Cloning And Expression Of A Laccase Gene From Bacillus Amyloliquefaciens

Laccases are the most important components of the lignolytic complex of wood-destroying responsible for decomposition of lignin. They can also break down lignin structure-like materials that are harmful to environment.As a results ,They were widely used in environment protection,delignification of paper pulp,food and biosensor industry.It has been became the new research topic. the majority of laccases are produced by white-rot-fungi,but the production is rather low,so it can hardly meet the need of industry. Bacterial laccases may perhaps display different enzyme properties and kinetics as compared with those of fungi.For instance,the glycosylation modification is not required in bacterial laccase. Most bacterial laccases are thermodynamic stable,and catalyze in extensive pH.The bacterial strain lac7 displaying laccase activity has been isolated from the soil in our laboratory.But it low yield. To resolve this problme,the gene encoding laccase in the strain lac7 has been cloned and overexpressed in E.coil host cells.The enzymatic properties have also been investigated.1. Clone of laccase gene. DNA fragement of lac7 laccase gene was obtained by PCR from the genomic DNA of Bacillus amyloliquefaciens. The result showed it has an ORF about 821bp2. The perdiction of laccase senior structure. We predicted the senior structure of laccase with the help of Bioinformatics. The secondary and tertiary structure are respectively displayed by the sofes of PSIPRED and pymol.3. The laccase was expressed in Escherichia coli : The gene encoded was inserted into the expression vector of pET21b and transformed into E.coli BL21（DE3） which was expressed successfully in Escherichia coli by inducing with IPTG. Overexpression of active enzyme encoded by the cloned gene Was achieved when the culture was induced for 4 hours at 30℃by using 0.6 mmol/L IPTG.After one step of purification by Ni2+affinity chromatography,the enzyme was purified to homogeneity.The apparent molecular weight of lac7 was about 30kDa according to SDS-PAGE.4. The study on recombinant laccase enzymatic properties The result show that at 37℃, The maximal enzyme activities were found at pH3.0 for ABTS oxidization,pH8.0 for DMP oxidization. Kinetic studies gave apparent Km value of 0.624mmol/L with a Kcat value of 1.32×10³s^-1.1 for DMP at pH8.0 and 37℃.apparent Km value of 6.01×10^-3mol/L with a Kcat value of 7.6×10³s^-1 for ABTS at pH3.0 and 37℃.It has well heat stability, it could retain 70% activity by treat up 30 minutes at80℃...