.The enzyme was lack of alkali resistance and enzyme activity loss was over 95%at pH 9.N 51003 laccase was acidic thermostable whose enzyme activity was 8.9 U/mg at 60oC,pH 4.5.It can work in80°C without alkali resistance.BliCotA/HOBT showed good delignification effect for kraft pulp at 50oC,but when the temperature was up to 80⁹0oC,the brightness of bleached pulp was significantly decreased.The molecular simulation analysis showed that Cu²⁺binding sites?for both Bli CotA and Mth Nov laccases?were linked with 4 highly conserved amino acid fragments.The alignment of BliCotA and B.subtilis CotA laccases was 65.4%with high homology and the sequence consistency of MthNov and Melanocarpus albomyces laccases was over 74.4%indicating the close relationship.Bli CotA laccase model was constructed based on BsuCotA laccase and QMEAN4 and GMQE gave scores as-0.5 and 0.81,respectively.Similarly,MthNov laccase model was constructed based on Mal laccase and QMEAN4 and GMQE gave scores as 0.27and 0.92,respectively.Ramachandran Plot analysis demonstrated that all of amino acid conformations are found in the reasonable region.Six lignin monomers were docked with BliCotA and Mth Nov into a a hydrophobic pocket near T₁ Cu.The hydrophobic region of BliCotA was a grooved structure surrounded by five rings and MthNov T₁ Cu region was a"gyrator"shape.As the degree of polymerization increases,the binding energy of the lignin model compound in the region became lower and lower and showed a linear relationship,indicating the region size limited the molecular weight of the laccase substrate.Alkyl imidazolium ionic liquids inhibited the laccase activity by competitive inhibition and showed increased inhibition with longer alkyl chain length.The red shift of ultraviolet spectrum verified that[Bmim]Cl and MthNov laccase were made to be a laccase/[Bmim]Cl complex.