【作者】 柳志强；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2008， 博士

【摘要】 从盐场分离到一株高产脂肪酶的酵母菌HN₂-3,经酵母常规鉴定方法和分子生物学方法鉴定为普鲁兰短梗霉。该酵母生产脂肪酶最佳培养基组分为3.0% （w/v）橄榄油、0.4% （w/v）葡萄糖、0.6% （w/v）硫酸铵、0.1% （w/v） K₂HPO₄和0.05% （w/v） MgSO₄.7H₂O,最佳培养条件为初始pH 7.0、培养温度25 oC和转速170rpm。我们发现橄榄油的添加时间对于脂肪酶的生产具有很大的影响,在接种培养6 h后加入橄榄油,继续培养96 h,脂肪酶产量达到最大,酶活力达到8.0 U/ml。普鲁兰短梗霉HN₂-3胞外脂肪酶通过硫酸铵沉淀、凝胶过滤层析和阴离子交换层析得到纯化,纯化倍数为3.4。SDS- PAGE显示该脂肪酶分子量约为63.5 kDa。纯化酶的最适作用pH和最适作用温度分别为8.5和35oC,该酶被Hg2+, Fe2+和Zn²⁺强烈抑制。同时苯甲基磺酰氟可以强烈抑制该脂肪酶的活性,乙二胺四乙酸对该纯化酶的影响不大,碘乙酸可以微弱抑制该酶的活性。纯化的脂肪酶对于花生油具有较强的水解活性。利用RACE的方法克隆了普鲁兰短梗霉HN₂-3胞外脂肪酶的基因。该基因含有一个1245bp的ORF框,同时发现从基因组克隆得到的脂肪酶基因序列中含有一个55bp的内含子。该基因编码一个414个氨基酸残基的蛋白质,在氨基端含有一个26个氨基酸残基的信号肽。推导氨基酸序列含有脂肪酶的保守序列（G-X-S-X-G）和3个推测的N-糖基化位点。通过脂肪酶系统进化树分析,普鲁兰短梗霉HN₂-3脂肪酶与Aspergillus fumigatu（sXP₇50543）和Neosartorya fischeri （XP₀01257768）脂肪酶亲缘关系最近,同源性分别为50%和51%。将脂肪酶基因克隆到pET-24a （+）表达载体上,并在大肠杆菌BL21（DE3）中进行了表达。SDS-PAGE和免疫印迹分析发现重组蛋白的分子量约为47kDa。测定了阳性克隆BL21（DE3）/pET-24a（+）LIP1细胞裂解液的脂肪酶活力,达到0.96 U/mg。重组脂肪酶最适作用pH和最适作用温度分别为8.0和35°C,重组脂肪酶依然对花生油具有较高的水解活性。更多还原

【Abstract】 A marine-derived yeast strain HN2-3 isolated from sediment of the salterns was found to secrete a large amount of lipase into the medium. This marine-derived yeast strain was identified to be a strain of Aureobasidium pullulans according to the results of routine yeast identification and molecular methods. The optimal medium for the crude lipase production was 3.0% （w/v） olive oil, 0.4% （w/v） glucose, 0.6% （w/v） ammonium sulfate, 0.1% （w/v） K₂HPO₄, 0.05% （w/v） MgSO₄.7H2O, while the optimal cultivation conditions for the crude lipase production were pH 7.0, 25 oC and 170 rpm. It was found that lipase production was dependent on the time when olive oil was added to the medium. When olive oil was added to 6 h old culture with 0.4% （w/v） glucose, the highest lipase activity was achieved. Under the optimal conditions, over 8.0 U/ml of lipase was produced within 96 h of the fermentation at shake flask level.The lipase in the supernatant of the yeast cell culture was purified to homogeneity with a 3.4-fold increase in specific lipase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography and anion-exchange chromatography. According to the data on SDS polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 63.5 kDa. The optimal pH and temperature of the purified enzyme were 8.5 and 35 oC, respectively. The enzyme was greatly inhibited by Hg²⁺, Fe²⁺ and Zn²⁺. The enzyme was strongly inhibited by phenylmethanesulphonyl fluoride, not inhibited by ethylene diamine tetraacetic acid （EDTA）, but weakly inhibited by iodoacetic acid. It was found that the purified lipase had the highest hydrolytic activity towards peanut oil.The extracellular lipase structural gene was isolated from cDNA of A. pullulans HN2-3 by using SMART^TM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the gene was interrupted by only one intron （55 bp）. It encodes 414 amino acid residues of a protein with a putative signal peptide of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus sequence （G-X-S-X-G） and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases, the lipase from A. pullulans was closely related to that from Aspergillus fumigatus （XP₇50543） and Neosartorya fischeri （XP₀01257768） and the identities were 50% and 52%, respectively.The mature peptide encoding cDNA was subcloned into pET-24a （+） expression vector. The recombinant plasmid was expressed in Escherichia coli BL21（DE3）. The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of 0.96 U/mg was obtained from cellular extract of E. coli BL21（DE3） harboring pET-24a（+）LIP1 . Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35°C, respectively and the crude recombinant lipase had the highest hydrolytic activity towards peanut oil.更多还原