【作者】 伍洛夫；

【导师】 周俊初；

【作者基本信息】 华中农业大学 ， 微生物学， 2005， 硕士

【摘要】 植酸及其盐类是植物性食品和饲料中磷的主要贮存方式,植酸酶是催化植酸及植酸盐水解成低磷酸化的肌醇衍生物和磷酸(或磷酸盐)的一类酶的总称。在饲料中添加植酸酶不但可以提高动物对磷的吸收,降低环境中磷的污染,而且能解除植酸对金属离子、氨基酸的螯合作用,大大提高饲料的利用效率,因而在国内外饲料工业中有着广泛的应用前景。 由于微生物野生菌株的产酶水平较低,制约了微生物植酸酶在生产上的广泛应用,因此提高菌株植酸酶的产酶水平是实现其产业化应用的关键。本研究的目的是克隆植酸酶基因并实现其在酵母和大肠杆菌中的异源表达。主要实验结果如下: 1.参照已发表的植酸酶基因phyA全序列设计一对引物,用PCR方法从土曲霉(Aspergillus terreus)总DNA中扩增到去除信号肽和内含子后约1.4 kb的phyA基因编码区片段。对该片段进行了亚克隆与序列测定。进一步用该基因构建了pPIC9K-phyA分泌型表达载体,并转化到毕赤酵母(Pichia pastoris)GS115。以重组酵母总DNA为模板进行PCR扩增以及采用含有植酸钙的选择性培养基的初步检测结果表明:植酸酶基因(phyA)在毕赤酵母中得到了表达。重组酵母摇瓶诱导发酵132 h发酵液中的植酸酶活性可达167 u/mL;表达植酸酶在pH 2.0～8.0均有活性,最适pH为5.5,最适温度为55℃。 2.参照已发表B.subtilsi的植酸酶基因序列设计了一对引物。采用PCR法,获得不含有信号肽序列的植酸酶基因phyC的非融合表达片段,并构建成带有T7启动子的大肠杆菌植酸酶基因表达载体pET17b—phyC,将其转入大肠杆菌BL21(DE3)后,以重组菌株总DNA为模板进行PCR扩增以及采用含有植酸钙的选择性培养基的验证试验结果表明:植酸酶基因phyC在重组大肠杆菌中得到了表达,表达产物具有正常的植酸酶生物活性。更多还原

【Abstract】 Phytate and phytic acid are the major phosphorus storage form contained in food or feeds of plant origin. Phytases are myo-inositol hexakisphosphate phosphohydrolase that catalyze the stepwise removal of inorganic orthophosphate from phytate. Addition of phytase into feed can result in the following advantages: increasing the absorptive efficiency of phosphate in animal body, decreasing the phosphate pollution in environment, eliminating the chelate reaction between phytate and metal ion so that phytase can improve the utilization efficiency of the feed. Hence the study on phytase, especially phytase produced by microorganism, has been paid much attention by scientists in China and abroad.Phytase production on a large scale are restricted by low level of phytase gene expression in wild strains. The objective of this study is to clone phytase gene from wild strains and then obtain its expression in heterogeneous yeast and escherichia strains. The main results of this study are as the following:1. About 1.4kb phyA fragment absent signal sequence and intron of Aspergillus terreus was amplified by PCR with primers designed according to published phyA sequences. It was further cloned and sequenced. The recombinant expression plasmid pPIC9K-phyA was constructed and transformed into Pichia pastoris GS115. The phytase gene was expressed in Pichia pastoris GS115 detected by PCR and hydrolytic zone on a solid phytate-containing medium . The enzyme activity in supernatant from flask fermentation after 132 hours induction reached to 167U/ml. Its enzyme activity was active under pH 2.0-8.0 and up to the the highest under pH5.5.The optimum temperature for enzyme activity was 55 ℃.2. A pair of primers were designed based on the phyC sequence of Bacillus stubilis. The phyC structure gene encoding phytase but without signal peptide sequences was obtained by polymerase chain reaction. The non-fusion fragment amplified was subcloned into a expression vector pET-17b with T7 promoter and then transformed into E. coli BL21(DE3). The phyC gene expression was detected by PCR and the hydrolytic zone observed on a solid phytate-containing medium.更多还原