【作者】 谢子文；

【导师】 王红宁；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 本课题组根据毕赤酵母的信号肽序列，按照酵母偏爱密码，人工合成毕赤酵母的新型信号肽MS，并构建了新型酵母表达载体pPIC9k-MS，构建重组质粒pPIC9k-MS-phyA^m-4，然后电击转化毕赤酵母，筛选获得了植酸酶毕赤酵母基因工程菌株pp-MS-NP^m-4-16。本研究以麦芽汁培养基、蚕蛹培养基为基础培养基，用于工程菌pp-MS-NP^m-4-16高密度培养，结果表明：40％的蚕蛹培养基，190mg╱L的营养盐使酶活比对照高出36.8％。通过对植酸酶毕赤酵母基因工程菌PP-MS-Np^m-4-16摇瓶培养条件的研究，实验结果表明，最佳转速为220r／min，最适生长pH和诱导pH分别为5.0和6.0，通过正交实验，结果表明：接种量4％，甲醇浓度4％，豆油浓度（生长阶段）1.3％，豆油浓度（诱导阶段）3.5％为工程菌最适产酶条件，诱导结束时最高酶活达到192.4 ku／ml，是培养条件优化前酶活力的1.6倍。对植酸酶毕赤酵母基因工程菌PP-MS-NP^m-4-16在5L发酵罐中的发酵条件包括补料方式、甲醇流速、甘油流速和诱导时间进行研究，确定了它的最佳培养条件。实验结果表明，恒溶氧补料为最佳补料方式，通过正交实验，结果表明：在温度28℃，搅拌750r／min条件下，接种量10％、甲醇流速8 ml／L／h、甘油流速1 ml／L／h、诱导时间84 h为发酵罐的最佳发酵条件，发酵结束时菌体浓度OD₆₀₀高达175.2，实现了工程菌的高密度发酵，最高酶活达到472.933 ku／ml，是摇瓶培养酶活力的2.458倍，比本课题组报道的植酸酶工程菌PP-NP^m-8最高酶活263 ku／ml提高了0.8倍。对植酸酶的耐热性及产品的配制进行了初步研究，发酵液经过冷冻离心除去菌体得到粗酶液，然后添加10mg／ml的甘露醇，采用细米糠作为载体，与发酵液以1：1进行混合，喷涂米糠质量分数20％的Na₂SO₄，用米糠质量分数0.5％的海藻酸钠进行包被，经过80℃空气热处理0.5h，剩余酶活92.6％。PP-MS-NP^m-4-16工程菌高密度发酵培养基成本与酶产量比价为0.005461元／10⁶U，获得5000 u／g的产品价格在18元╱kg左右。本研究得到了植酸酶工程菌的高密度发酵条件和产品配制方法，提高了植酸酶表达量，提高了植酸酶的耐热性，为降低植酸酶生产成本和大规模发酵生产提供了科学依据。更多还原

【Abstract】 Abstract: Synthesizing new signal peptide MS which was based on signal peptide of Pichia pastoris and codes of yeast, and new Pichia pastoris vector pPIC9k-MS and the expression plasmid pPICgk-MS-phyA^m-4 were constructed and were transformed into GS115 strain, one high activity strains of transformant PP-MS-Np^m-4-16 was selected. In this research, the media of PP-MS-NP^m-4-16 was studied, the result indicated that the 40% chrysalis culture, 190mg/ml nutrition, salt added, phytase activity was increased 36.8% on the base ofcomparision.In baffled flask the single element experiment indicated that the relevance rotation speeds was 220r/min, initial pH of cell growth 5.0, initial pH of methanol inducing 5.5. by using orthogonal experiment, the result indicated that inoculate quantity 4%, methanol inducing concentration 4%,bean oil concentration during growth phase 1.3%, bean oil concentration during inducement phase 3.5%. when it was finished inducing the hignest phytase activity reached 192.4 ku/ml, which was 1.6 times as high as that of before optimizing.In the 5L automatic fermenter, the fermentation condition was optimized as the follows: the temperature 28℃, stirring speed 750r/min, inoculate quantity 4%, methanol velocity 8ml/L/h, glycerol velocity during inducing phase lml/L/h, induce time 84 h, when fermentation end, the thalli density attain 175.2, the paytase activity reached to 472.933KU/ml, which was 2.458 times as high as that of flask and increased 0.8 times than the activity of PP-Np^m-8.The preparation of phytase product and its thermostability were studied, the coarse enzyme was obtained by freeze centrifugal of fermentation liquid, then the 10mg/ml mannitol was added, coat of rice was used as carrier, mixed the coat of rice and fermentation according to the proportion 1: 1 , Na₂SO₄ was 20% w/w to coat of rice, sodium alginate was 0.5% w/w to coat of rice, the result indicated that after coating, the relative retaining activites of coated phytase under the temperature of 80℃reached 92.6%.The ratio of substrate cost of PP-MS-Np^m-4-16 strain and enzyme yield was 0.005461yuan/10⁶u, the price of product of 5000u/g was about 18yuan/kg.In this research we brought out phytase fermentation and product procession, which increased phytase activity、increased thermostability of phytase and provided scientific base for its fermentational production and cost decreasing as well.更多还原