The gene encoding dipeptidyl aminopeptidase BI from Pseudomonas sp WO24:: cloning, sequencing and expression in Escherichia coli
The gene encoding dipeptidyl aminopeptidase BI from Pseudomonas sp WO24:: cloning, sequencing and expression in Escherichia coli
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DOI：
10.1016/s0378-1119(97)00590-8
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发表时间：
1998-01-12
期刊：
影响因子：
3.5
通讯作者：
Morikawa, Y
中科院分区：
文献类型：
--
作者：
Ogasawara, W;Kobayashi, G;Morikawa, Y
用显色底物酶平板法从假单胞菌WO 24染色体DNA质粒文库中分离到二肽氨基肽酶BI（DAP BI）基因。将命名为dap bl的DAP BI基因进一步亚克隆并测序。序列分析的一个近似。3-kb片段的开放阅读框为2169个核苷酸，经N-末端和内部氨基酸序列分析，该片段属于dap b1基因。含有丝氨酸蛋白酶Gly-X-Ser-X-Gly共有基序的DAP BI的预测氨基酸序列显示出与属于脯氨酰寡肽酶家族的几种蛋白酶的广泛同源性，脯氨酰寡肽酶家族是一种新的丝氨酸蛋白酶家族，其具有催化三联体，具有特定的Ser、Asp和His序列，这是包括DAP IV在内的该家族成员的标志。在大肠杆菌中表达dap bl基因，表达的酶纯化约230倍，无细胞提取物的回收率为2.6%。该酶的分子量、底物特异性和抑制剂的作用等性质与假单胞菌WO 24的天然酶相似。(C)1998年Elsevier Science B.V.
We have isolated the dipeptidyl aminopeptidase BI (DAP BI) gene from the plasmid library of Pseudomonas sp. WO24 chromosomal DNA by the enzymatic plate asaay using a chromogenic substrate. The DAP BI gene, designated dap bl, was further subcloned and sequenced. Sequence analysis of an approx. 3-kb fragment revealed an open reading frame of 2169 nucleotides, which was assigned to the dap bl gene by N-terminal and internal amino acid sequences. The predicted amino acid sequence of DAP BI containing a serine protease Gly-X-Ser-X-Gly consensus motif displays extensive homologies to the several proteases belonging to the prolyl oligopeptidase family, a novel serine protease family possessing the catalytic triad with a specific array of Ser, Asp and His in this order, which is the hallmark of the member of this family including DAP IV. The dap bl gene was expressed in Escherichia coli and the expressed enzyme was purified about 230-fold with 2.6% recovery from the cell-free extracts. The enzymatic properties such as molecular mass, substrate specificity and effect of inhibitor were similar to the native enzyme from Pseudomonas sp. WO24. (C) 1998 Elsevier Science B.V.