Screening Of Laccase-producing Strains And Expression Of Laccase Gene Fragments

The main component of straw is cellulose, hemicellulose and lignin, and the most important issue for straw utilizing is degradation of lignin at the outside of the cellulose and hemicellulose. Laccase (EC 1.10.3.2) could react to various substrates, and it could catalyze phenolic compounds, aromatic compounds and lignin. It has very broad application prospects, including environmental remediation, Food Industry, degradation of lignin and other areas. Microbial fermentation is used to produce laccase, but the periods of fermentation is too long and poor stability. In this paper, the laccase gene was cloned and expressed for increasing activity and stability, the results are as follows:Sixteen laccase-producing bacteria strains and six laccase-producing fungus strains were isolated from the samples. The strain with high laccase activity observed, then the laccase-producing strains QM11 and Z6 were finally confirmed, in which the activity of laccase were 4.36 U/L and 62.78 U/mL, respectively. The QM11 strain was identified as Bacillus subtilis based on the results of morphological, physiological characteristics and the homological analysis of 16S rDNA sequence. The Z6 strain was identified as Trametes versicolor based on the results of morphological and the homological analysis of ITS-rDNA sequence.CotA gene and lacl gene was cloned from QM11 and Z6 genome DNA by specific primers. The cotA gene sequence was 1542 bp, coding 513 amino acids, molecular weight was 58 kDa with an isoelectric point of 5.91. The lacl gene sequence was 1560 bp, coding 519 amino acids, molecular weight was 56 kDa with an isoelectric point of 5.87.The lac1 gene was inserted into the multiple cloning site of pPIC9k, so the recombinant plasmid pPIC9K-lac1 was used to transform P. pastoris GS115. After induction with methanol for 72 h, the recombinant P. pastoris GS115/pPIC9K-lac1 expressed active lac1 protein. The maximum activity of laccase reached 5.44 U/L.The cotA gene was inserted into the multiple cloning site of pET22b, so the recombinant plasmid pET22b-cotA was used to transform E.coli BL21. After induction with IPTG at 16 ?, the recombinant E.coli BL21/pET22b-cotA expressed active cotA protein. The molecular weights of the lacl protein was about 70 kDa by SDS-PAGE.The recombinant cotA protein was purified by nickel ion affinity chromatography. The specific activity of recombinant cotA protein was 136.73 U/mg, the purification fold was 7.83 and recovery was 40.29%. The properties of the recombinant cotA protein were characterized. The results showed that the optimum pH and temperature of recombinant cotA protein was 3.0 and 70 ?, respectively. The recombinant cotA protein was quite stable at the temperature from 60? to 80 ?, when it was treated at 80 ? for 1 h, the enzyme activity still remained above 40% and quite stable at the pH from 2 to 4, when it was treated at pH 2 for 1 h, the enzyme activity still remained above 70%. In addition, the influence of metal ions on recombinant cotA protein was investgated. The enzyme activity could be activated by Fe3+ and Cu2+, they were improved by 2.7 times and 4.5 times, respectively. However, Fe2+ could inhibit its activity. Kinetic studies gave apparent Km value of 0.48 mmol·L-1 with a Vmax value of 0.448 mmol·L-1·min-1 for ABTS.