海洋酵母普鲁兰类酵母碱性蛋白酶发酵生产、特性研究及基因克隆-微生物专业论文.docxVIP

长度为576bp。反向PCR法扩增得至忸三P1全长及两侧部分基因序列，共2562bp。 长度为576bp。反向PCR法扩增得至忸三P1全长及两侧部分基因序列，共2562bp。 推导出了碱性蛋白酶基因4工尸l ORF框，共1349bp。RT．PCR得到碱性蛋白酶基因 ALPl eDNA，共1245bp，包括3个外显子，分别为31lbp、364bp、588bp。根据eDNA 推导出普鲁兰类酵母碱性蛋白酶ALPl前体肽的氨基酸序列，共415个氨基酸，分 子量为42887Da。分析碱性蛋白酶基因丘￡P1及其上下游碱性序列，推导出其启动 子位置、终止子结构特点等。分析碱性蛋白酶ALPl前体氨基酸序列，推测出它 具有枯草蛋白酶N端特性和肽酶s8家族序列特征；含有丝氨酸蛋白酶枯草杆菌蛋 白酶家族丝氨酸活性位点和丝氨酸蛋白酶枯草杆菌蛋白酶家族组氨酸活性位点 两个蛋白模块；氮端有18个氨基酸残基的信号肽：含有3个N．糖基化位点。分析 了碱性蛋白酶ALPl前体肽氨基酸序列与其它碱性蛋白酶氨基酸序列的同源性， 建立了碱性蛋白酶ALPl系统发育树。 关键词海洋酵母普鲁兰类酵母碱性蛋白酶固体发酵分离纯化基因克隆 VI Production，Purification，Characterization Production，Purification，Characterization and Gene Cloning ofAlkaline Protease from Marine Yeast Aureobosidium pullulans Abstract Alkaline protease is one of the most important commercial enzymes． Microorganisms represent an attractive source of proteases as they call be cultured in large quantities in a relatively short time by established fermentation methods，and they produce an abundant，regular supply of the desired product．Furthermore， proteases fxom microorganism have a longer shelf life and Can be stored under less than ideal conditions for weeks without significant loss of activity．in general， microbial proteases ale extracellular in nature and are directly secreted into the fermentation broth by the producer，thus simplifying downstream processing of the enzyme as compared to proteases obtained from plants and animals． Production of alkaline protease employing nlarine yeast Aureobosidium pullulans 10 under solid state fermentation Was optimized．The optimum medium is soybean meal with 5％rice hull and 50％NaN03(2％)solution．The optimum inoculation size is 1．0×107 yeast cells per gram dry medium．The optimum quantity ofdry medium in 250-ml flask is 10 gram．The optimum temperature and initial pH ale 28。C and 10．0， respectively．Maximum yields of 196．9U／g dry medium were achieved after 72 h under the optimum medium and optimum conditions。 The extracellular protease secreted by Aureobosidium pullulans 10 Was purified to homo