High-Level Expression Of A Bacterial Laccase, CueO From Escherichia Coli K12 In Pichia Pastoris GS115 And Its Application On The Decolorization Of Synthetic Dyes

Laccases?EC 1.10.3.2?are multicopper-containing oxidases,capable of degrading a large variety of phenolic compounds,during one-electron transfer among copper atoms within active center,molecular oxygen is concomitantly reduced to water,it was discovered in fungi?especially white-rot fungi?,plant,insects and bacteria.Laccases exhibit broad substrates?about 250 kinds?,which mainly are phenolic and aromatic compounds.They have broad application prospects in numerous fields such as textile industry,paper pulp industry,environment protection.Recently,it is found that the production of laccases is a long-term and low level,it is too hard to meet the demand of industrial application.So it becomes an appropriate target model for large scale application in industrial production.The DNA sequence coding CueO?Gene Bank Accession No.P36649?was obtained from National Center of Biotechnology Information,coding sequence of yacK gene from Escherichia coli K12 was 1470 bp,encoding 490 amino acid residues,then cloned into the expression vector pHBM905BDM,which harbord d1+2×201 AOX1 promoter and MF4I leader sequence.The recombinant plasmid was transformed into P.pastoris GS115 competent cells by electroporation,and transformants were screened on MD plates.Positive colonies were further screened by the appearance of a yellow halo when incubated on BMMY plate containing 2 m M of 2,6-DMP and 0.5 m M of CuSO₄.Preparing CueO within a 5 L fermentor can reach high level,the yield of target protein reached 556 mg/L and the enzyme activity was about 41,000U/L.Through Ni²⁺-NTA affinity chromatographic purification,target protein was obtained.Its optimumpHandtemperaturewas3.0and55°Cwith2,2'-azino-bis-?3-ethylbenzothazoline-6-sulfonic acid??ABTS?as the substrate,respectively.This recombinant protein was thermostable and its half life at 70°C was about 60 min.The recombinant CueO was applied in degrading three kinds of dyes with different structures?azo,triphenyl methane and anthraquinone?.In the presence of natural redox mediator acetosyringone,the purified recombinant laccase decolorized about 98.1%,98.5%of Congo red,malachite green,respectively and it also decolorized about 90.03%of Remazol brilliant blue R without this mediator.In addition,this enzyme was applied on the decolorization of waste-water from a textile printing factory and showed an obvious bleaching effect.