| Laccases(benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are multicopper oxidases. Laccase is able to oxidize a wide variety of substrates, including phenols, arylamines, carboxylic acids, steroid hormones, biochromes, and so on. Laccase has applications of biodegradation of lignin and pulp and paper industry, but also potential applied value of wastewater treatment, enzyme bleaching, environmental pollutants degradation, soil bioremediation, biological monitoring, food processing and organic synthesis.We have recently obtained a laccase gene lac1338 by codon optimization synthesis. The laccase gene was induced high-level expression in Escherichia coli, and the enzymatic properties and the environmental pollutants degradation of the laccase were studied. Moreover, the enzyme protein was reformed by directed evolution technology. The main findings were as follows:This laccase gene was designated lac1338, which encoded 445 amino acid peptides with the full length of 1338 bp. Its predicted molecular mass is 50 k Da.The new laccase gene lac1338 was amplified via PCR, and then was ligated into expression vector p ET-32a(+), and transformed into E. coli BL21(DE3). The transformants was incubated at 30 °C for 16 h, with 0.2 mmol/L IPTG and 0.5 mmol/L Cu SO4 of the final concentration added to induce target protein expression. The highest expression level of lac1338 was about 450 mg/L, which was one of highest expression level of laccase gene reported. The molecular mass of recombinant Lac1338 purified was 67 k Da by SDS-PAGE analysis(including an N-terminal fusion of 156 amino acids, about 18 k Da).With ABTS as a substrate, the optimal temperature for the enzyme was 55 °C, and the enzyme was stable(70% for 120 min) under 50 °C(towards ABTS). The optimal p H for the enzyme was 6.0, the enzyme was stable(50% for 240 min) under(towards ABTS). In total, Lac1338 has excellent p H and temperature stability. For ABTS, Km value and Vmax value were 567μmol/L and 2.8 mmol/(min·g protein), respectively.The degradation of recombinant Lac1338 to industry dyes was also studied. With addtion of Ca2+, Lac1338 enhanced its degradation rate effectively; Moreover, with Ca2+ and ABTS added, Lac1338 could decolourize congo red and indigo carmine more than 95%, bromophenol blue 20%, Coomassie Brilliant Blue more than 20%, Acid Violet 7 10%.Because enzymatic activity of Lac1338 was lower, lac1338 gene was reformed by directed evolution technology. Random mutations were introduced through two rounds of error-prone PCR, and mutants were screened by assaying their activities towards the substrate ABTS. Among them, a mutant(Lac2-9) with the highest activity showed four mutations(D232V、V281A、P309L、S318G). Compared with the wild type, the specific activity of the mutant enzyme increased to 3.5 folds(79.8 U/mg, towards ABTS) of that of the wild type. Moreover, the mutant Lac2-9 showed maximum activity at 60 °C, higher than that of wild type to 5 °C, and the p H value for optimal activity was increased to 6.5 from 6.0(towards ABTS). Lac2-9 showed better temperature stability, the relative activity raised from 30% to 50% under 55 °C for 240 min, but p H stability was not improved. In addition, the degradation ability of Lac1338 and its mutant enzyme for several industrial dyes were studied, and the results showed that the activity of mutant enzyme Lac2-9 towards acid violet 7, bromophrnol blue, coomassie brilliant blue and amaranth was increased to 90.5%, 67.8%, 85% and 14.5% from 10.9%, 20%, 25% and 13.7%. The results showed that error-prone PCR is an effective method to increase enzymatic activity and degradation ability to industrial pollutants. The statistical optimization by response surface methodology resulted in 22.22% increase in the production of laccase, up to 550 mg/L. The constructed mutant laccase Lac2-9 is an appropriate candidate for biotechnological applications due to its high expression level and high activity in decolorization of industrial dyes. |
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