Study On The Purification And Characters For Extracellular Laccse From Lenzites Tricolor

This paper researched the characters of crude extracellular laccase from Lenzetis tricolor at first. We described the orthogonal experiment to study the effects of carbon resource, nitrogen resource, lignin-like substances and initial pH value on the production of extracellular laccase by Lenzetis tricolor. Then we validated the effect of different concentration of carbon resource and nitrogen resource on laccase activity. Finally, the optimum producing laccase medium was (g/L) : starch 20, beef peptone 2, Na₂HPO₄.12H₂O 0.47, KH₂PO₄ 0.45, MgSO₄.7H₂O 0.5, CaCl₂ 0.01,, MnSO₄.4H₂O 0.001, FeSO₄.7H₂O 0.001, ZnSO₄.7H₂O 0.001, CuSO₄.5H₂O 0.001, guaiacol 0.062, VB₁ 50μg and pH 5.0. On the basis of above results, we investigated the change of the laccase activity, the dry weight of mycelium, the residual sugar content and the pH value respectively by different cultivation means. We obtained also the best cultivation means was seed and fermentation liquid were grown by static means.The laccase was purified by two series of methods. The one set means were composed of these aspects which are (NH₄)₂SO₄ salting-out, dialyzing, polyethylene glycol 20000 (PEG 20000) concentrating then, SephadexG-150 gel chromatotography (1.6x60) and EDAE-SephadexA25 ion exchange chromatotography (2.6x40) . We obtained that purification multiple was 9.36 and the activity reclamation rate was 8.6% by this purification method. Another group method included following parts also, which embody (NH₄)₂SO₄ salting-out, dialyzing, ultrafiltration concentrating then DEAE-Scpharose FF ion exchange chromatotography (1.6x50) and then SephadexG-75 gel chromatotography (1.6x50) . The second method presented the results that purification multiple was 18.54 and the activity reclamation rate was 37.17%. The second purification method is more scientific than the first one to compare the curve of 280 nm absorption value and laccase avtivity during the chromatotography course.The reaction process curve showed that this laccase accord the dynamical character of enzyme catalytic reaction. Following purification, we mensurated some dynamical parameter of this laccase with syringaldazine (3,5- dimethoxy-4-hydroxybenzaldehyde azine) substrate. The optimum reaction temperature of crude laccase is 40 degrees and the purified laccase is 60 degrees. The optimum reaction pH value of crude laccase is 6.0, while the purified laccase is 4.6. Metal ion has a very important influence over laccase. This paper mainly involved that sodium and barium ion have no obvious effect, bivalent and trivalence iron ion restrain entirely laccase activity, while calcium promote evidently the enzyme reaction. Within a certain concentration rang of inhibitors, the test result is unexpected. EDTA and EDTA.Na₂ advanced absolutely the laccase reaction. The advantageous effect is the strongest when the concentrate of EDTA reached 10 mmol/L and the activity is 9.78 times in contrast to control. Thiourea, sodium azide and DTT have definite stimulative function in the sufficientlow concentrate, but they restrain the laccase reaction under the upper concentrate. The Michaelis constant is 26.62yUmol/Lthat this laccase oxidize syringaldazine.