铜绿假单胞菌PAO1中三个γ-谷氨酰转肽酶的鉴定

IF 0.8 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Journal of General and Applied Microbiology Pub Date : 2023-12-05 Epub Date: 2023-01-18 DOI:10.2323/jgam.2023.01.001
Yuuki Nonomura, Xinjia Wang, Takeshi Kikuchi, Daisuke Matsui, Yosuke Toyotake, Kazuyoshi Takagi, Mamoru Wakayama
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引用次数: 1

摘要

铜绿假单胞菌菌株PAO1具有三个假定的γ-谷氨酰转肽酶(GGT)基因:ggtI、ggtII和ggtIII。本研究分析了这些基因在P. aeruginosa PAO1中的表达,并研究了相应的GGT蛋白的特性。本文首次报道了假单胞菌GGT相似物的生化特性。铜绿假单胞菌PAO1粗提物水解和转肽酶活性分别为17.3和65.0 mU/mg,并通过RT-PCR证实了各基因对mRNA的转录。克隆所有基因,将构建的表达质粒导入大肠杆菌表达系统。系统中未检测到ggtI表达蛋白(PaGGTI)的酶活性,而检测到ggtII和ggtIII衍生蛋白(分别为PaGGTII和PaGGTIII)的酶活性。然而,PaGGTII酶活性很低,很容易降低。具有c端his-tag的PaGGTII (PaGGTII25aa)的活性和稳定性增强,纯化后的酶由40 kDa的大亚基和28 kDa的小亚基组成。PaGGTIII由37 kDa的大亚基和24 kDa的小亚基组成。PaGGTII25aa的水解和转肽酶活性在40ºC-50ºC时达到最大值,PaGGTIII的水解和转肽酶活性在50ºC-60ºC时达到最大值。这些酶在50ºC孵育10分钟后仍保持约80%的水解和转肽酶活性,反映出良好的稳定性。PaGGTII25aa和PaGGTIII在碱性条件下的水解和转肽酶活性均高于酸性条件下。然而,它们在较宽的pH范围内(5-10.5)具有很高的稳定性。
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Characterization of three γ-glutamyltranspeptidases from Pseudomonas aeruginosa PAO1.

The Pseudomonas aeruginosa strain, PAO1, has three putative γ-glutamyltranspeptidase (GGT) genes: ggtI, ggtII, and ggtIII. In this study, the expression of each of these genes in P. aeruginosa PAO1 was analyzed, and the properties of the corresponding GGT proteins were investigated. This is the first report on biochemical characterization of GGT paralogs from Pseudomonas species. The crude extracts prepared from P. aeruginosa PAO1 exhibited hydrolysis and transpeptidation activities of 17.3 and 65.0 mU/mg, respectively, and the transcription of each gene to mRNA was confirmed by RT-PCR. All genes were cloned, and the expression plasmids constructed were introduced into an Escherichia coli expression system. Enzyme activity of the expressed protein of ggtI (PaGGTI) was not detected in the system, while the enzyme activities of the expressed proteins derived from ggtII and ggtIII (PaGGTII and PaGGTIII, respectively) were detected. However, the enzyme activity of PaGGTII was very low and easily decreased. PaGGTII with C-terminal his-tag (PaGGTII25aa) showed increased activity and stability, and the purified enzyme consisted of a large subunit of 40 kDa and a small subunit of 28 kDa. PaGGTIII consisted of a large subunit of 37 kDa and a small subunit of 24 kDa. The maximum hydrolysis and transpeptidation activities of PaGGTII25aa were obtained at 40ºC-50ºC, and the maximum hydrolysis and transpeptidation activities of PaGGTIII were obtained at 50ºC-60ºC. These enzymes retained approximately 80% of their hydrolysis and transpeptidation activities after incubation at 50ºC for 10 min, reflecting good stability. Both PaGGTII25aa and PaGGTIII showed higher activities of hydrolysis and transpeptidation in the alkali range than in the acidic range. However, they were highly stable at a wide pH range (5-10.5).

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来源期刊
Journal of General and Applied Microbiology
Journal of General and Applied Microbiology 生物-生物工程与应用微生物
CiteScore
2.40
自引率
0.00%
发文量
42
审稿时长
6-12 weeks
期刊介绍: JGAM is going to publish scientific reports containing novel and significant microbiological findings, which are mainly devoted to the following categories: Antibiotics and Secondary Metabolites; Biotechnology and Metabolic Engineering; Developmental Microbiology; Environmental Microbiology and Bioremediation; Enzymology; Eukaryotic Microbiology; Evolution and Phylogenetics; Genome Integrity and Plasticity; Microalgae and Photosynthesis; Microbiology for Food; Molecular Genetics; Physiology and Cell Surface; Synthetic and Systems Microbiology.
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