Cloning And Expression Of Phellinus Igniarius Laccase In Pichia Pastoris

Phellinus igniarius is a kind of precious medicinal fungi.It is reported that Phellinusigniarius have the founction to resist the cancer、oxidation and could protect the liver.As theresult of Phellinus igniarius is a rare fungi, the large-scale industrial production of Phellinusigniarius is limited, it difficult to make full use of the medicinal value of Phellinus igniarius. Laccase is a polyphenol oxidaset that belong to the multi-copper enzymes. Most of themproduced by the white rot fungus. Laccase play an important role in ligin degradation, phenolsand the phenoxy herbicides. So the experts of environment protection, pharmaceutical, paper,food and other industries begin to pay attention to the laccase in recent years. Applications oflaccase have made remarkable achievements and good resμLts in some sectors up to now. Laccase showed great research value and potential applications in these industries. Theapplications of laccase have made some progress, but the yield of phellinus igniarius is scarce,the submerged fermentation on laccase of Phellinus igniarius still in its infancy, so the researchon the laccase gene of Phellinus igniarius and the preparation of engineering bacteria plays animportant role in the large-scale industrial production of Phellinus igniarius and the applicationof laccase in industries.In this paper, the sequence of laccase gene have been cloned and analyzed from thePhellinus igniarius by the degenerate primer PCR and genome walking.The recombinantplasmid pPICZαA-lac was made and the recombinant plasmid pPICZαA-lac was transformedinto Pichia pastoris GS115.Pichia pastoris GS115was induced by methanol.The purificationand character of the laccase were also studied.Main resμLts as followed:1. Cloning and analyzing of the sequence of laccase gene from the Phellinus igniarius-A.The degenerate primers were designed according to the conserved amino acids sequencesof the copper-binding region in fungal laccases. Degenerate PCR was performed using the DNAof Phellinus igniarius-A as the template. A1554bp fragment was obtained. The special nestedprimers used for genome walking was designed according the1554bp DNA fragment. To get the5’and3’-flanking sequence of the laccase gene, genome walking was used. Then the fragmentof5’-flanking and3’-flanking were obtained, the length of them are642bp and288bp. By theDNAstar, put the three DNA fragments together, a2329bp genomic DNA of Phellinusigniarius-A laccase was obtained. Using the total RNA of Phellinus igniarius-A as template, a1575bp cDNA of Phellinus igniarius-A was got by RT-PCR. The TATA box and CAAT frame was found at the5’-flangking of the laccase gene. Theylocated66bp and198bp nucleotides upstream of initiation codon, respectively.But,The singlePoly(A) was not found at the3’-flangking of the laccase gene,the reason maybr the length ofgenome walking’s production is to short to reach the structure. The laccase gene of Phellinusigniarius-A contains nine introns and ten exons according to contrast the DNA and cDNA. Theexons of the laccase gene for a protein with520amino acids including a21-residue signalpeptide sequence, the laccase molecμLar weight is probably55.7KD.The laccase amino acidssequence homology between Phellinus igniarius-A and other fungus up to60%by the Blastp.2. Construction of recombinant vector and heterogeneous expression in Pichia pastoris GS115oflaccase gene from Phellinus igniarius-A.The special nested primers were designed according to the cDNA sequence of Phellinusigniarius-A laccase without signal peptide. Then the purified PCR production was digested by EcoR Iand Xba I. The digested gene fragment was inserted into the vector pPICZαA and therecombinant vector pPICZαA-lac was constructed successfully.Sac I was used to linearize the recombinant vector pPICZαA-lac,then the vector wastransformed into Pichia pastoris GS115according to the electroporation method. Laccase activity wasdetected after4days induced by methanol. At the8th day, the activity of laccase up to the maximum739U/L.3. The purification and character of the laccaseAfter purified of the laccase, The purification factor is3.06and the recovery is41.52%.The laccase was detected by SDS-PAGE, a specific protein band was found at55.7KD.The optimum temperature and pH of phellinus igniarius-A laccase is55℃and3.2;The besttemperature and pH of the laccase stability is45℃and6. Fe3+、Fe2+etc. ions can inhibit theactivity of the laccase and Ca2+、Zn2+etc. ions can promote it.The Cu2+don’t have notablecontribution to the laccase activity though the laccase is a polyphenol oxidaset that belong to themulti-copper enzymes.