Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement
Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement
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DOI：
10.1128/aem.70.5.3041-3046.2004
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发表时间：
2004-05-01
影响因子：
4.4
通讯作者：
Gray, KA
中科院分区：
文献类型：
--
作者：
Garrett, JB;Kretz, KA;Gray, KA
在单胃动物饲料中添加植酸酶的好处是水解难消化的植物植酸（肌醇1,2,3,4,5,6-六磷酸二氢盐），为家禽和猪提供膳食磷。理想的植酸酶补充剂应具有耐高温性，使其能够在饲料制粒过程中存活，在低 pH 下具有高比活性，以及足够的胃性能。在本研究中，通过使用基因位点饱和诱变技术优化了细菌植酸酶的性能。从大肠杆菌的 appA 基因开始，生成了一个克隆文库，该克隆文库在序列的 431 个残基中包含所有 19 个可能的氨基酸变化和 32 个可能的密码子变化，并筛选了表现出改善的耐热性的突变体。发现了 14 个单位点变体，在加热孵育方案后保留了野生型酶残余活性的 10 倍。在单个构建体 (Phy9X) 中添加八个单独的突变产生了最大适应性的蛋白质，即高活性植酸酶，在 62℃ 加热 1 小时后活性没有损失，在 85℃ 加热 10 分钟后活性为初始活性的 27%，这比 appA 亲本植酸酶有显着改进。 Phy9X 的胃稳定性也提高了 3.5 倍。
The inclusion of phytase in monogastric animal feed has the benefit of hydrolyzing indigestible plant phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) to provide poultry and swine with dietary phosphorus. An ideal phytase supplement should have a high temperature tolerance, allowing it to survive the feed pelleting process, a high specific activity at low pHs, and adequate gastric performance. For this: study, the performance of a bacterial phytase was optimized by the use of gene site saturation mutagenesis technology. Beginning with the appA gene from Escherichia coli, a library of clones incorporating all 19 possible amino acid changes and 32 possible codon variations in 431 residues of the sequence was generated and screened for mutants exhibiting improved thermal tolerance. Fourteen single site variants were discovered that retained as much as 10 times the residual activity of the wild-type enzyme after a heated incubation regimen. The addition of eight individual mutations into a single construct (Phy9X) resulted in a protein of maximal fitness, i.e., a highly active phytase with no loss of activity after heating at 62degreesC for 1 h and 27% of its initial activity after 10 min at 85degreesC, which was a significant improvement over the appA parental phytase. Phy9X also showed a 3.5-fold enhancement in gastric stability.