植酸酶基因在毕赤酵母中高效表达及工程菌培养基初步优化
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摘要
植酸酶是催化植酸及植酸盐水解成肌醇和无机磷酸盐的一类酶，将其添加到单胃动物植物性饲料中，可以释放植酸中的无机磷以及被植酸络合的Ca~(2+)、Mg~(2+)、Zn~(2+)等矿物元素和一些蛋白质，从而解除植酸抗营养作用、提高动物饲料的营养价值，同时也减少了动物粪便中植酸磷的含量，降低了磷对环境的污染程度。研究表明，饲料中添加植酸酶可使其中磷利用率提高60％，粪便中磷排出量减少40％，还可以提高饲料转化率，改善动物的生产性能，因此对提高养殖业的经济效益及环境保护均具十分重要意义。
植酸酶主要存在于微生物和植物中，动物体内也有少量存在，但真正具有开发价值的主要是微生物产生的植酸酶，尤其是曲霉产生的酸性植酸酶。国外早在1991年就成功地利用DNA重组技术获得第一株酸性植酸酶的工程菌，并已投入商品化生产。目前已开始从事植酸酶的植物和动物基因工程的研究，已成功将植酸酶基因导入苜蓿、烟草、小鼠和猪的唾液腺中。国内对植酸酶的研究起步较晚，到目前为止，植酸酶野生菌株产酶水平低下的问题仍然没有得到彻底解决，目前市场上应用的植酸酶制剂多属进口产品，且价格昂贵。因此，提高植酸酶野生菌株产酶水平，降低其生产成本，对实现植酸酶产品国产化并获取自己专利具有十分重要意义。而提高野生菌株产酶水平的主要思路集中在通过基因工程手段克隆植酸酶基因或进行适当改造，然后将这些优良基因导入适当的宿主菌，进一步筛选出高产工程菌株，优化发酵工艺条件从而实现植酸酶基因的高效表达。
因此，本实验着力于将已构建的可进行诱导调控分泌表达的毕赤酵母整合型表达载体转化毕赤酵母受体菌，进而筛选出高产稳产植酸酶的工程菌，为植酸酶的规模化生产提供优良菌种，并对工程菌株的产酶培养基进行初步优化从而为规模化生产提供科学依据。主要实验结查如下：
西南农业大学硕士学位论文
1、用乃aI酶切携带植酸酶基因表达片段的重组质粒pPICgK夕句)A，回收DNA，用Gene
Pulser电击转化毕赤酵母，涂布MD平板，又用含不同浓度G418的YPD平板进行抗性筛选，
得到98个可检测到植酸酶活力的阳性转化子，它们在MD、MM平板上均表现快速生长，说
明其甲醇利用表型是Mut干。用特异性引物，对转化菌株进行PcR检测，扩增出了特异条带，
表明外源基因整合进了宿主细胞。挑选转化子经过BMGY摇瓶培养、BMMY诱导发酵后，用
钒铝酸按法测定了表达产物的酶活性，结果表明重组菌株可有效表达具有生物学活性的植酸
酶。经摇瓶复筛后得到l株产酶活性为143958.3UZmL发酵液，并且具有良好遗传稳定性的高
产工程菌株(E22)，其产酶活性是出发菌株酶活性(422U/mL)的341 .13倍。
2、重组酵母E一22在诱导培养168h之内，植酸酶的表达随时间的延长而增加，到144h
接近高峰，之后基本保持稳定。菌体湿重在诱导培养72h之内有所增加，随后基本保持稳定。
重组植酸酶部分酶学性质的研究结果表明:重组酶在pH值2.5一3.0和5.0一5.5时酶活性最高，
且在pH4.5一6.5之间均有较高的酶活性，最适作用温度为55℃。sDs一PAGE的结果显示重组
植酸酶的分子量介于70kDa~97kDa之间。重组植酸酶粗酶比活力为461575.8 u/mg。
3、用5因素的均匀设计试验对工程菌株的产酶培养基进行了初步优化，酵母提取物、甲
醇、蛋白陈、YNB、生物素在培养基中的浓度分别取2%，4%，1%，0，0.0001%;在此条件
下发酵液的酶活力达到108758.3 U/mL发酵液，高于均匀设计中各试验点的酶活力值，是使用
BMMY培养基同条件(诱导培养96h，37℃测酶活)下酶活力(97666.7U/mL)的1 1 1.4%。
Phytases are a class of phosphatases that can catalyze the hydrolysis of phytate into myo-inositol and phosphate. It can relieve anti-nutrition of phytate and improve the nutritional value of animal feed from plant.The study on phytase, especially acidic phytase produced by micro-organism, has drawn much attention in home and abroard. However, its wide application in manufacture has always been restricted by such a question as low phytase-producing level of wild strains, so before the wild strains which can produce acidic phytase are widely used, their phytase activities must be improved by various means, in which using an efficient expression system to express heterogenous phytase is a main consideration. The objective of this research is to transform the cloned phytase gene into Pichia pastoris in order to obtain high-yield phytase-producing strain and to optimize the engineered yeast media recipes for the scale-up production of phytase. Main results of this research are as follow:
1 ,Xba I-linized recombinant plasmid pPIC9K-phyA was transformed into Pichia pastoris by Gene Pulser .98 positive transformants showing measurable phytase activities were screened on MD plates and YPD plates containing G418. They all grew quickly on both MD plates and MM plates, which proved their phenotypes of methanol utilization were Mut+. Specific Pichia clony PCR product showed that foreign phytase gene was integrated into the host cell. The experimental results from flask fermentation and phytase activity assay indicated that phytase gene was effectively expressed by the recombinant Pichia. One highly productive and genetically stable recombinant strain named E-22, which produced phytase with 143958.3U/mL under the condition of flask cultivation, was selected through further screening. The phytase activity of E-22 was 341.13 times as high as that of the original strain (422U/mL).
2,During the inducing cultivation, the phytase expressed by recombined yeast E-22 keeps
improving in 168h, approaches maximum at 144h and then stays stable. Its cell wet weight increases in 72h and then keeps constant. Studies on the partial enzyme characters of recombinant phytase showed that the recombinant phytase had two pH optima (2.5-3.0 and 5.0-5.5) and a temperature optimum 55℃, and with higher activity under the pH ranged from 4.5 to 6.0. The result of SDS-PAGE indicated that the molecular weight of the recombinant phytase is between 70kDa-97kDa. Specific activity of the rude recombinant phytase is 461575.8 U/mg.
3, A five-factor Uniform Design experiment was conducted to optimize the engineered yeast media recipes for phytase production.Phytase activity of 108758.3 U/mL was observed under the condition of flask cultivation in which the contents of yeast exract, methnol, peptone, YNB, biotin in the media were 2%, 4%, 1%, 0, 0.0001% respectively. The phytase activity in fermentation broth was 1.114 times as high as that obtained from native BMMY (97666.7U/mL) under the same condition (inducing time 120h, phytase activity measured at 37℃).
植酸酶主要存在于微生物和植物中，动物体内也有少量存在，但真正具有开发价值的主要是微生物产生的植酸酶，尤其是曲霉产生的酸性植酸酶。国外早在1991年就成功地利用DNA重组技术获得第一株酸性植酸酶的工程菌，并已投入商品化生产。目前已开始从事植酸酶的植物和动物基因工程的研究，已成功将植酸酶基因导入苜蓿、烟草、小鼠和猪的唾液腺中。国内对植酸酶的研究起步较晚，到目前为止，植酸酶野生菌株产酶水平低下的问题仍然没有得到彻底解决，目前市场上应用的植酸酶制剂多属进口产品，且价格昂贵。因此，提高植酸酶野生菌株产酶水平，降低其生产成本，对实现植酸酶产品国产化并获取自己专利具有十分重要意义。而提高野生菌株产酶水平的主要思路集中在通过基因工程手段克隆植酸酶基因或进行适当改造，然后将这些优良基因导入适当的宿主菌，进一步筛选出高产工程菌株，优化发酵工艺条件从而实现植酸酶基因的高效表达。
因此，本实验着力于将已构建的可进行诱导调控分泌表达的毕赤酵母整合型表达载体转化毕赤酵母受体菌，进而筛选出高产稳产植酸酶的工程菌，为植酸酶的规模化生产提供优良菌种，并对工程菌株的产酶培养基进行初步优化从而为规模化生产提供科学依据。主要实验结查如下：
西南农业大学硕士学位论文
1、用乃aI酶切携带植酸酶基因表达片段的重组质粒pPICgK夕句)A，回收DNA，用Gene
Pulser电击转化毕赤酵母，涂布MD平板，又用含不同浓度G418的YPD平板进行抗性筛选，
得到98个可检测到植酸酶活力的阳性转化子，它们在MD、MM平板上均表现快速生长，说
明其甲醇利用表型是Mut干。用特异性引物，对转化菌株进行PcR检测，扩增出了特异条带，
表明外源基因整合进了宿主细胞。挑选转化子经过BMGY摇瓶培养、BMMY诱导发酵后，用
钒铝酸按法测定了表达产物的酶活性，结果表明重组菌株可有效表达具有生物学活性的植酸
酶。经摇瓶复筛后得到l株产酶活性为143958.3UZmL发酵液，并且具有良好遗传稳定性的高
产工程菌株(E22)，其产酶活性是出发菌株酶活性(422U/mL)的341 .13倍。
2、重组酵母E一22在诱导培养168h之内，植酸酶的表达随时间的延长而增加，到144h
接近高峰，之后基本保持稳定。菌体湿重在诱导培养72h之内有所增加，随后基本保持稳定。
重组植酸酶部分酶学性质的研究结果表明:重组酶在pH值2.5一3.0和5.0一5.5时酶活性最高，
且在pH4.5一6.5之间均有较高的酶活性，最适作用温度为55℃。sDs一PAGE的结果显示重组
植酸酶的分子量介于70kDa~97kDa之间。重组植酸酶粗酶比活力为461575.8 u/mg。
3、用5因素的均匀设计试验对工程菌株的产酶培养基进行了初步优化，酵母提取物、甲
醇、蛋白陈、YNB、生物素在培养基中的浓度分别取2%，4%，1%，0，0.0001%;在此条件
下发酵液的酶活力达到108758.3 U/mL发酵液，高于均匀设计中各试验点的酶活力值，是使用
BMMY培养基同条件(诱导培养96h，37℃测酶活)下酶活力(97666.7U/mL)的1 1 1.4%。
Phytases are a class of phosphatases that can catalyze the hydrolysis of phytate into myo-inositol and phosphate. It can relieve anti-nutrition of phytate and improve the nutritional value of animal feed from plant.The study on phytase, especially acidic phytase produced by micro-organism, has drawn much attention in home and abroard. However, its wide application in manufacture has always been restricted by such a question as low phytase-producing level of wild strains, so before the wild strains which can produce acidic phytase are widely used, their phytase activities must be improved by various means, in which using an efficient expression system to express heterogenous phytase is a main consideration. The objective of this research is to transform the cloned phytase gene into Pichia pastoris in order to obtain high-yield phytase-producing strain and to optimize the engineered yeast media recipes for the scale-up production of phytase. Main results of this research are as follow:
1 ,Xba I-linized recombinant plasmid pPIC9K-phyA was transformed into Pichia pastoris by Gene Pulser .98 positive transformants showing measurable phytase activities were screened on MD plates and YPD plates containing G418. They all grew quickly on both MD plates and MM plates, which proved their phenotypes of methanol utilization were Mut+. Specific Pichia clony PCR product showed that foreign phytase gene was integrated into the host cell. The experimental results from flask fermentation and phytase activity assay indicated that phytase gene was effectively expressed by the recombinant Pichia. One highly productive and genetically stable recombinant strain named E-22, which produced phytase with 143958.3U/mL under the condition of flask cultivation, was selected through further screening. The phytase activity of E-22 was 341.13 times as high as that of the original strain (422U/mL).
2,During the inducing cultivation, the phytase expressed by recombined yeast E-22 keeps
improving in 168h, approaches maximum at 144h and then stays stable. Its cell wet weight increases in 72h and then keeps constant. Studies on the partial enzyme characters of recombinant phytase showed that the recombinant phytase had two pH optima (2.5-3.0 and 5.0-5.5) and a temperature optimum 55℃, and with higher activity under the pH ranged from 4.5 to 6.0. The result of SDS-PAGE indicated that the molecular weight of the recombinant phytase is between 70kDa-97kDa. Specific activity of the rude recombinant phytase is 461575.8 U/mg.
3, A five-factor Uniform Design experiment was conducted to optimize the engineered yeast media recipes for phytase production.Phytase activity of 108758.3 U/mL was observed under the condition of flask cultivation in which the contents of yeast exract, methnol, peptone, YNB, biotin in the media were 2%, 4%, 1%, 0, 0.0001% respectively. The phytase activity in fermentation broth was 1.114 times as high as that obtained from native BMMY (97666.7U/mL) under the same condition (inducing time 120h, phytase activity measured at 37℃).
引文
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48、Ha NC, Oh BC, Shin S, et al.Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states. Nat Struct Biol 2000,7(2): 147-53
49、Maraz A. From yeast genetics to biotechnology Acta Microbiol Immunol Hung. 2002; 49(4):483-91.
50、Sudbery PE. The expression of recombinant proteins in yeasts[J], Curr Opin Biotechnol. 1996, 7(5):517-24.
51、Cregg J M, Cereghino J L, Shi J, et al. Recombinant protein expression in Pichia pastoris.Mol biotechenol, 2000, 16(1): 23-52.
52、丁云菲，刘勇，戴长柏．巴斯德毕赤酵母研究进展，[J]生命科学，2003，15(1)：26-30
53、李艳，王正祥，诸葛健．酵母作为外源基因表达系统的研究进展，生物工程学报[J]，2001，22(2)：10-14
54、韦宇拓，甘凤菊，苏华波等。巴斯德毕赤酵母新型分泌表达载体构建，广西农业生物科学[J]，2003，22(1)：37-40
55、Sunga A J, Cregg JM.The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris. Gene. 2004, 330:39-47
56、Soderhoim J, Bevis BJ, Giick BS. Vector for pop-in/pop-out gene replacement in Pichia pastoris. Biotechniques. 2001,31 (2):306-312.
57、Wan H, Sjolinder M, Schairer HU, et al.A new dominant selection marker for transformation of Pichia pastoris to soraphen A resistance.J Microbiol Methods. 2004,57(1):33-39.
58、Wu S, Letchworth G J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques. 2004, 36(1): 152-154.
59、Eiden-Plach A, Zagorc T, Heintel T, et al.Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastods, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Appl Environ Microbiol. 2004,70(2): 961-966.
60、C. M. J. Sagt, B. Kleizen, R. Verwaal, et al, Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts.Appl Environ Microbiol, 2000,66(11): 44940-4944
61、聂东宋，梁宋平．李敏．外源蛋白在巴氏毕赤酵母中高效表达的策略[J]。吉首大学学报(自然科学版)，2001，22(3)：40-44．
62、Carol A S,The intracellular prodction and secretion of HIV-1 Envelope in methylotrophic yeast Pichia[J],Gene,1993, (136):111-119
63、Clare JJ,Romamos M A. Production of EGF in Yeast: High-level Secretion Using Pichia Pasoris
Strains Containing Mutiple Gene Copies.[J], Gene, 1991, 105:205-212
64、赵翔，霍兢，李育阳．毕赤酵母的密码子用法分析[J]，生物工程学报，2000，16(3)：308-311
65、Sreekrishna K, Brankamp R G, Kr0pp K E, et al.Strategy for Optimal Synthesis and secretion of Heteroguious Proteins in the Mythy Trophic Yeast Pastoris pichia, Gene 1997,191(1): 55-62
66、李欣，郭树华．影响外源基因在巴氏毕赤酵母中表达的因素[J]，生物技术通讯，2000。11(2)：132-134
67、藏秀玉，王恂，周坚．毕赤氏酵母Paox2突变化序列分析[J]．微生物学报，1999，39(6)：559—561
68、杨晟，黄鹤，章如安等．重组人血清白蛋白在Pihia pastoris中分泌表达影响因素的研究[J]，生物工程学报，2000，16(6)：675-678
69、Waterham H R, Digan M E, Koutz P J, et al. Isolation of the Pichia Pastoris Glyceraldehyde-3-phospate and Rgulation and Use of Its Promomoter [J],Gene, 1997, 186(1): 37-44.
70、Score C A, Clare JJ, MC Combie W R, et al. Rapid selection of high Copy Number transformants of pastoris pichia for high-level foreign gene expression[J]. Bio/Technology, 1994 12(2):181-184
71、Vassileva A,Chugh D A ,Swaminathan S, et al, Effect of copy number on the epression levels of hepatitis B surface antigen in the ethyiotrophic yeast Pichia pastoris [J], Protein Expr Purif, 2001, 21(1):71-80
71、Menendez J, Gania B, Hidalgo Y, New method for the selection ofmulticopy transformants of Pichia pastoris using 3-amino-1,2,4triazoi [J]. Biotechniques, 2002, 29(5): 1094-1099
73、欧阳菁，杨林，龙綮新等．猪生长激素在巴斯德毕赤酵母中高效分泌表达及产物的 N-糖基化分析[J]，生物工程学报，2001，17(5)：520-524
74、Lopes T S, Hakkaart G, Koertsb L, et al. Maehenisms of high-copy-number integration of pMIRY-Type vectors into the ribosomal DNA of Saecharomyees cerevisiae [J]. Gene, 1991, 105(1):83-90
75、唐南筠，霍競，李育阳．乳酸克鲁维酵母高拷贝整合载体的构建和应用[J]．生物化学与生物物理学报，1996，28(5)：540-545
76、Gregg J M, Tschopp J F, Stiman C, et al.High-level expression and efficient asssembly of hepetitis B surface antigen in methyltrophic yeast Pichia pastoris [J]. Bio/Technoiogy, !987,(5): 479——486.
77、Hohenblum H, Gasser B, Maurer M, et al. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris [J].Biotechnol Bioeng. 2004, 85(4):367-375
78、MEI M, WHITTAKER,JAMESW, et al. Expression of recombinant galactose oxidase by pichia
pastoris [J]. Protein Expr. Purif. 2000, 20:105-111
79、Clare JM, Udevick T S, Raschke W C, Curecent advances in the expression of foreign genes in Piehia pastoris[J],Bio/Technology. 1993,11(8): 905-910
80、Pen J,Verwoerd T C,Paridon P A Van.Phytase-containing transgenic seed as a novel feed additive for improved phosphorus utilization Bio/technol, 1993,11:811～814.
81、王红宁，马孟根，吴琦等．黑曲霉N25植酸酶phyA基因在巴斯德毕赤酵母中高效表达的研究[J]，菌物系统，2001．20(4)：486～493
82、贝锦龙，陈庄，杨林等．人工合成的黑曲霉NRRL3135菌株植酸酶基因在毕赤酵母系统中的高效表达[J]．生物工程学报，2001，17(3)：254～258
83、张若寒，植酸酶活性的检测方法[J]，中国饲料，1997，5：30-32
84、金冬雁，黎孟枫，侯云德等译．分子克隆实验指导(第二版)，北京，科学出版社，2002
85、Brethauer R K,Castellino F J, Review: Glycosylation of Pichia pastoris-derived proteins. bioteehnol Appl bioehem, 1999,30:193-200
86、丁云菲，刘勇，刘红岩等．分泌型乙型肝炎病毒包膜M蛋白在毕赤酵母中的表达[J]，中国生物化学与分子生物学报，2002，18(3)：365—271
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43、Golovan SP, Hayes MA,Phillips JP. et al,Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control,[J] Nature biotechn01ogy,2001, 19(5): 429-433
44、Golovan SP, Meidinger RG, Ajakaiye A, et al. Pigs expressing salivary phytase produce low-phosphorus manure, [J] Nature biotechnology ,2001, 19(8):741-745
45、王文兵，姚斌，肖庆利等．植酸酶基因在家蚕．杆状病毒表达系统中的表达及酶学特性[J]，生物工程学报，2003，19(1)：112-116
46、Lehmann M, Loch C, Middendorf A, et al. The consensus concePt for thermostability
engineering of proteins: further proof of concept. Protein Eng, 2002, 15(5):403-411
47、Kerovuo J, Tynkkynen S, Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755, Lett Appl Microbiol 2000, 30(4):325-329
48、Ha NC, Oh BC, Shin S, et al.Crystal structures of a novel, thermostable phytase in partially and fully calcium-loaded states. Nat Struct Biol 2000,7(2): 147-53
49、Maraz A. From yeast genetics to biotechnology Acta Microbiol Immunol Hung. 2002; 49(4):483-91.
50、Sudbery PE. The expression of recombinant proteins in yeasts[J], Curr Opin Biotechnol. 1996, 7(5):517-24.
51、Cregg J M, Cereghino J L, Shi J, et al. Recombinant protein expression in Pichia pastoris.Mol biotechenol, 2000, 16(1): 23-52.
52、丁云菲，刘勇，戴长柏．巴斯德毕赤酵母研究进展，[J]生命科学，2003，15(1)：26-30
53、李艳，王正祥，诸葛健．酵母作为外源基因表达系统的研究进展，生物工程学报[J]，2001，22(2)：10-14
54、韦宇拓，甘凤菊，苏华波等。巴斯德毕赤酵母新型分泌表达载体构建，广西农业生物科学[J]，2003，22(1)：37-40
55、Sunga A J, Cregg JM.The Pichia pastoris formaldehyde dehydrogenase gene (FLD1) as a marker for selection of multicopy expression strains of P. pastoris. Gene. 2004, 330:39-47
56、Soderhoim J, Bevis BJ, Giick BS. Vector for pop-in/pop-out gene replacement in Pichia pastoris. Biotechniques. 2001,31 (2):306-312.
57、Wan H, Sjolinder M, Schairer HU, et al.A new dominant selection marker for transformation of Pichia pastoris to soraphen A resistance.J Microbiol Methods. 2004,57(1):33-39.
58、Wu S, Letchworth G J. High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques. 2004, 36(1): 152-154.
59、Eiden-Plach A, Zagorc T, Heintel T, et al.Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata, Pichia pastods, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Appl Environ Microbiol. 2004,70(2): 961-966.
60、C. M. J. Sagt, B. Kleizen, R. Verwaal, et al, Introduction of an N-Glycosylation Site Increases Secretion of Heterologous Proteins in Yeasts.Appl Environ Microbiol, 2000,66(11): 44940-4944
61、聂东宋，梁宋平．李敏．外源蛋白在巴氏毕赤酵母中高效表达的策略[J]。吉首大学学报(自然科学版)，2001，22(3)：40-44．
62、Carol A S,The intracellular prodction and secretion of HIV-1 Envelope in methylotrophic yeast Pichia[J],Gene,1993, (136):111-119
63、Clare JJ,Romamos M A. Production of EGF in Yeast: High-level Secretion Using Pichia Pasoris
Strains Containing Mutiple Gene Copies.[J], Gene, 1991, 105:205-212
64、赵翔，霍兢，李育阳．毕赤酵母的密码子用法分析[J]，生物工程学报，2000，16(3)：308-311
65、Sreekrishna K, Brankamp R G, Kr0pp K E, et al.Strategy for Optimal Synthesis and secretion of Heteroguious Proteins in the Mythy Trophic Yeast Pastoris pichia, Gene 1997,191(1): 55-62
66、李欣，郭树华．影响外源基因在巴氏毕赤酵母中表达的因素[J]，生物技术通讯，2000。11(2)：132-134
67、藏秀玉，王恂，周坚．毕赤氏酵母Paox2突变化序列分析[J]．微生物学报，1999，39(6)：559—561
68、杨晟，黄鹤，章如安等．重组人血清白蛋白在Pihia pastoris中分泌表达影响因素的研究[J]，生物工程学报，2000，16(6)：675-678
69、Waterham H R, Digan M E, Koutz P J, et al. Isolation of the Pichia Pastoris Glyceraldehyde-3-phospate and Rgulation and Use of Its Promomoter [J],Gene, 1997, 186(1): 37-44.
70、Score C A, Clare JJ, MC Combie W R, et al. Rapid selection of high Copy Number transformants of pastoris pichia for high-level foreign gene expression[J]. Bio/Technology, 1994 12(2):181-184
71、Vassileva A,Chugh D A ,Swaminathan S, et al, Effect of copy number on the epression levels of hepatitis B surface antigen in the ethyiotrophic yeast Pichia pastoris [J], Protein Expr Purif, 2001, 21(1):71-80
71、Menendez J, Gania B, Hidalgo Y, New method for the selection ofmulticopy transformants of Pichia pastoris using 3-amino-1,2,4triazoi [J]. Biotechniques, 2002, 29(5): 1094-1099
73、欧阳菁，杨林，龙綮新等．猪生长激素在巴斯德毕赤酵母中高效分泌表达及产物的 N-糖基化分析[J]，生物工程学报，2001，17(5)：520-524
74、Lopes T S, Hakkaart G, Koertsb L, et al. Maehenisms of high-copy-number integration of pMIRY-Type vectors into the ribosomal DNA of Saecharomyees cerevisiae [J]. Gene, 1991, 105(1):83-90
75、唐南筠，霍競，李育阳．乳酸克鲁维酵母高拷贝整合载体的构建和应用[J]．生物化学与生物物理学报，1996，28(5)：540-545
76、Gregg J M, Tschopp J F, Stiman C, et al.High-level expression and efficient asssembly of hepetitis B surface antigen in methyltrophic yeast Pichia pastoris [J]. Bio/Technoiogy, !987,(5): 479——486.
77、Hohenblum H, Gasser B, Maurer M, et al. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris [J].Biotechnol Bioeng. 2004, 85(4):367-375
78、MEI M, WHITTAKER,JAMESW, et al. Expression of recombinant galactose oxidase by pichia
pastoris [J]. Protein Expr. Purif. 2000, 20:105-111
79、Clare JM, Udevick T S, Raschke W C, Curecent advances in the expression of foreign genes in Piehia pastoris[J],Bio/Technology. 1993,11(8): 905-910
80、Pen J,Verwoerd T C,Paridon P A Van.Phytase-containing transgenic seed as a novel feed additive for improved phosphorus utilization Bio/technol, 1993,11:811～814.
81、王红宁，马孟根，吴琦等．黑曲霉N25植酸酶phyA基因在巴斯德毕赤酵母中高效表达的研究[J]，菌物系统，2001．20(4)：486～493
82、贝锦龙，陈庄，杨林等．人工合成的黑曲霉NRRL3135菌株植酸酶基因在毕赤酵母系统中的高效表达[J]．生物工程学报，2001，17(3)：254～258
83、张若寒，植酸酶活性的检测方法[J]，中国饲料，1997，5：30-32
84、金冬雁，黎孟枫，侯云德等译．分子克隆实验指导(第二版)，北京，科学出版社，2002
85、Brethauer R K,Castellino F J, Review: Glycosylation of Pichia pastoris-derived proteins. bioteehnol Appl bioehem, 1999,30:193-200
86、丁云菲，刘勇，刘红岩等．分泌型乙型肝炎病毒包膜M蛋白在毕赤酵母中的表达[J]，中国生物化学与分子生物学报，2002，18(3)：365—271
87、Ostapehuk P, Hearing P,Ganem D,A dramatic shift in transmemberane topology of a viral envolope glycoprotein accompanies bepatitis B viral morphogenesis, EMBO J,1994,13:1048-1057
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