Acera Felipe, Carmona María Isabel, Castillo Francisco, Quesada Alberto, Blasco Rafael
Departamento de Bioquímica, Biología Molecular y Genética, Facultad de Veterinaria, Universidad de Extremadura, Cáceres, Spain.
INBio G+C Research Institute, Universidad de Extremadura, Cáceres, Spain.
Appl Environ Microbiol. 2017 Apr 17;83(9). doi: 10.1128/AEM.00089-17. Print 2017 May 1.
CECT 5344 is a bacterium able to assimilate cyanide as a sole nitrogen source. Under this growth condition, a 3-cyanoalanine nitrilase enzymatic activity was induced. This activity was encoded by , one of the four nitrilase genes detected in the genome of this bacterium, and its expression in enabled the recombinant strain to fully assimilate 3-cyanoalanine. CECT 5344 showed a weak growth level with 3-cyanoalanine as the N source, unless KCN was also added. Moreover, a knockout mutant of CECT 5344 became severely impaired in its ability to grow with 3-cyanoalanine and cyanide as nitrogen sources. The native enzyme expressed in was purified up to electrophoretic homogeneity and biochemically characterized. Nit4 seems to be specific for 3-cyanoalanine, and the amount of ammonium derived from the enzymatic activity doubled in the presence of exogenously added asparaginase activity, which demonstrated that the Nit4 enzyme had both 3-cyanoalanine nitrilase and hydratase activities. The gene is located downstream of the cyanide resistance transcriptional unit containing genes, whose expression levels are under the positive control of cyanide. Real-time PCR experiments revealed that expression was also positively regulated by cyanide in both minimal and LB media. These results suggest that this gene cluster including and could be involved both in cyanide resistance and in its assimilation by CECT 5344. Cyanide is a highly toxic molecule present in some industrial wastes due to its application in several manufacturing processes, such as gold mining and the electroplating industry. The biodegradation of cyanide from contaminated wastes could be an attractive alternative to physicochemical treatment. CECT 5344 is a bacterial strain able to assimilate cyanide under alkaline conditions, thus avoiding its volatilization as HCN. This paper describes and characterizes an enzyme (Nit4) induced by cyanide that is probably involved in cyanide assimilation. The biochemical characterization of Nit4 provides a segment for building a cyanide assimilation pathway in This information could be useful for understanding, and hopefully improving, the mechanisms involved in bacterial cyanide biodegradation and its application in the treatment of cyanide-containing wastes.
CECT 5344是一种能够将氰化物作为唯一氮源进行同化的细菌。在这种生长条件下，诱导出了一种3-氰基丙氨酸腈水解酶的酶活性。这种活性由该细菌基因组中检测到的四个腈水解酶基因之一的 编码，其在 中的表达使重组菌株能够完全同化3-氰基丙氨酸。CECT 5344以3-氰基丙氨酸作为氮源时生长水平较弱，除非同时添加KCN。此外，CECT 5344的 基因敲除突变体在以3-氰基丙氨酸和氰化物作为氮源生长的能力上严重受损。在 中表达的天然酶被纯化至电泳纯，并进行了生化特性分析。Nit4似乎对3-氰基丙氨酸具有特异性，在外源添加天冬酰胺酶活性的情况下，酶活性产生的铵量增加了一倍，这表明Nit4酶同时具有3-氰基丙氨酸腈水解酶和水化酶活性。 基因位于包含 基因的抗氰转录单元的下游，其表达水平受氰化物的正调控。实时PCR实验表明，在基本培养基和LB培养基中， 基因的表达也受氰化物的正调控。这些结果表明，包括 和 的这个基因簇可能既参与了CECT 5344的抗氰作用，也参与了其对氰化物的同化作用。氰化物是一种剧毒分子，由于其在一些制造过程中的应用，如金矿开采和电镀行业，存在于一些工业废物中。从受污染废物中生物降解氰化物可能是物理化学处理的一种有吸引力的替代方法。CECT 5344是一种能够在碱性条件下同化氰化物的细菌菌株，从而避免其以HCN形式挥发。本文描述并表征了一种由氰化物诱导的可能参与氰化物同化的酶（Nit4）。Nit4的生化特性为在 中构建氰化物同化途径提供了一个片段。这些信息可能有助于理解并有望改进细菌氰化物生物降解所涉及的机制及其在含氰废物处理中的应用。
