High Throughput Screening And Cloning Of Laccase Gene Fragments From The Pesticide-contaminated Soil

Laccase(laccase, ECl. 10.3.2) is a muti-copperoxidasewhich oxidizes phenolic substrates such as para-phenylene diamine, hydroquinone, polyphenols and ascorbic acid accompanied byreducing O2 to water. It plays vital role in lignindegradation and humification process. Laccase has been widely applied in various areas, mainly concentrated in the following aspects: organic compounds synthesis, biological sensor detection, the degradation of nonsteroidal anti-inflammatory drugs, treatment of industrial wastewater, biological bleach of pulp,food industry and the biological energy battery.The pesticide-contaminated soil samples were collected from a primary aerobic pool inHubei QiChun pesticide plant. In order to analysis of the diversity of laccase in the soil sample, metagenomic DNA of soil was extracted, the degenerate primers was designed and the fragment of laccase gene(about 140bp) was amplified by PCR amplification. The result of sequencing was indicated that 61 different laccase gene fragments were obtained among 66 clones containing 140 bp laccase gene fragment. It proved that the soil sample showed abundantdiversity of laccase.Then,the soil metagenomic fosmid library was constructed.More than 6000 clones were obtained in the library.Six different laccase genes(Lac2A3, Lac2E6, Lac13H9, Lac13B22, Lac15A5, Lac15P1) were screened out from the library.The signal peptides of six laccase geneswere analysized by the Signal P 4.1 Server software. The resultindicatedthat the laccase gene Lac2A3, Lac13H9 and Lac15A5 contained 36, 17 and 30 amino acids, respectively.All six laccase genes without signal peptideswereinserted into vector pET-22 b and pET-30 to construct recombinant expression vectors. It was found that only recombinant laccaseLac13H9 can be expressed from pET22b-Lac13H9. Analysis of laccase genelac13H9 showed that it has a total length of 1389-bp and encoded 462-amino acids polypeptidewith a calculated molecularmass of 50 kDa.Basedon analysis by Signal P 4.1, Lac13H9 containeda 17 amino acid residue signal peptide. The deduced amino acid sequence wasaligned with available protein sequences held in theGenBank. It showed 67% and 61%, similarity with laccases fromMyxococcusstipitatus and Sorangiumcellulosum, respectively.The recombinant lac13H9 heterologously expressed in Escherichia coli Transseta(DE3) and was purified by the affinity chromatography on a Ni-NTA column. Maximal activity of laccase was observed at a temperature of 40℃ and pH 6.0. It has good thermal stability. About 60% enzyme activity remained after being held for 90 min under 50℃. It was found that low concentration of Fe2+ can promote laccase enzyme activity, otherwise a high concentration inhibiting enzyme activity.The enzymatic properties of Lac13H9 are good for its application.