Jones B V, Sun F, Marchesi J R
Alimentary Pharmabiotic Centre, Department of Microbiology, University College Cork, Cork, Ireland.
Lett Appl Microbiol. 2007 Oct;45(4):418-20. doi: 10.1111/j.1472-765X.2007.02202.x.
Functional screens using skimmed-milk agar to obtain protease activity is a common approach. The aim of this study was to determine the efficacy of this screen to obtain protease activity from a metagenomic library.
A distal gut metagenomic library was functionally screened using a skimmed-milk agar. The functional screen provided 231 clones generating the characteristic clear halo indicative of protease production. Clone analysis revealed that they were not protease-positive, but expressed glycosidic hydrolases and produced acid, which was responsible for the clear halos.
The current skimmed-milk agar method to obtain proteases is not sufficiently robust to provide a definitive screen. Other- non-protease activities will also give the same clear halo and these would be interpreted as protease positive clones without further analysis. Hence a more robust buffered medium or a specific protein should be used.
Functional screens are a powerful approach to obtaining enzymes from large metagenomic libraries and proteases are a particularly interesting target. The skimmed-milk agar is not sufficiently robust to ensure that only proteases are isolated and in order to save time and money this study has shown that better designed media can aid in the process.
使用脱脂乳琼脂进行功能筛选以获得蛋白酶活性是一种常用方法。本研究的目的是确定这种筛选方法从宏基因组文库中获得蛋白酶活性的有效性。
使用脱脂乳琼脂对远端肠道宏基因组文库进行功能筛选。功能筛选得到了231个产生特征性透明晕圈的克隆，这表明有蛋白酶产生。克隆分析表明它们并非蛋白酶阳性，而是表达糖苷水解酶并产生酸，正是酸导致了透明晕圈的出现。
当前使用脱脂乳琼脂获取蛋白酶的方法不够可靠，无法提供确定性的筛选。其他非蛋白酶活性也会产生相同的透明晕圈，而这些在未经进一步分析的情况下会被解读为蛋白酶阳性克隆。因此，应使用更可靠的缓冲培养基或特定蛋白质。
功能筛选是从大型宏基因组文库中获取酶的一种有效方法，蛋白酶是一个特别有趣的目标。脱脂乳琼脂不够可靠，无法确保仅分离出蛋白酶，为节省时间和金钱，本研究表明设计更好的培养基有助于这一过程。
