Ni Xiumei, Chi Zhenming, Ma Chunling, Madzak Catherine
Unesco Chinese Center of Marine Biotechnology, Ocean University of China, Qingdao, China.
Mar Biotechnol (NY). 2008 May-Jun;10(3):319-27. doi: 10.1007/s10126-007-9067-4. Epub 2008 Jan 3.
The alkaline protease structural gene (ALP1 gene) was isolated from both the genomic DNA and cDNA of Aureobasidium pullulans 10 by inverse PCR and RT-PCR. An open reading frame of 1248 bp encoding a 415 amino-acid protein with calculated molecular weight of 42.9 kDa was characterized. The gene contained two introns, which had 54 bp and 50 bp, respectively. The promoter of ALP1 gene was located from -62 to -112 and had two CCAAT boxes and one TATA box. The terminator of ALP1gene contained the sequence with a hairpin structure (AAAAAGTT TGGTTTTT). The protein sequence deduced from ALP1 gene exhibited 55.24%, 50.35%, and 31.68% identity with alkaline proteases from Aspergillus fumigatus, Acremonium chrysogenum, and Yarrowia lipolytica, respectively. The protein was found to have the conserved serine active site and histidine active site of serine proteases in the subtilisin family. The recombinant A. pullulans alkaline protease produced in Y. lipolytica formed clear zones on the double plates with 2% casein and alkaline protease activity in the supernatant of the recombinant Y. lipolytica culture was detected, suggesting that the cloned ALP1 gene is expressed in Y. lipolytica and the expressed alkaline protease is secreted into the medium.
通过反向PCR和RT-PCR从出芽短梗霉10的基因组DNA和cDNA中分离出碱性蛋白酶结构基因（ALP1基因）。鉴定出一个1248 bp的开放阅读框，编码一个415个氨基酸的蛋白质，计算分子量为42.9 kDa。该基因包含两个内含子，分别为54 bp和50 bp。ALP1基因的启动子位于-62至-112，有两个CCAAT框和一个TATA框。ALP1基因的终止子包含具有发夹结构的序列（AAAAAGTT TGGTTTTT）。从ALP1基因推导的蛋白质序列与烟曲霉、产黄顶孢霉和解脂耶氏酵母的碱性蛋白酶分别具有55.24%、50.35%和31.68%的同一性。发现该蛋白质具有枯草杆菌蛋白酶家族丝氨酸蛋白酶的保守丝氨酸活性位点和组氨酸活性位点。在解脂耶氏酵母中产生的重组出芽短梗霉碱性蛋白酶在含有2%酪蛋白的双平板上形成透明圈，并且检测到重组解脂耶氏酵母培养上清液中的碱性蛋白酶活性，这表明克隆的ALP1基因在解脂耶氏酵母中表达，并且表达的碱性蛋白酶分泌到培养基中。
