| Laccase is a group of copper-containing polyphenol oxidases which can oxidize lignin and some compounds whose structure similar to lignin.The complexity of the lignin structure endows laccase with diversity of substrate,and make it can oxidize various phenolic and non-phenolic compounds.Currently,laccase has been used in various areas,such as pulp and paper industry,textile and dye industry,food industry,bio-synthesis and bioremediation etc.The major producer of laccase is white rot fungi,there universally exist several isoforms with different kinetic or physicochemical features even in the same fungus strain,and the expression of those isoforms exist spatial and temporal specificity.In the previous study,our laboratory got a thermotolerant fungus called Trametes trogii S0301.It has not only high thermal stability but also high tolerance to alkali,acid,organic solvents and heavy metal ions,owning some application prospects.At present there are only two laccase genes(lcc1 and lcc2)cloned in T.trogii.Besides the purified laccase of T.trogii has the problem of low thermal stability andionic tolerance.Based on the above,this study was carried out as following:1.The cloning and expression analysis of the 6 laccase genes in thermotolerant T.trogii S0301.Designed the degenerate primers(AsP1 and AsP2)based on the amino acid sequence of the laccase copper-binding conserved region(CuI and CuIV).Degenerate-PCR was performed using the genomic DNA of T.trogii S0301 as template.Six different partial laccase genes lac1,lac7,lac13,lac 16,lac50 and lac53 of T.trogii S0301 was obtained.lac7 shared 100%identity with T.trogii 201 lcc2,showing that the two laccase enzyme are the same one.As the laccase gene structure prediction showed that the lac1,lac50 and are lac53 had the similar gene structure;lac7 and lac16 also shared the similar gene structure;however,there existed large discrepancy between the intron and exon structure of lac13 and other laccase gene from T.trogii S0301.Quantitative analysis found that laccase genes from thermotolerant fungus T.trogii S0301 showed discrepant induction by external conditions.The maximum relative expression level of lac1,lac7,lac13,lac16,lac50 and lac53 were 1155.8,506.1,9661.6,349.8,809.3 and 3071.7,respectively.The expression of six laccase genes was induced by temperature.2mM Cu2+ can induce the expression of lac1 and lac50,instead inhibit lac13 expression.The induction effect of 2 mM Cu2 on lac7,lac16 and lac53 was not obvious.However,the lac1 and lac50 expression was inhibited under both temperature and 2 mM Cu2+.It may be due to the antagonism caused by the presence of both temperature and 2 mM Cu2+ at the same time.The expression of the six laccase genes varied under the regulation of the external conditions which implied that these six laccase genes might differ in biological function.2.Cloning of lac1、lac13 and lac50 full-length gene and gene sequence analysislac1,lac13 and lac50 full-length gene were cloned by TAIL-PCR,and obtained the genes length with 2142 bp,2081 bp and 2246 bp,respectively.lac1 full-length gene contained 10 introns whose length was 53-131 bp;lac13 full-length gene contained nine introns whose length was 54-73 bp;lac50 full-length gene contained 12 introns whose length was 48-67 bp.The His,Cys and predicted N-glycosylation sites contained in the predicted amino acid sequences of the three laccases existed difference.Besides,there were cis-acting elements with different species,number and location in the 5’ upstream regions of lacl,lac 13 and lac50.This implies that the three laccase genes might be differently regulation by the external condition.The laccase gene expression analysis on the T.trogii S0301 lac1,lac13 and lac50 also showed that these three genes expressed quite differently under different external conditions.3.Research of purification,enzyme analysis and deys decolorization on thermotolerant T.trogii S0301.The crude laccase of T.trogii S0301,gained from the optimized enzyme production medium,had high laccase activity,high thermal stability,and high tolerance to alkali,acid,organic solvents and heavy metal ions.Laccase crude enzyme had complex composition,which would affect the accuracy of the enzyme characteristics determination.So laccase isozymes of T,trogii S0301 were purified,however,only one laccase protein of T.trogii S0301 was isolated.The purified laccase activity was 352.1 U/mg.The laccase molecular weight was about 56 kDa,and the optimum temperature and pH were 45℃ and 3.0 respectively.The purified laccase had high thermal stability with T1/2 at 60℃ and 75℃ for 3 h and 10 min,respectively.What’s more,it was highly resistant to 100 mM Cu2+,Zn2+,Mn2+,Co2+ and other metal ions.Compared with other purlfied fungal laccase,the purified laccase was effective to decolorize bromophenol blue,acid red,malachite green and crystal violet.After reflecting for 4 hours,the decolorization rate of the crystal violet(25 mg/L),acid red(25 mg/L),malachite green(10 mg/L)and bromophenol blue(50 mg/L)were detected to be 68.9%,77.3%,79.8%and 96.4%in the absence of the oxidizing media,respectively.The laccase could effectively decolorize high concentration of bromophenol blue(125 mg/L)too.The MALDI-TOF MS and the homology analysis of amino acid sequence was shown that the seven peptides of purified laccase shared 100%identity with the predicted amino acid sequence of lacl,and covered 94.9%,74.8%,73.4%and 65.5%of lcc1,lcc2,lac13 and lac50,respectively.So we inferred that the purified laccase maybe was lac1.4.Heterologous expression of lac13 gene from thermotolerant T.trogii S0301 in yeast.As the temperature increased,the lac13 expression level increased and was the highest of all the gene expression levels.No laccase protein lac13 was isolated during T.trogii S0301 laccase isozymes purification.So in order to gain a large number of the lac13 and verify its fuction,heterologous expression of lac13 in yeast was tried.After the G418 resistance screening,a strain of recombinant yeast resistant to G418 at a concentration of 3 mg/mL of was gained.The molecular identification of the recombinant yeast showed that it had lac13 cDNA approximately 1.5 kb.Laccase activity detection of the recombinant yeast extracellular fluid showed that there was laccase activity in the recombinant yeast extracellular fluid,however with low laccase production.This study was the research on the thermotolerant fungal T.trogii S0301 laccase isozymes expression,enzymatic properties and its function,and it might has an impact on promoting the research on the genetic diversity of fungi expression and the expression regulation mechanism of fungal laccase.Also provides a theoretical basis for the industrial applications of laccase.The purified laccase in this study not only had high thermal stability and highly resistant to metal ions,but also was effective to decolorize bromophenol blue,acid red,malachite green and crystal violet,which showed that the laccase in industrial applications had a certain prospects. |
