Screening Of Bacterial Laccase Strain And Cloning Of Laccase Gene Fragments

Laccase, capable of oxidizing a wide range of aromatic compounds, is a type of multicopper polyphenol oxidases.The enzyme plays an important role in the biodegradating of lignin and formatting of humus in composting. Recently, laccase is widely applied in organic syntheses, bio-detection, industrtial wastewater treatment and so on.In this experiment, a bacterial strain with laccase activity was isolated from the dunghill samples. The physicochemical properties of this srain were studied. In addition, the laccase gene has been cloned by constructing a genomic library. The detail results as followed.1. Laccase activity of L-1 strain was 32.3U/L respectively.2. By Biolog test and 16S rDNA sequence analysis, the L-1 strain was identified as Pseudomonas sp., it's a gram-negative bacteria.3. The optimum temperature of L-1 strain is 27℃, and the optimum pH is 7.0. Additionally, its growth pH range was 6.0 to 8.0. It confers salt-tolerance of 2%, besides it can resist Penicillin.4. In this experiment, we constructed the genomic library of the bacteria, with the E.coli JM109 as the receptor strain, and pGEM-3Z as the vector. We got the genomic library with the insert fragments among 2~7kb, and the capacity is 104. A recombinant bacterial strain with laccase activity was screened.Then sequenced and analysed, the results showed that the full-length insert fragment composed 2458bp, including an integrant open reading frame which is 1193bp at length, composed 397 amino acid, with 13-amino-acid signal petide. The ORF was 5' integrity, 3' missed, and its expected promoter sequence was located at position -50 to -100. The sequence fragment has been submitted to the GenBank, and the accession number is EF067317.5. The laccase activity of recombinant strain is 20.4 U/L.At present, there is no report about the laccase activity of Pseudomonas sp., this is the first time that the gene encoding an active laccase has been cloned from Pseudomona sp.