【作者】 马春玲；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 微生物学， 2007， 博士

【摘要】 碱性蛋白酶是世界工业酶制剂中最重要酶制剂之一,其用途非常广泛,受到了大家的广泛关注,特别是微生物发酵产碱性蛋白酶由于其产量大、周期短、操作简单种类多等优点而在短短几十年内迅速发展。本论文主要讨论了海洋酵母普鲁兰类酵母A. pullulans 10碱性蛋白酶的发酵生产及酶的分离纯化、特性研究及基因克隆。优化了海洋酵母普鲁兰类酵母A. pullulans 10固体发酵生产碱性蛋白酶培养基主要成分和发酵条件。在豆饼粉和稻糠以4:1比例混合,含水量50%,添加含有2%硝酸钠溶液酶活较高。最适固体发酵条件,接种量107cells/g培养基,装量10 g/250ml三角瓶,温度28°C,最适初始pH10.0。在优化的培养基和发酵条件下,3天后酶活达到196.9U/g干培养基。普鲁兰类酵母10胞外蛋白酶通过分子筛SephadexTM G-75和阴离子交换柱DEAE Fast Flow分离纯化,纯化倍数和回收率分别为2.1和18.8%。纯化酶SDS- PAGE凝胶电泳显示分子量约为32kDa。纯化酶的最适反应pH和最适反应温度分别为9.5和45°C,酶对温度比较敏感,广泛pH内都有活性,确定普鲁兰类酵母10胞外蛋白酶属于碱性蛋白酶,命名为ALP1。存在Mn²⁺和低浓度Cu²⁺（1 mM）时,蛋白酶酶活有一定程度的增加;当存在Hg²⁺、Fe²⁺、Fe³⁺、Zn²⁺、Co²⁺、和高浓度的Cu²⁺时,蛋白酶酶活明显降低,纯化酶活性被苯甲基磺酰氟（PMSF）强烈抑制,因此分离纯化的碱性蛋白酶ALP1属于丝氨酸蛋白酶。蛋白酶对底物酪蛋白的Km值和Vmax分别是0.25mg/ml和0.0286μmol/min/mg蛋白。用纯化的碱性蛋白酶ALP1水解不同来源蛋白,能产生具有生物活性的多肽。水解海洋酵母蛋白、螺旋藻蛋白、虾蛋白、牛奶蛋白和酪蛋白,所得到的水解产物都具有抑制ACE活性和抗氧化活性,但来自虾蛋白的水解产物抑制ACE活性最高,达85.3%,来自螺旋藻蛋白的水解产物的抗氧化活性最高,达40.6%。没有观察到蛋白水解产物的抗菌活性。简并引物法扩增了编码普鲁兰类酵母10碱性蛋白酶ALP1的部分基因,片段长度为576bp。反向PCR法扩增得到ALP1全长及两侧部分基因序列,共2562bp。推导出了碱性蛋白酶基因ALP1 ORF框,共1349bp。RT-PCR得到碱性蛋白酶基因ALP1 cDNA,共1245bp,包括3个外显子,分别为311bp、364bp、588bp。根据cDNA推导出普鲁兰类酵母碱性蛋白酶ALP1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ALP1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ALP1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ALP1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ALP1系统发育树。更多还原

【Abstract】 Alkaline protease is one of the most important commercial enzymes. Microorganisms represent an attractive source of proteases as they can 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 from 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 are 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 marine yeast Aureobosidium pullulans 10 under solid state fermentation was optimized. The optimum medium is soybean meal with 5% rice hull and 50% NaNO₃ （2%） solution. The optimum inoculation size is 1.0×10⁷ yeast cells per gram dry medium. The optimum quantity of dry medium in 250-ml flask is 10 gram. The optimum temperature and initial pH are 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 homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ultrafiltration, gel filtration chromatography （SephadexTM G-75）, and anion-exchange chromatography （DEAE Sepharose Fast Flow）. According to the sodium dodecyl sulfate-polyacryl- amide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0kDa. The optimal pH and temperature of the purified enzyme were 9.5 and 45°C, respectively. The enzyme was activated by Cu²⁺ （at a concentration of 1.0 mM） and Mn²⁺ and inhibited by Hg²⁺,Fe²⁺, Fe³⁺, Zn²⁺, and Co²⁺. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride. The Km and Vmax values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 mmol/min/mg of protein, respectively.After digestion of shrimp protein, spirulina （Arthospira platensis） protein, proteins of marine yeast strains N3C （Yarrowia lipolytica） and YA03a （Hanseniaspora uvarum）, milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme （ACE） inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.A genomic DNA fragment for the full length of gene encoding the alkaline protease（ALP1） from marine yeast Aureobosidium pullulans 10 has been cloned and sequenced by degenerate PCR and inverse PCR. ORF frame in the genomic DNA fragment consisting of 1349 bp is presumed by BLASTx in NCBI. The cDNA fragment is amplified by RT-PCR.After sequencing and blasting with ALP1 genomic ORF, the results show that ALP1 genome DNA fragment consists of three extrons of 311bp, 346bp, 588bp and two introns of 54bp and 50bp, respectively. The cDNA fragment of ALP1 encoded 415 amino acids. The molecular weight of alkaline protease precursor is 42887 Da.A putative promoter at–111 from the start codon is observed. A TATA box and two CAAT are observed at -101, -82, -25, respectively. Terminator at the 3′-noncoding region is observed with clip structure. A consensus sequence of a signal peptide consisting of 18 amino acids is found at the N-terminus and three N-glycosylation sites are found. The primary structure of the mature region shares extensive homology with that of subtilisin families and peptidase S8, and the two active sites （Histidine active site and serine active site） are preserved. Multiple alignment and phylogeny analysis of deduced amino acid sequence of pre-protease with other alkaline protease from representative species are done by using the software of Clustal X and PHYLIP-NP respectively.更多还原