Aberystwyth University Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome
Aberystwyth University Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome
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发表时间：
2015
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通讯作者：
C. J. Newbold;N. Kaderbhai;S. Girdwood;O. Golyshina;P. Golyshin;N. Scollan;S. Huws
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作者：
C. J. Newbold;N. Kaderbhai;S. Girdwood;O. Golyshina;P. Golyshin;N. Scollan;S. Huws
提高肉类和牛奶中有益健康的脂肪酸含量是一项重大挑战，需要对瘤胃脂质代谢有更多的了解。在这项研究中，我们使用功能宏基因组学分离和表征了瘤胃细菌脂肪酶/酯酶。从培养的瘤胃液（SRF）、固体附着细菌（SAB）和液体相关瘤胃细菌（LAB）中提取DNA，构建宏基因组文库，连接到fosmid载体中，随后转化为大肠杆菌宿主。Fosmid图书馆包括7,744个；8448年;SRF、SAB和LAB的平均插入尺寸分别为30 ~ 35 kbp，为7680个无性系。转化体在含有三丁酸甘油酯的灵蓝琼脂板上筛选脂肪酶/酯酶活性。5个SAB克隆和4个LAB克隆表现出溶脂活性，SRF文库中未发现阳性克隆。对阳性克隆的Fosmids进行焦磷酸测序，检索到12个推测的脂肪酶/酯酶基因和2个磷脂酶基因。尽管衍生蛋白聚集在不同的酯酶和脂肪酶家族中，但在BlastP搜索后，发现了一定程度的新颖性，同源性在40%到78%之间。分离的脂肪酶/酯酶在一定温度和ph范围内对大多数短至中链底物具有活性。这些新酶在瘤胃代谢中的功能需要进一步研究，以及它们潜在的工业用途。
Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78 % following BlastP searches. Isolated lipases/esterases exhibited activity against mostly shortto medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.