UNLABELLED 6-Chloro-2-benzoxazolinone (CDHB) is a precursor of herbicide, insecticide, and fungicide synthesis and has a broad spectrum of biological activity. Pigmentiphaga sp. strain DL-8 can transform CDHB into 2-amino-5-chlorophenol (2A5CP), which it then utilizes as a carbon source for growth. The CDHB hydrolase (CbaA) was purified from strain DL-8, which can also hydrolyze 2-benzoxazolinone (BOA), 5-chloro-2-BOA, and benzamide. The specific activity of purified CbaA was 5,900 U · mg protein(-1) for CDHB, with Km and kcat values of 0.29 mM and 8,500 s(-1), respectively. The optimal pH for purified CbaA was 9.0, the highest activity was observed at 55°C, and the inactive metal-free enzyme could be reactivated by Mg(2+), Ni(2+), Ca(2+), or Zn(2+) Based on the results obtained for the CbaA peptide mass fingerprinting and draft genome sequence of strain DL-8, cbaA (encoding 339 amino acids) was cloned and expressed in Escherichia coli BL21(DE3). CbaA shared 18 to 21% identity with some metal-dependent hydrolases of the PF01499 family and contained the signature metal-binding motif Q127XXXQ131XD133XXXH137 The conserved amino acid residues His288 and Glu301 served as the proton donor and acceptor. E. coli BL21(DE3-pET-cbaA) resting cells could transform 0.2 mM CDHB into 2A5CP. The mutant strain DL-8ΔcbaA lost the ability to degrade CDHB but retained the ability to degrade 2A5CP, consistent with strain DL-8. These results indicated that cbaA was the key gene responsible for CDHB degradation by strain DL-8. IMPORTANCE 2-Benzoxazolinone (BOA) derivatives are widely used as synthetic intermediates and are also an important group of allelochemicals acting in response to tissue damage or pathogen attack in gramineous plants. However, the degradation mechanism of BOA derivatives by microorganisms is not clear. In the present study, we reported the identification of CbaA and metabolic pathway responsible for the degradation of CDHB in Pigmentiphaga sp. DL-8. This will provide microorganism and gene resources for the bioremediation of the environmental pollution caused by BOA derivatives.

中文翻译：

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Pigmentiphaga sp。参与的代谢途径涉及6-氯-2-苯并恶唑啉酮的降解。菌株DL-8和新型金属依赖性水解酶CbaA的鉴定。

UNLABELLED 6-氯-2-苯并恶唑啉酮（CDHB）是除草剂，杀虫剂和杀真菌剂合成的前体，具有广泛的生物活性。Pigmentiphaga sp。菌株DL-8可以将CDHB转化为2-氨基-5-氯苯酚（2A5CP），然后将其用作生长的碳源。从菌株DL-8纯化CDHB水解酶（CbaA），该菌株还可以水解2-苯并恶唑啉酮（BOA），5-氯-2-BOA和苯甲酰胺。纯化的CbaA的CDHB比活性为5,900 U·mg蛋白（-1），Km和kcat值分别为0.29 mM和8,500 s（-1）。纯化的CbaA的最佳pH为9.0，在55°C时观察到最高活性，并且无活性的无金属酶可以通过Mg（2 +），Ni（2 +），Ca（2+），或Zn（2+）基于获得的DL-8菌株CbaA肽质量指纹图谱和基因组草图序列的结果，克隆了cbaA（编码339个氨基酸）并在大肠杆菌BL21（DE3）中表达。CbaA与PF01499家族的一些金属依赖性水解酶具有18至21％的同一性，并包含签名性金属结合基序Q127XXXQ131XD133XXXH137。保守的氨基酸残基His288和Glu301用作质子供体和受体。大肠杆菌BL21（DE3-pET-cbaA）静息细胞可将0.2 mM CDHB转化为2A5CP。突变菌株DL-8ΔcbaA丧失了降解CDHB的能力，但保留了降解2A5CP的能力，与菌株DL-8一致。这些结果表明cbaA是负责菌株DL-8降解CDHB的关键基因。重要事项2-苯并恶唑啉酮（BOA）衍生物被广泛用作合成中间体，并且也是对禾本科植物的组织损伤或病原体侵袭作出反应的重要的化感物质。但是，微生物对BOA衍生物的降解机理尚不清楚。在本研究中，我们报道了负责色素沉着菌中CDHB降解的CbaA和代谢途径的鉴定。DL-8。这将为BOA衍生物引起的环境污染的生物修复提供微生物和基因资源。我们报道了鉴定CbaA和代谢途径的作用，该代谢途径可降解Pigmentiphaga sp。中的CDHB。DL-8。这将为BOA衍生物引起的环境污染的生物修复提供微生物和基因资源。我们报道了鉴定CbaA和代谢途径的作用，该代谢途径可降解Pigmentiphaga sp。中的CDHB。DL-8。这将为BOA衍生物引起的环境污染的生物修复提供微生物和基因资源。