Gene Cloning, Heterologous Expression, Protein Purification And Enzyme Properties Of Ochrobactrum Sp.531Laccase

Laccase (EC1.10.3.2) is a member of the muticopper oxidase family which widely exists in nature. It catalyzes the oxidization reaction of a category of phenol and non-phenolic compounds into the benzoquinone and the reduce reaction of an oxygen molecule into the water, accompanying with four electronic transfer. Laccases were mainly found in plants and fungus, however, they are also found recently in prokaryotic bacteria and insects. The bacterial strain Ochrobactrum sp.531displaying laccase activity has been isolated from the forest soil in our laboratory. Due to its low yield, the enzyme can’t be purified and studied. To resolve this issue, the gene encoding laccase in the strain Ochrobactrum sp.531was cloned and expressed in E. coil host cells in this study. The enzymatic properties and kinetic parameters were also investigated. This work sets up a good platform for improving the enzyme activity and stability via protein engineering. In order to fermentate bacteria laccase in a large scale, we have tried to use the yeast expression system to express Ochrobactrum sp.531laccase, and indeed realized the secretary expression in Pichia. pasteur.According to the conserved amine acids of Cu2-binding site, the degenerate primers designed specifically were used to amplify the partial laccase gene from Ochrobactrum sp.531genomic DNA. Searched by blastP in GenBank with a target of partial laccase gene, we found a laccase gene in Ochrobactrum anthropi genome DNA, Sequencing analysis indicated the DNA segment amplified came probably from Ochrobactrum, coincide with previous result of the16S DNA molecule identification. Based on10amino acids of the amino and carboxyl termini of Ochrobactrum and Brucella laccases, the rostrocaudal degenerate primers was designed and an intact ORF of1602bp was finally coloned. The coloned gene encodes533amino acids with a molecular mass of57,812dalton and a pl value of5.58. The speculated protein contains10.51%Ala,9.76%Gly,8.82%Leu and7.88%Asp, which are richer than other amino acids. SingalP predicted that30amino acids of the amino terminus are a signal peptide. The gene cloned was inserted into the expression vector pET23a, and then expressed in E.coli BL21(DE3) plysS. One step of purification by Ni2+-affinity chromatography was used to purify the target enzyme, and the enzyme was purified to homogeneity.8.0mg pure enzyme was obtained from1L bacterial culture. DMP, ABTS and SGZ were used as substrates for Ochrobactrum sp.531laccase. At37℃, maximal enzyme activities were obtained at pH8.0for DMP, pH3.6for ABTS and pH7.5for SGZ. The enzyme was found stable in a wide range of acidic and alkaline pHs, especially pH6.5. As compared to pH6.5, the enzyme remains about60%activity after incubation at pH3or pH9.5for16hours. As compared with that at4℃, the enzyme remains about40%activity after incubation at80℃for30minites,or65℃for1hour, or50℃for2.5hours. Kinetic studies gave apparent Km value of8.98×10-5mol/L with a kcat value of7.94s-1for DMP at pH8.0and37℃7.22×10-5mol/L with a kcat value of2.95s-1for ABTS at pH3.6and37℃, and1.49×10-5mol/L with a kcat value of2.40s-1for SGZ at pH7.5and37℃. Obviously, the largest kcat/Km value for SGZ (1.61×105mol-1L s-1) suggests that the enzyme catalyzes SGZ-oxidation more effectively than those for other two substrates DMP (8.84×104mol-1L s-1) and ABTS (4.09×104mol-1L s-1).Under the optimal conditions. Enzyme expression of the cloned gene in a soluble form was realized when the culture was induced at20by0.5mmol/L IPTG. for7hours. We also found that the maltase binding protein (MBP) was able to markedly improve the solubility of Ochrobactrum sp.531laccase, but the culture additives sorbitol, betaine and glycylglycine didn’t have apparent effect on the soluble yields.The gene with or without DNA region encoding singal peptide was inserted into the yeast expression vector pHBM905A, the linearized DNA with Sail was then electrotransformed into Pichia. Pasteur GS115. Two recombinant colonies with the high activities were screened by the measureing the enzyme activity in yeast culture. After optimization of the induced conditions by the orthogonal design, the highest enzyme activity reaches3,300U/L when cultivated at20℃and pH6.8, and induced by0.8%methanol for4days.