【作者】 李晓宇；

【导师】 池振明；

【作者基本信息】 中国海洋大学 ， 微生物学， 2008， 博士

【摘要】 采用单因子试验和表面响应的方法相结合,对产植酸酶培养基组分及条件进行了优化,结果如下:燕麦1.0% （w/v）,葡萄糖2.0% （w/v）,硫酸铵2.3% （w/v）氯化钠2.0% （w/v）,初始pH6.3,培养温度28 oC,培养72 h植酸酶的产量达到最高575.5 U/mL,这同软件预测的569.16 U/mL基本相一致。K. ohmeri BG3胞外植酸酶酶通过分子筛SephadexTM G-75和阴离子交换层析柱DEAE Fast Flow分离纯化,纯化倍数和回收率分别为7.2和10.4%。纯化后植酸酶SDS- PAGE凝胶电泳显示分子量约为98.2 kDa。纯化酶的最适反应pH和最适反应温度分别为5.0和65℃,该酶的温度稳定性较好,在pH3-9范围内稳定性也较好。Mn²⁺、Ca²⁺、Mg²⁺、Li⁺、K⁺、Na⁺、Ba²⁺和Co²⁺在浓度5.0mM时对纯化植酸酶有激活作用;而Zn²⁺、Fe²⁺、Fe³⁺、Hg²⁺、Ag⁺、和Cu²⁺在浓度5.0 mM时,对纯化的植酸酶有抑止作用。纯化的植酸酶活性被Phenylgloxal hydrate强烈抑制,因此分离纯化的植酸酶属于组氨酸酸性磷酸酶。纯化的植酸酶对底物植酸的动力学常数Km值和Vmax分别是1.45 mM和0.083 mM/min。用简并引物法扩增了编码海洋酵母K. ohmeri BG3植酸酶的部分基因,片段长度为1023 bp,NCBI登录号为EU009483。用RACE的方法扩增出了海洋酵母K. ohmeri BG3植酸酶基因5’和3’两端的区域,推导出了该植酸酶基因ORF框,共为1389 bp（NCBI登录号:EU082006）。通过比对海洋酵母K. ohmeri BG3植酸酶的基因序列和cDNA序列,发现两者完全一致,说明该基因没有内含子。K. ohmeri BG3植酸酶基因中含有两个酸性磷酸酶所特有的保守区域RHG X RX P和HD区域,这也说明该植酸酶属于酸性磷酸酶家族的一员。根据基因推导出K. ohmeri BG3植酸酶的氨基酸序列,共462个氨基酸,分子量为51.9 kDa。有一段含有15个氨基酸的信号肽,有6个糖基化位点。分析了K. ohmeri BG3植酸酶的氨基酸序列与其他植酸酶氨基酸序列的同源性,发现其与C. albicans （XP₇13452）和P. stipitis （XP₀01385108）植酸酶亲缘关系最近,同源性分别为61%和58%。利用pET表达系统成功实现了植酸酶基因PHY1在大肠杆菌中的表达,重组蛋白部分以活性蛋白形式存在,而大部分以包涵体形式存在。SDS-PAGE显示重组蛋白的分子量约为51 kDa。重组酶的最适反应pH和最适反应温度分别为5.0和65oC,该酶的热稳定性较好,在pH 3-8范围内稳定性也较好。重组酶在较短时间内可以将植酸水解为不同大小的水解产物,但不能将植酸彻底水解为肌醇。更多还原

【Abstract】 Statistical experimental designs were applied for the optimization of phytase production by a marine yeast Kodamaea ohmeri BG3 in a cost-effective oats medium. Using Plackett–Burman design, oats, ammonium sulfate and initial pH were identified as significant factors and these factors were subsequently optimized using a central composite design. The optimum variables that supported maximum enzyme activity were with oats 1.0%（w/v）, ammonium sulfate 2.3%（w/v）, glucose 2.0% （w/v）, NaCl 2.0% （w/v） and initial pH 6.3. The validity of the optimized variables was verified in shake-flasks level. An overall 9-fold enhancement in phytase activity （62.0–575.5 U/mL） was attained due to optimization.The extracellular phytase in the supernatant of cell culture of the marine yeast K. ohmeri BG3 was purified to homogeneity with a 7.2-fold increase in specific phytase activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography （Sephadexi G-75）, and anion-exchange chromatog- raphy （DEAE Sepharose Fast Flow Anion-Exchange）.According to the data from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular mass of the purified enzyme was estimated to be 98.2 kDa and the enzyme was shown to be a monomer according to the results of gel filtration chromatography. The optimal pH and temperature of the purified enzyme were 5.0 and 65oC, respectively. The enzyme was stimulated by Mn²⁺, Ca²⁺, K⁺, Li⁺, Na⁺, Ba²⁺, Mg²⁺ and Co²⁺ （at a concentrations of 5.0 mM）, but it was inhibited by Cu²⁺, Hg²⁺, Fe²⁺, Fe³⁺, Ag⁺, and Zn²⁺ （at a concentration of 5.0 mM）. The enzyme was also inhibited by phenylmethylsulfonyl fluoride （PMSF）, iodoacetic acid （at a concentration of 1.0 mM）, and phenylgloxal hydrate （at a concentration of 5.0 mM）, and not inhibited by EDTA and 1,10-phenanthroline（at concentrations of 1.0 mM and 5.0 mM）. The Km and Vmax, values of the purified enzyme forphytate were 1.45 mM, 0.083 mM / min, respectively.The extracellular phytase structural gene was isolated from cDNA of marine yeast K. ohmeri BG3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1389 bp long encoding a phytase. The coding region of the gene had no intron. It encoded 462 amino acid residues of a protein with a putative signal peptide of 15 amino acids. The protein sequence deduced from the extracellular phytase structural gene contained the consensus motifs （RHG XRX P and HD） which are conserved among histidine acid phosphatase and six conserved putative N-glycosylation sites. According to the phylogenetic tree of the phytase, the phytase from K. ohmeri BG3 was closely related to Candida albicans （XP₇13452） and Pichia stipitis （XP₀01385108） phytase protein and more distantly related to other phytases, 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 51.9 kDa was found. Enzyme activity assay verified the recombinant protein as a phytase. A maximum activity of 16.5 U/mg was obtained from cellular extract of E. coli BL21 （DE3） harboring pET-24a （+）PHY1. Optimal pH and temperature of the crude recombinant lipase were 5.0 and 65°C, respectively and the crude recombinant phytase had the hydrolytic activity towards phytate.更多还原