| Laccase is a copper polyphenol oxidase and also is one of the enzymes in lignin-cellulose degration.In the process of edible fungus cultivation,laccase can promote the keratinization of mycelium and form more fruiting body primordia,as a result the edible fungus production could be improved.Based on the data of Pleurotus ostreatus,the coding genes of fungal lignin-degradtion enzyme would be counted and annotated at the genomic level.But,there are still some problems to be solved,such as the inaccurate gene annotation and the unclear mechanism of transcriptional and regulation of laccase gene.This study analyzed the eleven laccase genes and their dedued amino acid sequences by using bioinformatics methods,to provide theoretical guidance for the experiment and the study of the transcriptional regulation and functional genomics in laccase gene.Simultaneously,the study of laccase which was isolated and purified from the heterosis-2 Pleurotus ostreatus would be provided a reference for edible fungus production,industrialapplication,further study of the Laccase and fully understand the physicochemical properties of heterosis-2 Pleurotus ostreatus.Pleurotus ostreatus gene may be regulated by N source and stress from external stress,but may not be affected by the concentration of glucose in the culture medium,the other laccase genes transcription(except LACC12)may be activated by co-respond to high concentrations of copper ions,the stability of the laccase of Heterosis-2 Pleurotus ostreatus was better under neutral and alkaline conditions,but the thermal stability was lower.The disulfide bond plays an important role in maintaining the spatial structure of the laccase stability.The results were as follows:1.The analysis of the promoter showed that LACC1-LACC12(except LACC5)have various cis-acting elements,such as heat stress responsiveness(HSE),stress-responsive element(STRE),metal-resposive element(MRE),xenobiotic-responsive element(XRE),antioxidant responsive element(ARE),CreA-binding site(CreA),nitrogen factor binding site(NIT),and core promoter elements CAAT-box,TATA-box.We took each 2000 bp upstream and downstream of the laccase gene coding region,and the result pointed that the number of restriction sites in LACC1-LACC12(except LACC5)ranges from twenty-one to fifty-six.2.The analysis of laccase gene system evolution and similarity of LACC1-LACC12(except LACC5)displayed that LACC1 similar to Pleurotus sajor-caju LACC2 was 99.59%,LACC3 similar to Pleurotus sajor-caju LACC5 was 87.90%,similar to Pleurotus pulmonarius LACC7 was 87.04%,LACC7 similar to Pleurotus eryngii LACC1 was 95.96%,LACC10 similar to Pleurotus sajor-caju LACC4 was 97.93%.The eleven laccase genes clustered in three branchs.3.The similarity of eleven dedued amino acid sequences displayed out that they clustered into four branchs,in evolutionary relationships,LACC6 and LACC8,LACC9 and LACC10 were on an evolutionary branch and they had the closest genetic relationship.The multiple sequence alignment and BLAST and RPS_BLAST results pointed out that the dedued amino acid sequences of LACC1-LACC12(except LACC5)were contained the laccase characteristic sequences L1-L4 and three conserved Cu-oxidase domains.SignalP4.1 and Protcomp9.0 results showed that each dedued amino acid sequence had signal peptide and they were exocrine proteins.4.The physicochemical properties of eleven dedued amino acid sequences by Protparam showed that AI ranged from 83.64 to 92.81,there had no difference between ExtCof1 and ExtCof2,in addition to LACC1 and LACC10,the GRAVY were negative value.II,MW,pI were ranged from 32.81 to 42.04,55.68 to 58.87 and 4.53 to 7.78.Except for LACC8,others had the higher TNNCR than TNPCR.5.The analysis of physicochemical properties and composition of the eleven dedued amino acid sequences dislayed that physicochemical properties clustered into three Clusters and there had significant difference between Cluster?and Cluster?,Cluster ? and Cluster ?(P < 0.05),Cluster ? and Cluster ? had no significant difference(P > 0.05);there were no significant dissimilarity differences of deduced amino acid physicochemical properties in each cluster among three groups(P > 0.05).The deduecd amino acid sequences composition of each laccase also clustered into three groups,of which Cluster?significantly distinguish from Cluster?and Cluster?(P < 0.05).Howerer,no significant statistical difference was detected between Cluster?and Cluster?(P >0.05).As same as the deduced amino acid physical properties,the deduced amino acid sequencescomposition dissimilarity of each laccase in each cluster also do not significantly differ between groups(P >0.05).6.Electrophoresis-purity laccase from heterosis-2 Pleurotus Ostreatus was obtained through fermentation broth,ammonium sulfate fractionation,DEAE-Sepharose fast flow chromatography and Superdex-200 prep grade chromatography.The result pointed out that it has the highest activity on the sixth day,6.96% of the Laccase activity was obtained,the enzymatic specific activity reached at 115 U/mg and the purification fold of the Laccase was 111.65.The relative molecular weight of the laccase was approximately 244.0 kD,in which the subunit molecular mass was roughly 85.6 kD.The enzymatic properties showed that the optimum pH and temperature for the laccase were 5.0 and 55 ?,respectively.The enzyme was stabled at p H 6.0~8.0 and 40-55 ?,and its apparent Km and Vmax were 2.1 mmol/L and 0.117 ?mol/min·L.Fe 2+ and ascorbic acid have a full inhibition for heterosis-2 Pleurotus Ostreatus laccase,whereas slightly affected of EDTA,Ag+,Mg 2+,Cu2+ and Li+,the Cu2+ has little activation for it.The enzyme activity of laccase could be activated by urea,ethanol,isopropanol,inhibited by oxalic acid,methanol,n-butanol,K+,Ca 2+,Ba 2+,Zn 2+,Cd 2+,Pb 2+,Mn 2+,Co 2+.7.Modified by chemical modification agent on the fuction of laccase from heterosis-2 Pleurotus Ostreatus results showed that two disulfide bonds and tryptophan residues constitute the essential groups of this enzyme activity center;serine residues,lysine residues and arginine residues are not directly related with laccase from heterosis-2 Pleurotus Ostreatus,it is presumed that they are not necessary group for the laccase activity center. |
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