真菌嵌合体植酸酶的构建及其抗蛋白酶水解特性研究

 在烟曲霉植酸酶（Afp）的基因上，通过PCR逐步将编码1-47、178-237及338-381多肽序列的DNA片段置换为黑曲霉NRRL 3135植酸酶（Anp）中的对应片段，构建了3个嵌合体（嵌合体Ⅰ、Ⅱ和Ⅲ）基因，并在毕赤酵母中将其成功表达及纯化。所有的嵌合体和亲本植酸酶均能抵抗胃蛋白酶。在m(胰蛋白酶)/m(植酸酶)为0.1时，Anp完全失活；但Afp与所有嵌合体的活性仅损失13%~23%。胰蛋白酶水解产物的SDS-PAGE结果显示嵌合体Ⅱ与Ⅲ也能被胰蛋白酶严重水解，但非变性PAGE结果却表明它们在水解后仍能保持结构完整。嵌合体也具有一些与Anp及Afp相异的新pH曲线及耐热性。实验表明，通过替换基因片段改良真菌植酸酶可行。

Construction of Chimerical Fungal Phytase and its Resistance to Proteolysis

Chimera Ⅰ,Ⅱ and Ⅲ were created by stepwise substitutions of DNA fragments encoding region 1-47, 178-237 and 338-381 from Aspergillus niger NRRL 3135 phytase (Anp) gene for their counterparts in Aspergillus fumigatus phytase (Afp) gene, expressed in Pichia pastoris, and then purified. All chimera and parent phytases resisted to pepsin. Afp and all chimeras lost 13%~23% phytase activities at m(trypsin)/m(phytase)ratio of 0.1, while Anp was thoroughly inactivated. However, SDS-PAGE of trypsin digestion results suggested serious proteolysis of Chimera Ⅱ and Ⅲ. But nondenaturing PAGE analysis showed they still kept structural intact in nature state after trypsin treatment. Chimera phtases also possessed novel pH profiles and thermo stability different from Anp and Afp. These results demonstrated the feasibility of improving fungal phytase via gene fragment repalcement.