金针菇漆酶基因的克隆及其在毕赤酵母中的表达研究

Cloning of A Laccase Gene from Flammulina velutipes and Study on Its Expression in Pichia pastoris

综合运用cDNA末端快速扩增 (RapidAmplificationofcDNAEnds ,RACE )和基因组步行等技术克隆到一个金针菇 (Flammulinavelutipes)的漆酶结构基因和其对应的全长cDNA ,经测序和BLAST比对分析表明该基因属于多铜氧化酶基因家族 ,与已发表的漆酶基因 (AF176 2 30 )的同源性最高 ,在氨基酸水平为 72 %。该结构基因命名为gl ccFv,cDNA命名为lccFv ,其序列提交GenBank ,登录号分别为AY4 85 82 6和AY4 5 0 4 0 6。将lccFv的开放阅读框克隆到毕赤酵母表达载体pHBM90 6 ,转化毕赤酵母GS115且实现了分泌表达。将重组毕赤酵母GS115 (pHBM5 6 5 )诱导产酶 ,在培养温度 2 0℃、甲醇流加量为 1 0 % (V V)的情况下 ,其分泌表达的LCCFv的最高酶活为 0 10 70U mL ,最适反应温度为 4 5℃ ,最适反应pH值为 3 9。

Laccase(EC1.10.3.2) can be used for enzymatic detoxification of lignocellulosic hydrolysates. By using molecular techniques such as RACE (rapid amplification of cDNA ends) and Genome-Walking, a laccase gene and its corresponding full-length cDNA were cloned from Flammulina velutipes and designated as glccFv and lccFv. The sequences were submitted to GenBank, and the accession numbers obtained were AY485826 and AY450406, respectively. Analysis of amino acids sequence suggested that one laccase from Polyporus ciliatus possessed the highest homology with the protein encoded by lccFv showing for 72%. The ORF (open reading frame) of lccFv was transformed into Pichia pastoris strain GS115 through the P. pastoris expression vector pHBM906, which contains both the promoter and transcription terminator of the AOX1 gene. The recombinant laccase LCCFv was detected from the engineering strain GS115(pHBM557) which was fermented with BMMY liquid medium and induced by 1.0%(V/V)methanol at 20℃ with the highest expression level (0.1070 U/mL). The optimal reaction temperature of LCCFv that secreted from P. pastoris GS115(pHBM557) was 45℃, the optimal reaction pH value was pH3.9 and the thermostability and pH stability were very well under the optimal conditions.