CAJ | 학술논문

以1株产弹性蛋白酶的铜绿假单胞菌(Pseudomonas aeruginosa)基因组DNA为模板,经PCR扩增得到的铜绿假单胞菌弹性蛋白酶(P.acruginosa elastase,PAE)基因,与GenBank中的序列对比发现同源性为99%.成功地构建了重组表达载体pPIC3.5K/PAE,莺组质粒Sac Ⅰ线性化后转化毕赤酵母(Pichia pastoris)菌株KM71中,通过PCR和表型鉴定表明,PAE基因已经整合到毕赤酵母染色体上.经大量筛选获得48株含高拷贝的重组毕赤酵母转化子.在甲醇诱导下,经过毕赤酵母高密度发酵进行PAE的表达,经SDS-PAGE分析.结果表明,在培养基上清中含有一明显特异性蛋白条带,大小为34kD.活性检测结果,酶活为1 060 U/mL,是出发菌株的26倍.
이1주산탄성단백매적동록가단포균(Pseudomonas aeruginosa)기인조DNA위모판,경PCR확증득도적동록가단포균탄성단백매(P.acruginosa elastase,PAE)기인,여GenBank중적서렬대비발현동원성위99%.성공지구건료중조표체재체pPIC3.5K/PAE,앵조질립Sac Ⅰ선성화후전화필적효모(Pichia pastoris)균주KM71중,통과PCR화표형감정표명,PAE기인이경정합도필적효모염색체상.경대량사선획득48주함고고패적중조필적효모전화자.재갑순유도하,경과필적효모고밀도발효진행PAE적표체,경SDS-PAGE분석.결과표명,재배양기상청중함유일명현특이성단백조대,대소위34kD.활성검측결과,매활위1 060 U/mL,시출발균주적26배.
The Pseudomonas aeruginosa elastase(PAE)gene fragment was amplified by PCR from the genome DNA of a strain of Pseudomonas aeruginosa.The nucleotide sequence of the cloned DNA shared 99%homology with lasB from P.aeruginosa PA01(GenBank accession No.M19472).The secreted recombinant expression plasmid pPIC3.5K/PAE was constructed and digested with Sac Ⅰ and introduced into Pichia pastoris KM71.PCR and phenotype analysis showed that PAE has been integrated into P.pastoris genome.After screening,the 48 high-copy recombinant P.pastoris transformants were obtained.The specific expression protein was secreted into the medium after inducing with methanol.SDS-PAGE results showed that there Was one main protein band with molecular weight 34 kD in the fermentation supemation.Activity tests revealed that the specific activity of the recombinant elastase was 1 060 U/mL,which was approximately 26-fold higher than that of elastase obtained from P.aeruginosa.