论文标题:植酸酶产生菌的选育及高表达工程菌的构建研究
Study on the Screening of Phytase Producing Strain and the Construction of High Phytase Producing Engineered Strains
论文作者 彭远义
论文导师 周泽扬,论文学位 博士,论文专业 特种经济动物饲养
论文单位 西南农业大学,点击次数 142,论文页数 108页File Size6637k
2004-05-15论文网 http://www.lw23.com/lunwen_77960367/ 植酸酶;黑曲霉N14;诱变;phyA基因;毕赤酵母
phytase,Aspergillus niger N14,mutation,phyA gene,Pichia pastoris
植酸酶是一类能催化植酸及植酸盐水解成肌醇和无机磷酸盐的酶，它具有解除植物性饲料(或食品)中植酸的抗营养作用、提高机体对蛋白质及多种微量元素的利用率、促进生长发育、提高动物生产性能、减少粪便中磷的排放量、降低磷对环境的污染等多种功能，因而受到国内外的广泛关注。植酸酶主要存在于植物和微生物中，其中以微生物产生的植酸酶具有良好的开发前景，但微生物天然菌株产酶水平较低，加上天然植酸酶的某些性质不能完全适合饲料加工和养殖业的要求，从而限制了植酸酶在生产中的推广应用。通过植酸酶产生菌的筛选和基因克隆，并利用分子生物学技术构建基因工程菌，或在分子水平上改造植酸酶基因，以提高植酸酶产量或改变植酸酶酶学性质，从而降低生产成本，提高其使用有效性，己成为世界性的研究热点之一。 本研究的目的旨在通过从土壤中筛选酸性植酸酶高产菌株，并进一步通过诱变选育提高其产量，对高产突变菌株植酸酶基因进行克隆和分析，在此基础上，构建植酸酶高产基因工程菌，从而为植酸酶产品的工业化生产奠定基础。主要结果如下： 1 植酸酶高产菌株的筛选 利用植酸钙选择性子板培养基从土样中筛选出102株酸性植酸酶产生菌，从中挑选出透明圈较大的菌株32株，经分离纯化后分别进行摇瓶复筛，28℃、220r/min发酵5天后，在37℃、pH2.5或pH5.5条件下用钒钼酸铵法检测其酶活，结果发现有3株菌产酶活性较高且产酶性能较为稳定。其中编号为03214号的菌株在37℃、pH2.5时酶活性最高，达345U/mL发酵液；编号为041和0324号的菌株在37℃、pH5.5时酶活性最高，分别达285U/mL和250U/mL发酵液。根据产酶活性情况，选取03214号菌株作进一步鉴定和诱变选育的出发菌株。经初步鉴定，03214号菌为黑曲霉，编号为Aspergillus niger03214。 2 植酸酶高产菌株黑曲霉N14的诱变选育 以Aspergillus niger03214为出发菌株，通过紫外线诱变处理不同时间，经平板初筛和摇瓶复筛，获得一株产酶活性较高的突变株03214-3，酶活为373U/mL发酵液，比出发菌株提高了8％左右。以突变株03214-3为出发菌，经0.8mg/mL的亚硝基胍诱变处理不同时间，最终获得了产酶活性较原始菌株Aspergillus niger03214提高了22.3％，达422.3U/mL发酵液的突变菌株，编号为Aspergillus nigerN14。 将黑曲霉N14在PDA斜面上连续传7代，分别测定各代菌株酶活，结果其酶活稳定在410～430U/mL发酵液之间，说明突变株黑曲霉N14具有良好的遗传稳定性，可作为下一步基因工程研究的出发菌株。 3 黑曲霉N14基因组DNA的提取及植酸酶phyA基因的克隆，，画.‘，，妇..圈.翻‘通~ 以A，e啥1111”nigerN14为出发菌，采用改良氯化节法和斑迹抽提法提取基因组DNA，经紫外分光光度计测得DNA的ODZ曰心D翔均在1.80左右，表明这两种方法均适合提取黑曲霉基因组DNA。但在所用菌丝体重量相同的条件下，以氯化节法提取的pNA浓度较高。 根据曲霉菌属植酸酶基因具有高度同源性的特点，结合已报道植酸酶神州基因序列，自行设计一对特异性引物(上游引物:5，月队GGG叼℃八TGGGCG子巳n刃口，;下游引物:5，CAGC以六GCA阳心咙火CTCCGC3，)，采用乃仰七。t DNA聚合酶(高保真DNA聚合酶)，通过PCR方法从黑曲霉N14基因组DNA中扩增出了预期的约1.skb的特异性产物，将其与pMD18.T载体连接后，转化大肠杆菌Dll5Q，.经质粒抽提、五之口月111、tl双酶切鉴定和PcR鉴定，确认该目的基因已得到成功克隆。4黑曲霉N14植酸酶夕句以基因序列分析 DNA序列测定表明，本试验所克隆的目的基因片段含有植酸酶户州基因的完整序列(GeneBank AcceSSIOn:AY4269”)，夕句讨基因全长15仅无p，其中包含一段长102bp的内含子序列，编码467个氨基酸，5’端有拼编码19个氨基酸的信号肤序列。植酸酶活性位点序列CRvTEAQvLsRHGA即n红DsKCK位于氨基酸序列的+71~+93。黑曲霉N14植酸酶夕勺讨基因中G+仁含量达54.7%，密码子第三位碱基的G+C含量高达67%。黑曲霉N14植酸酶助州基因与报道的产植酸酶活性最高的天然黑曲霉标准株N刊心乃135(来源于孟，己心iuus niger介uum夕va:a~ori)的尸句“基因(GeneBankA以尤SSion:M94550)及A.nigerN25PhyA基因(GeneBankA。戈SSion:AF21如13)、A.nigervar~夕句“基因(枷eBank Aa笼ssfon:ID2421)、A.niger5K-57夕hJ讨基因(G闭eBaDk八口戈SSion:川叨227(X))同源性分别为%.7%、%.8%、%.4%和91.7%，而编码的氨基酸序列同源性分别为98%、97.8%、%.7%和95.7%。进一步采用公pa叮IProtParam等软件对黑曲霉N14植酸酶夕句“基因编码氨基酸序列及编码的蛋白质二级结构预测进行分析，结果表明，黑曲霉Nl助h)“基因所编码氨基酸序列存在10个潜在的N-糖基化位点，与黑曲霉NRRU 135和黑曲霉N25的p勺讨基因所编码氨基酸序列上的N-糖基化位点个数及位置完全相同，而糖基化对微生物表达的植酸酶的生物合成和热稳定性至关重要;理论PI为4.95，分子量为5 n42.0，分子式为q6eH蝴3Ns990，消。;负电荷氨基酸残基 (As尹Glu)有51个，正电荷氨基酸残基(A犯+切s)有34个。实验结?
Phytases catalyze the hydrolysis of phytic acid or phytate(myo-inositol hexakisphosphate) to inositol and inorganic phosphate. Their roles in eliminating anti-nutritional effects of phytate in plant fodder or foods, in increasing the efficiencies of protein and various microelement utilizations in the animal body and the animal productivities, in reducing the amount of phosphorus in animal excretions and thus reducing environmental pollutions, etc, have been the research focuses domestically and internationally. Phytases are found mainly in plants and microorgnisms. Although microbial phytases have great developmental potentials, its producing ability in natural microbial strains is limited, impeding its further utilization as phytase-producers. In addition, some unfavorable properties of phytases such as heat instabilities can"t completely meet the requirements of fodder processing and rearing industries. All these could be improved by means of gene engineering. Through the screening of phytase-producing microbial strains, cloning and modifying of phytase gene and constructing engineered strains by molecular biology methods, it is possible to increase the production of phytases, modify its properties, reduce costs and increase its efficiencies. Presently it"s one of the research hot points the worldwide.This investigation aims to screen out the high-yield acidic phytase-producing microbial strains and men further to select for high yield strains by mutagenesis and still then to clone and analyze the phytase gene in the high-yield phytase-producing mutant strains. On these bases, to construct high yield phytase gene engineered microbial strains for the industrialized production of phytase.Main results are as follows. 1. The screening of high yield phytase producing microbial strains.In total 102 strains of acidic phytase producing strains were selected from soil by selective plate containing calcium phytate. Among them 32 strains with relatively large clear circle were purified and re-selected by shaking-culturing. After fermented for 5days at 28 C and shaking at 220r/min, the activity of phytase was determined by NH4VO4 -(NH4)6Mo7O24 method at 37 C and pH2.5 or pH5.5. Results show that all together three strains with relatively high production and stable phytase were selected. Strain 03214 showed the highest enzyme activity at 37 C and pH2.5, reaching 345U/mLfermentation. Strain 041 and 0324 showed the highest enzyme activity at 37"C and pH5.5, reaching 285U/mLand 250U/mLfermentation respectively. Strain 03214 was used for further characterization and as starting strains for selection. Preliminary result show that strain 03214 was Aspergillus niger, designated Aspergillus niger 03214.2.Selection of high yield phytase producing strain Aspergillus niger N14 by complex mutagenesisThe starting Aspergillus niger 03214 was induced with UV treatment at various time periods and then plating screened and shaking-culture re-screened. A high phytase activity mutant strain 03214-3 was obtained with 373U/mL fermentation, up by about 8% compared with the starting strain. And then the mutant strain 03214-3 was induced with N-methyl-N-nitro-N-nitrosoguanidine (0.8mg/mL) at various time periods; and finally a mutant strain, designated Aspergillus nigerN14, with increased phytase activity, 22.3% more than the original strain Aspergillus niger03214, reaching 422.3345U/mL fermentation, was obtained.Aspergillus nigerN14 was sub-cultured on PDA medium for 7 generations, and the activity of each generation was monitored respectively. Results show that the activity was stabilizes somewhere between 410~430U/mLfermentation, suggesting good genetic stability of the mutant Aspergillus niger N14, which could be used for further gene engineering investigation. 3. Extraction of genomic DNA and cloning of the gene phyA from Aspergillus nigerN14 The genomic DNA of the mutant strain Aspergillus nigerN14 was extracted by benzyl chloride and trace method. UV spectrophotometer determination of the preparations show the ra
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