Characterization,gene Cloning And Dye Decolorization Of Laccase From Bacillus Amyloliquefaciens

Synthetic dyes are aromatic compounds with complex structures,which are usually resistant to degradation.Most of the synthetic dyes and their intermediates are toxic,mutagenic or carcinogenic.Therefore,the treatment of dye effluents are difficult due to its characteristics such as high COD,poor biodegrability,deep chromaticity,high alkalinity and changeable water quality.Laccases,which are copper-containing polyphenol oxidases,can catalyze the oxidation of various organic compounds,and can be widely used in pulp and paper industry,textile industry,pollutants degradation,organic synthesis,and so on.Laccases are able to degrade various synthetic dyes and can be efficiently applied in the treatment of dye effluents.In the present work,we isolated two bacterial strains exhibiting laccase activities from the forest soil.The isolated strains were identified,and their laccase activities were studies in different conditions.We also cloned the laccase gene of the isolated strains and analyzed their laccase gene sequences.Finally,the bacterial laccases and their immobilized counterparts were tested for their abilities in the decolorization of synthetic dyes and simulated dye effluents.The main results and conclusions are listed as follows:(1)Two bacterial strains exhibiting laccase activity were isolated from forest soil by enrichment from copper-containing medium.Laccase substrates were also used to detect laccase activity from the enrichment culture.The isolated strains were named as LC02 and LC03,and they can oxidize the common laccase substrates ABTS,syringaldazine(SGZ)and 2,6-dimethoxyphenol(DMP).Strain LC02 and LC03 were identified by morphology observation,physiological and biochemical tests and 16S rDNA sequence analysis.The results indicated that the two strains are rod,G+and sproe producing bacteria,and they demonstrated sequence identities of higher than 99%with several Bacillus amyloliquefaciens strains.Finally,the two isolated strains are identified as B.amyloliquefaciens.(2)The isolated B.amyloliquefaciens strain LC02 and LC03 could catalyze substrates in a wide pH range.The oxidation of SGZ,DMP and ABTS showed optimum pH in acid,neutral and alkaline pH,respectively.The stability of the spore laccase are low in pH 3,0 and rather high in alkaline conditions(pH 9,0 and 10.0).The spore laccase also demonstrated high thermal stability,with optimum temperature of 60 and 70? for laccase from LC02 and LC03,respectively.Furthermore,the spore laccases from the two strains are even retain activity at 100?.The reducer cysteine and dithiothreitol strongly inhibited the spore laccase activity,whereas EDTA only showed obvious effects in higher concentrations.Most tested metal ions showed activation effects to a certain extent on the spore laccase activity,except for Hg2+,Ag+and Mn2+,which demonstrated a remarkable inhibition effect.NaCl could only inhibit the laccase activity with a concentration higher than 0.2 mM.The spore laccase of the isolated strains were also resistant to organic solvents and surfactants with low concentrations.The high stability of spore laccases in alkaline and high temperature conditions as well as their resistance to inhibitors,metal ions and organic solvents suggests they have more advantages in the treatment of industrial wastewater than fungal laccases.(3)The spore laccase genes were cloned by PCR amplification using specific primers designed from the cotA gene of B.amyloliquefaciens,and the sequences have been submitted to GenBank.The Blast results suggested a high similarity of more than 98%of spore laccases gene sequences with the other corresponding Bacillus strains.The amino acid sequences of spore laccases from strain LC02 and LC03 were phylogenetically related with to that of B.amyloliquefaciens and B.subtilis.High homology in laccase protein sequences were also found between the isolated strains and Arabidopsis thaliana,Streptomyces lavendulae and Streptomyces pristinaespiralis,while the phylogenetic distances were relative low with the other bacterial laccases and fungal laccases.The spore laccase contained three conserved copper-binding domains like laccases with different origins.8 histidines appeared in the protein sequence with 4 highly conserved His-X-His regions.The protein sequeces of spore laccase were analyzed using a series of bioinformatical softwares.The results showed that:the spore laccases were similar in amino acid length,the molecular weights were about 58 kDa,the isoelectrical points were 6.19.All spore laccase contained no signal peptide sequnces and 3 hydrophobic regions.The secondary structures of three different spore laccases were rather similar,with a high composition of coils and a low composition of a-helixs.In addition,high similarities were also found in three-dimensional structures of the three kinds of spore laccases.The tertiary structure of all the spore laccases was composed of three domains with a spherical shape.(4)The capacities of spore laccases from B.amyloliquefaciens LC02 and LC03 in dye decolorization were tested using synthetic dyes with different structures(RBBR,reactive black 5,indigo carmine and crystal violet)as well as simulated dye effluents containing these dye mixtures.Both the spore laccases could efficiently decolorize crystal violet in the absence of mediators,and the decolorization percentage after 6 h were about 75%and 65%for laccase from LC02 and LC03,respectively.The other three tested dyes(RBBR,reactive black 5 and indigo carmine)were not decolorized without the addition of mediators.Laccase mediators were able to improve both the rate and extent of decolorization process for RBBR,reactive black 5 and indigo carmine.Acetosyringone(ACE)was found to be the most effective mediator in enhancing the decolorization process for the tested dyes.Almost complete decolorization for indigo carmine was observed in the presence of ACE by spore laccases from strain LC02 and LC03,and more than 65%of RBBR and reactive black 5 were also achieved under the same conditions.The other tested mediators such as 2,2,6,6-Tetramethyl-l-piperidinyloxy(TEMPO)and 1-Hydroxybenzotriazole(HBT),and violuric acid(VA)demonstrated little promotion effect on dye decolorization.The spore laccases from the two isolated strains remained high decolorization ability towards the four tested synthetic dyes in alkaline conditions(pH9.0)with ACE as the mediator.The spore laccase-mediator system was able to decolorize the simulated dye effluents in a broad pH range when ACE was used.The promotion effect of ACE in the pH 3.0 was not obvious,however,the decolorization efficiency remarkably increased in the other pH conditions.The effect of 0.5 mM and 1 mM of ACE was similar in enhancing the decolorization process.The highest decolorization percentage for the simulated dye effluents was found in alkaline conditions.The decolorization extents for the simulated dye effluents in pH 9.0 were 78.34%and 73.21%for laccase from strain LC02 and LC03.The spore laccases were immobilized by calcium alginate gel,and were successfully used in the decolorization of synthetic dyes and simulated dye effluents.The immobilized laccase could be repeatedly used for at least 4 times without obvious decrease in dye decolorization efficiency.More than 97%of indigo carmine was decolorized by the immobilized laccase after 4 times usage in the presence of 0.1 mM of ACE.Meanwhile,the decolorization percentage of the simulated dye effluents was higher for the immobilized laccase than the spore laccase alone.The results suggested the potential applications of spore laccase from B.amyloliquefaciens LC02 and LC03 in the treatment of alkaline dye effluents.