植酸酶毕赤酵母基因工程菌PP-MS-NP～（m-4）-16高密度发酵条件研究
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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OD_(600)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Na_2SO_4，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／10~6U，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。
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 , Na_2SO_4 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/10~6u, 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.
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OD_(600)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Na_2SO_4，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／10~6U，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。
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 , Na_2SO_4 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/10~6u, 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.
引文
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