Efficient Production Of Thermostable Bacteria Laccase And Its Application In Pulp Biobleaching

Pulp bleaching is an essential step of industrial paper production. The residual lignin in pulp needs to be cleared by a multi-step bleaching process to whiten the ?nal paper products. Biobleaching, a novel pulp bleaching technology that uses enzymes instead of polluting chlorine-based chemicals, is now a research hotspot as enzymatic treatment of pulp can provide an environmentally-friendly bleaching strategy.Thermostability and resistance ability to a variety of physical and chemical inactivation factors are critical for enzymes applied in industrial catalysis. The use of thermostable enzymes in industrial bleaching processes can offer many biotechnological advantages, e.g., better robustness, higher enzyme reaction rates at the elevated temperatures that can lead to better bleaching effects, shorter process time, and even the less consumption of enzymes. Until now, research mainly focused on enzymes of fungal origins, the applications of prokaryotic thermophilic enzymes in pulp bleaching sector are still very limited. Searching enzymes with application potential from extreme thermophilic bacteria provide new ideas and may open up a direction that bring breakthrough in application of enzymatic bleaching technology in pulp industry sector. Thermophilic bacteria Thermus thermophilus and hyperthermophilic bacteria Thermotoga maritima are important sources of valuable thermophilic enzymes. The content of this study are:1. A simple selective codon optimization strategy was devised to quickly improve the expression of a hyperthermophilic laccase from Thermus thermophilus HB27(named as Tth-laccase) in E. coli using p Hsh expression vector. After the region A(from amino acid 2(Gln) to amino acid 84(Val)) and region B(GC-rich area; from amino acid 278(Gly) to amino acid 303(Pro)) of Tth-laccase coding sequence were codon optimized through site-directed mutagenesis, laccase activity in the cell extract was successfully increased from 20.7±1.5 U/L to 1790.2±81.6 U/L after heat-shock induced expression, the enzyme activity was elevated by 86-fold. This is the first report on over expression of Tth-laccase in Escherichia coli by using p Hsh expression system.2. Cu2+ is founded to be critical to thermostability of recombinant Tth-laccase.Taking advantage of remarkable thermostability of holoenzyme, a simple and quick protein purification procedure including heat treatment of cell supernatant at 85°C and hydrophobic interaction chromatography resulted in the purified protein of 14U/mg with a yield of 62%.3. The recombinant laccase from Thermus thermophilus was applied to the biobleaching of wheat straw pulp. The best bleaching effect was when the pulp was treated with 3 U laccase g-1 dry pulp at 90°C, p H 4.5, 8% consistency for 1.5 h. Under these conditions, the pulp brightness was increased by 3.3% ISO, and the pulp kappa number was decreased by 5.6 point. Enzymatic treatment improved the bleachability of wheat straw pulp but caused no damage to the pulp ?bers. The use of enzyme-treated pulp saved 25% H2O2 consumption in subsequent peroxide bleaching without decreasing the ?nal brightness. Pulp biobleaching in the presence of 5 mmol/L 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate)(ABTS) further increased the pulp brightness by 1.5% ISO. This is the ?rst report on the application of laccase from T. thermophilus in the pulp and paper sector.4. Recombinant laccase from the hyperthermophilic bacteria Thermus thermophilus HB27 and recombinant xylanase from hyperthermophilic bacteria Thermotoga maritima MSB8(named as Xyn B) were applied jointly to biobleaching of wheat straw pulp. The best bleaching effect was boosted when the pulp was treated with xylanase at the optimized condition(15 U xylanase g-1 dry pulp at 90°C, p H 5.8, 8% consistency for 2 h) preceded the laccase bleaching stage(3 U laccase g-1 dry pulp at 90°C, p H 4.5, 8% consistency for 1.5 h.), Under these conditions, the brightness of bleached pulp was increased by 11.5% ISO, and the kappa number of pulp was decreased by 6.9. Enzymatic treatment improved the bleachability of wheat straw pulp but caused no damage to the pulp fibers. The use of enzyme-treated pulp saved nearly 50% H2O2 consumption in subsequent peroxide bleaching without decreasing the final brightness.