Study On Laccase Activity And Mn²⁺-oxidizing Activity Of The Multicopper Oxidases CumA And CopA From Pesudomonas

Bacterial laccases have advantageous properties compared to eukaryotic laccases,such as gene sequences are simple and don't have exon-intron structure of eukaryotic laccase genes,enzyme proteins also don't have glycosylation of eukaryotic laccase,and so on.Meanwhile,some bacterial laccases are highly active with wide substrate specificities and more stable at elevated temperatures and high pH conditions.All of these make bacterial laccases could be an interesting biocatalyst in applications for which classical laccases are unsuitable.In our study,we cloned two multiplecopper oxidases?MCOs?genes cumA593 and cop A from Pseudomonas sp.593.Each genes was expressed in E.coli BL21?DE3?pLysS and their corresponding products were purified to homogeneity through Co-affinity chromatography.The purified two MCOs were characterized about their laccase activity.The optimum pH and temperatures of CumA593 towards laccase substrates 2,6-dimethoxyphenol?DMP?,syringaldazine?SGZ?and 2,2'-Azino-bis?3-ethylbenzthiazoline-6-sulfonic acid??ABTS?were at pH 5.0,55?;pH7.5,60?;and pH 5.0,60 ?,respectively.Effect of pH on enzyme stability was also investigated and relative activity of the CumA593 remained 60%activity at pH 9.5 after 24 h.In contrast,when the CumA593 was incubated in the pH 3.0 buffer for 12 h,no enzyme activity was detected.These data suggested that the CumA593 was more stable in weak alkaline pH than in acidic pH.The thermal stability of the CumA593 was not good.When the CumA593 was incubated at 60?for 2 h,the enzyme only remained 10%relative activity.Cu²⁺ remarkably enhanced the enzyme activity of the CumA593.By contrast,the enzyme activity of the CumA593 was inhibited by addition of Fe²⁺.Kinetic studies of CumA593 gave the Km,Vmax and kcat values of 4.38×10-4 mol/L,1.187×10-6 mol·L-1·min-1 and 0.056 s-1 for DMP;1.714×10-5 mOl/L,0.705×10-6mol·L-1·min-1 and 0.03 s-1 for SGZ and 1.06×10-4 mol/L,2.807×10-6mol·L-1·Kmin-1 and 0.393 s-1 for ABTS.The amino acid sequences of the CopA displayed an identity of 21.75%with the CumA593,and the protein structures and laccase activity of the two MCOs were also different.The optimum pH and temperatures of the CopA towards laccase substrates DMP,SGZ and ABTS were at pH 7.5,50?;pH7.5,42?;and pH 3.5,50?,respectively.Effect of pH on enzyme stability was also investigated and the CopA was more stable in neutral pH.The thermal stability of the CopA was not good.When the CopA was incubated at 60? for 0.5 h,the enzyme only remained 40%relative activity,and continued to save for 3 h at 60 ?,the enzyme activity could not been detected.Cu²⁺ remarkably enhanced the enzyme activity of the CopA,and the CopA displayed weak laccase activity when the other divalent metal ions were separately added or without any divalent metal ions.Kinetic studies of CopA gave the Km,Vmax and kcat values of 1.41×10-4 mol/L,4.54?10-6mol·L-1·min-1 and 2.2 s-1 for DMP;0.25×10-4 mol/L,0.7×10-6 mol·L-1·min-1 and 0.87 s-1 for SGZ and 2.81×10-4 mol/L,3.02×10-6mol·L-1·min-1 and 1.8 s-1 for ABTS.In order to investigate whether the CumA itself could oxidize manganese?II?or not.We cloned separately two MCOs genes cumA593 and cumA27853 from Pseudomonas sp.593 with Mn²⁺-oxidizing activity and Pseudomonas aeruginosa 27853 without Mn²⁺-oxidizing activity.Each genes was expressed in E.coli BL21?DE3?pLysS and their corresponding products were purified to homogeneity through Co-affinity chromatography.Both CumA27853 and CumA593 displayed Mn²⁺-oxidizing activity in vitro,no matter whether the host bacteria could oxidize manganese?II?or not.The purified two MCOs were characterized about their Mn²⁺-oxidizing activity.Although the amino acid sequences of the CumA27853 displayed an identity of 81%with the CumA593,both the MCOs were the same enzyme characteristics.The optimum pH of the CumA27853 and CumA593 were pH7.5,the enzyme activities were relatively better in 30-37 ?,and Cu²⁺ remarkably enhanced the enzyme activity.Kinetic studies gave the Km,Vmax and kcat values of 27.27 ?mol/L,0.124?xmol·L-1·min-1 and 0.016 s-1 for CumA27853 and 42.75 ?mol/L,0.153 ?mol·L-1·min-1 and 0.035 s-1 for CumA593.