Fermentation, Purification And Characterization Of Laccase From Paraconiothyrium Variabile GHJ-4

Laccase is a type of multi copper-containing polyphenol oxidases capable of oxidizing a wide range of polyphenols and other lignin-derived aromatic compounds. Laccases can degrade lignin and phenoxy containing herbicide and waste water in petroleum industry and affect toxic phenolic materials; they can thus be widely used in paper pulp industry, food industry, medication and sanitation, biosensor and envir-onment protection. Hence, this study t a strain of high laccase producing microorganism was obtained from my laboratory. It was termed as Paraconiothyrium variabile GHJ-4 that was be called for GHJ-4 short. The fermentation medium and fermentantion conditions were statistical optimized. Lastly purification and characterization of laccase was operated from GHJ-4. The main contents were as follows.1. Through the cultivation in shake-flask fermentation, the effects of environmental conditions and nutrition on production of laccase by GHJ-4.Refering to the effects of nutrition on production of laccase, the optimum carbon, nitrogen, C/N and inorganic salt were confirmed by one-factor at a time and orthogonal test experiment. Firstly, we screened the important parameters by Plackett-Burman design. It showed that three factors playing the important roles in the medium, including soluble starch, beef extract and CuSO₄. After steepest ascent experiment approaching the optimal level of the three factors, the center composite design and response surface analysis were applied to establish the second-order equation model for the yield of laccase. On the last step, the optimal concentrations of components were determined by central composite design and response surface analysis.The optimal medium for GHJ-4 was composed of (in g/L): wheat bran 30, soluble starch 58.08, beef extract 3.39, NaNO₃ 1, KH₂PO4 3, CuSO₄ 0.554.Based on the optimal medium, statistical analysis methods were applied to optimize the fermentation conditions. Firstly, Plackett-Burman design was used to investigate the main factors affecting laccase yield of GHJ-4. The results showed that four factors playing the important roles in the fermentation conditions, including inoculation volume, culture time, broth content and rotating speed. After steepest ascent experiment approaching the optimal level of the four factors, central composite design and response surface analysis were applied to establish the second-order equation model for the yield of laccase. On the last step, the optimal fermentation conditions were determined by central composite design and response surface analysis.The optimal fermentation conditions were obtained as following: 6.43% of inoculation, 28℃of culture temperature, initial pH value 4, 182 h of culture time, 72.5 mL of broth content, 157 r/min of rotating speed, and 96 h of seed age. Under this optimum conditions, the activity of laccase increased nearly 16.4 times to 710.44 U/mL and the experimental values argreed with the predicted values. These results suggested that the predicted model was reliable and available for the optimization of laccase fermentation conditions.2. Purification and characterization of laccase from GHJ-4.The laccase produced by strain GHJ-4 in liquid-state fermentation was isolated and purified by a three-step process: ammonium sulfate precipitation, DEAE-sepharose Fast Flow aion-exchange chromatography, Sephadex G-75 gel penetration chromatography. After the three-step purification, we obtained a electrophoresis pure laccase, its purity and yield were 8.1 folds and 24.2% of the crude extract respectively. The molecular mass of the laccase was about 53 kDa.The laccase was stable within the range of pH between 3.5 and 6.5 and at the temperature lower than 55℃with the maximal activity at 50℃, pH 5.0. Laccase could be markedly activated by Cu²⁺, Mg²⁺ and Zn²⁺ while be inhibited by Hg²⁺, Fe³⁺ and Ag⁺. The apparent Km of guajacolum was 62.07μmol/L, the Vmax was 68.97 mmol/(L·min).