Gene cloning, identification, and characterization of the multicopper oxidase CumA from Pseudomonas sp. 593

Abstract

The cumA, a gene encoding a multicopper oxidase (MCO), was cloned from the soil-dwelling bacterium Pseudomonas sp. 593. Its corresponding product was overexpressed in Escherichia coli BL21 (DE3) pLysS and purified to homogeneity through Ni-affinity chromatography. The amino acid sequence of the CumA of Pseudomonas sp. 593 was strongly homologous to that of CumA as previously reported. The CumA was quite stable in neutral pH and had poor thermostability. Meanwhile, its optimum pH and temperature toward laccase substrates 2,6-dimethoxyphenol (DMP), syringaldazine (SGZ), and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) were 5.0 and 55 °C, 7.5 and 60 °C, and 5.0 and 60 °C, respectively. Cu²⁺ remarkably enhanced the activity of the CumA. By contrast, the activity of the CumA was inhibited by the addition of Fe²⁺ . Kinetic studies gave the K_m , k_cat , and k_cat /K_m values of 0.438 mmol·L^-1 , 0.056 Sec^-1 , and 0.128 Sec^-1 ·mmol^-1 ·L for DMP; 0.017 mmol·L^-1 , 0.031 Sec^-1 , and 1.824 Sec^-1 ·mmol^-1 ·L for SGZ; and 0.101 mmol·L^-1 , 0.393 Sec^-1 , and 3.891 Sec^-1 ·mmol^-1 ·L for ABTS. To our knowledge, this is the first report of cloning, expressing in E. coli of the cumA from Pseudomonas and characterization of the CumA.

Keywords: Pseudomonas sp. 593; bacterial laccase; enzyme kinetics; multicopper oxidase; protein structure.