從水牛瘤胃微生物多源基因庫篩選新穎脂解酵素基因
Other Title
Screening of novel lipolytic genes from a metagenomic library of rumen microflora in Taiwan Water Buffalo
Journal
畜產研究
Journal Volume
45
Journal Issue
3
Start Page
265
End Page
274
ISSN
0253-9209
Date Issued
2012
Author(s)
Abstract
本研究目的在以多源基因體學方式建構水牛瘤胃多源基因庫，再以功能導向的篩選方式分離出含有脂解活性的株系。水牛瘤胃中細菌之多樣性頗為豐富，以質體系統建構由水牛瘤胃液樣品萃取DNA之基因庫，利用活性篩選的方式，直接從基因庫中篩選出具脂解活性之株系4株，經DNA定序與比對後，此4株系的插入片段具有4個完整相對應脂解酵素基因的ORFs。且此4個預測的脂解蛋白質序列經與資料庫比對分析後，發現其相同性介於27－59%，依據Arpigny & Jaeger之細菌脂解酵素分類，其中EstR1屬於第七類，EstR3比較明顯屬於第八類，EstR4可能屬於第六類，而EstR2則可能成為新的類別。依據培養盤上之功能分析，此4個預測的脂解酵素應為酯酶而非脂肪酶，又以資料庫比對分析結果顯示，其中EstR1與EstR2可能另具有聚醣分解能力。
The purpose of this study was to construct a metagenomic library from the rumen contents of a Taiwan Water Buffalo and to screen for lipolytic clones using activity based screening methods. By screening the metagenomic library of rumen microflora of Taiwan Water Buffalo, a total of 4 unique clones conferring lipolytic activities were obtained using tributyrin plate assay. DNA analysis revealed that there were 4 putative lipolytic ORFs of 4 clones. Based on the BLASTP analysis, the 4 putative lipolytic enzymes showed 27-59% identity to the closest matches in protein database. According to the classification of bacterial lipolytic enzymes proposed by Arpigny & Jaeger, EstR1 and EstR3 might belong to Family VII and Family VIII, respectively. EstR2 might be classified into Family VI, and EstR4 might represent a new family. On the base of the plate assay with different substrates of tributyrin and olive oil, the lipolytic enzymes may be recognized as esterases not lipases. Moreover, EstR1 and EstR2 might be suggested to be bifunctional enzymes with lipolytic and polysaccharide degrading activities after comparison with the protein database.
Subjects
多源基因庫
脂解酵素基因
瘤胃微生物
Lipolytic genes
Metagenomic library
Rumen microflora
水牛
Water buffalo
Type
journal article