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首页> 外文期刊>Applied and Environmental Microbiology >Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide
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Characteristics of a New Enantioselective Thermostable Dipeptidase from Brevibacillus borstelensis BCS-1 and Its Application to Synthesis of a d-Amino-Acid-Containing Dipeptide

机译：博氏短杆菌BCS-1对映体选择性热稳定的新型二肽酶的特性及其在合成含d-氨基酸的二肽中的应用

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A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an Mr of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (kcat/Km) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 ± 2.2 [E = (kcat/Km)l,d/(kcat/Km)d,d], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 ± 0.5 [E = (kcat/Km)l,d/(kcat/Km)l,l] at 55°C. The enzyme was stable up to 55°C, and the optimal pH and temperature were 8.5 and 65°C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.
机译：通过在含有d-Ala-d的平板上对d-Glu营养缺陷型大肠杆菌 WM335进行遗传互补，从嗜热性芽孢杆菌BCS-1中克隆了一个新的热稳定的二肽酶基因。 -胶核苷酸序列分析显示该基因包含一个编码307个氨基酸序列的开放阅读框，该序列的 M r 为35,000。推导的二肽酶氨基酸序列与单核细胞增生李斯特菌的二肽酶具有52％的相似性。将该酶从重组E中纯化至同质。大肠杆菌 WM335，带有来自 B的二肽酶基因。 borstelensis BCS-1。对Ala-Ala的P 1 和P 1 '位的对映选择性（ E ）的研究表明，特异性常数之比（ k cat / K m ）对P 1 位点的l对映选择性丙氨酸的丙氨酸为23.4±2.2 [ E =（ k cat / K m ） l，d /（ k cat / K m ）< sub> d，d ]，而对Ala-Ala P 1 '位的d对映选择性为16.4±0.5 [ E =（ k cat / K m ） l，d /（ k cat / K m ） l，l ]在55°C。该酶在高达55°C的温度下稳定，最佳pH和温度分别为8.5和65°C。该酶能够水解l-Asp-d-Ala，l-Asp-d-AlaOMe， Z -d-Ala-d-AlaOBzl和 Z -l -Asp-d-AlaOBzl，但是它不能水解d-Ala-1-Asp，d-Ala-1-Ala，d-AlaNH 2 和1-AlaNH 2。该酶还表现出类似于人肾二肽酶的β-内酰胺酶活性。二肽酶成功合成了二肽甜味剂 Z -1-Asp-d-AlaOBzl的前体。

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