| Laccases (( P -diphenol oxidase EC 1.10.3.2)are blue multi coppper-containing phenol oxidases widely distributed in plants and fungi. Laccases have been assigned several different biological roles. In higher plants, laccases are involved in lignification of xylem tissues. In fungi, laccases are linked to pigment biosynthesis , fruitbody morphogenesis , lignin degradations condial pathogenicity and detoxification. The thesis study the screening for high yield laccase producing strains from Auricularia, purification and properties of laccase and cloning of laccase gene.These studies may supply the theory bases for the further study on the biological roles played by laccases during the growth of Auricularia.Using different methods to detect superoxidase and laccase activity produced by 27 strains belonging to three isolate in Auricularia, we got a high yield producing laccase strain-AP4. Then we studied the fermentation conditions produing laccase from AP4. The results showed that: the composition of optimal fermentation medium was CMC5g/L, NH4NO3+L-Asparagine24mmol/L; pH of medium was 4.0, and incubation temperature was 25'C. The experiment supplied the better strain and optimal fermentation conditions producing laccase for the future study on laccase characteristics and molecular biology .The result of PAGE for laccase crude extract showed that three kinds of isoenzymes exist in the Auricularia polytricha AP4. The molecular weight of the three isoenzymes was determined by Native SDS-PAGE : LacA(110KD), LacB (84KD) ;LacC(65KD). The laccase LacB was purified from AP4 by ammonium sulphate fractionation , DEAE_ sepharose fast flow chromatography. The optimum pH and temperature for LacB activity were 4.0 and 30 C, respectively. The LacB catalyzed the oxidation of ABTS with a Michaelis constant of 6. 64 10-5mol/L. Various metal ions showed different effects on the LacB activity. The activity was enhanced by Ga2+, Mg2+, Zn2+, Na2+, Ag2+, and was strongly inhibited by Co2+, Hg2+,Fe3+ Fe2*, Ba2\ LacB also was able to decolorize Remazol brilliant blue R efficiently.Based on the conserved Cu-bind domains in the Fungal laccases, the degenerated primers were designed to amplify the genomic DNA. Sequeencing of the amplification product revealed a fragment of 204bp encoding an amino acid sequence corresponding to laccase.On the basis of the specific 204bp fragment of laccase gene ofA.Pol'ytricha two specific primes were designed to get the complete cDNA sequences of the lacl gene by using the RACE technique. The full length of the lacl cDNA sequence is 1972bp, which includes a complete Open Rading Frame (ORF)of 1860bp, from N0.33 to No. 1890, encoding 619 amino acides, the molecular weight is about 68kD , and isoelectric point is about 5.15.In comparison with the amino acid sequence of laccase of other species, there is higher homologous similarity : the highest identity is 41% and positive is 58% respctively.The genomic sequence of A. Polytricha laccase gene was amplified by the primers designed according to the 5' and 3'ends of the full length sequence of lac I cDNA. The length of genomic DNA is 2514bp, which contains 14exons and 13introns based on the comparison of cDNA and genomic DNA sequence.A signal peptide sequence exists in the N-terminal of the deduced amino acids, which also contains three multi-copper oxidase domains:KOG1263, Sufi and pfam00394. and it also includes four conserved Cu-bind domains. Based on the analysis of the sequence and structure , we can conclude the deduced amino acids encoded by the lacl gene possess the typical characteristics of fungal laccases.The full length cDNA sequence of A.polytricha lacl gene was digested with BamHI and NotI restriction enzyme and Iigated to the pPIC9K digested by the same enzyme to construct expression vectorpYH3660, the recombinant plasmid pYH3660 was transformed into Pichia pastoris, laccase activity was detected after 2 days induced by methanol. The results further domenstrates that lacl we got is laccase gene.
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