Studies On Laccase Liquid Fermentation By Shiraia Sp. SUPER-H168

Laccase （EC 1.10.3.2）, a kind of polyphenol oxidase, can be widely used in wine clarification, kraft bleaching, dye decolorization, wasterwater treatment and biosensor, and thus has been recognized as an important and industrial value enzyme. Therefore, it is important to screen unique and high-yield laccase producing strains and study their fermentation for laccase production.In this study, a new laccase producing strain, Shiraia sp. SUPER-H168, was used to produce laccase. The medium and fermentation conditions for laccase production were optimized. The optimal medium composition was as follows: potato 200 g/L, soluble starch 20 g/L, yeast extract 8 g/L, and copper ion 2.4 mmol/L. With the optimum medium, the activity of laccase could reach to 120 U/mL at initial pH 5.0, liquid volume 50 mL/250 mL, 30℃, and 200 r/min, shaking for 64 h. It was 17 times higher than that of non-optimized conditions.The purification methods of Shiraia sp. SUPER-H168 laccase were studied. The laccase was purified by ammonia sulfate precipitation, anion exchange chromatography on DEAE.FF and gel filtration on Superdex G-200. The purification factor and recovery of laccase activity were 10.20 and 19.08%, respectively.Enzymatic properties of Shiraia sp. SUPER-H168 laccase were studied. By using DMP as the substrate, the optimum temperature and pH for laccase activity were 60℃and 3.5, respectively. For the substrate ABTS, the optimum temperature and pH for laccase activity were 55℃and 2.5, respectively. The purified laccase showed Km values of 11.06 and 0.496 mmol/L, Vmax values of 1227 and 1866 U/mg, and Kcat values of 1411.05 and 2145.9 S-1, when the DMP and ABTS were respectively utilized as substrate.The effect of metal ions and several chemical compounds on the activity of Shiraia sp. SUPER-H168 laccase was investigated. Cu²⁺, Mg²⁺, Mn²⁺, Zn²⁺, K+, Ca²⁺, Ba²⁺ and EDTA had no obvious effection on laccase activity; Cd²⁺, Ni²⁺ and Co²⁺ showed some inhibition and Ag+（10 mmol/L）, Fe²⁺（5 mmol/L）, Fe³⁺（10 mmol/L）, SDS and NaN3（5 mmol/L） strongly inhibited the activity of laccase.The decolorization of the anthraquinone dyes and azo dyes with Shiraia sp. SUPER-H168 laccase was studied. Laccase could decolorize the anthraquinone dyes directly without adding small molecular mediators. After 240 min, the decolorization rate could reach 49% at the optimal temperature of 40℃. However, the small molecular mediator （HOBT） should be added for decolorization of the azo dyes by this laccase. The decolorization rate could reach 60.50% at the optimal temperature of 50℃after 140 min treatment.