Production, Purification And Characterization Of Laccase From Paecilomyces Species

Laccases (p-diphenol: dioxygen oxidoreductase; EC 1.10.3.2) belong to the group of copper-containing polyphenol oxidases, which catalyze the oxidation of a wide range of polyphenols, aminophenols, polyamines, lignin and some inorganic ions. Laccases have attracted increasing attention in the recent years due to their potential application in diverse industrial sectors such as in food, textile and pulp industries; however, industrial applications of laccases are usually hindered by their long fermentation period and low laccase yield. Although fungal laccase has been extensively investigated for decades, the production of laccase was still performed mostly in flask scale. To improve laccase productivity, screening of new laccase producing strains for further understanding of laccase location in cells, the function of laccase and the laccase-secretion properties were necessary.In this study, a novel laccase-producing strain was isolated from soil and was identified as Paecilomyces species. An novel approach of microbial breeding was employed to improve laccase production and enzyme properties. The laccase-secretion characteristics of the mutant and the laccase fermentation control strategies were investigated. Besides, the purification and properties of laccase from Paecilomyces species were also investigated. The main results are as follows:1. A novel laccase-producing strain was isolated from a lignin-containing sample by selective plates and flask fermentation, and the newly isolated strain was identified as Paecilomyces sp. WSH-L07 according to the morphological characteristics and analysis of internal transcribed spacer (ITS) rDNA gene sequences. The optimal fermentation parameters for enhanced laccase secretion were found to be: 250 ml flasks containing 100 ml medium (initial pH 6.5) and cultivated at 30°C and 150 r/min for 8 days. Under the optimal fermentation conditions, a maximal laccase activity of 820 U/l was obtained. Moreover, detectable laccase was only found in the copper-supplemented medium, suggesting copper was essential for laccase production by Paecilomyces sp. WSH-L07. Further enhanced laccase production was achieved by complex inducers methylene blue and CuSO4. The addition of 20μmol/l methylene blue and 50μmol/l CuSO4 after cultivation for 12 and 24 h, respectively, gave the maximum laccase production with an activity of 1650 U/l.2. Low-energy ion implantation was employed to breed laccase producing strain Paecilomyces sp. WSH-L07, and a genetically stable mutant S152 was obtained with four times laccase activity compared with that of the wild strain. The optimum substrate of both the wild and mutant laccases was 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and followed by guaiacol with optimal pH at 3.4 and 5.0, respectively, while the mutant laccase exhibited a broader active pH range. The mutant laccase had a higher optimal catalytic temperature (60oC to 65oC) than the wild one (55oC), and the wild laccase deactivated rapidly when temperature increased above 55oC. Furthermore, the mutant laccase was more stable under neutral and alkaline conditions. A thermostability experiment revealed that the mutant laccase was superior to wild laccase. Both the wild and mutant laccases were almost not affected by the tested metal ions, and mildly inhibited by SDS (0.5 mmol/l), EDTA (1 mmol/l) and DTT (0.5 mmol/l). The activities were almost completely inhibited by 0.1 mmol/l NaN3. The zymogram analysis of laccase from the mutant S152 indicates that the activity profiles obtained were similar to that from the wild strain with at least three ABTS-active bands (Lac 1, Lac 2 and Lac 3) but only two guaiacol-active bands (Lac 1 and Lac 3).3. The culture medium containing maltose (20 g/l), bean peptone (10 g/l) and yeast extract (1 g/l) was found to favor laccase production by Paecilomyces sp. S152 with an enzyme activity of 5064 U/l. The zymogram analysis indicated that nutrition conditions had no effect on the types of laccase isozymes, but regulated the secretion ratio among the different isozymes. The optimal addition concentration and addition time of complex inducers were determined by orthogonal experiment and multiple linear regressions. The maximum laccase activity of 5730±150 U/l was obtained in the fermentation broth, which was supplemented with 15.4μmol/l methylene blue and 76μmol/l copper sulphate after incubation for 11 and 12 h, respectively. Methylene blue was almost completely degraded during cultivation, suggesting methylene blue could be degraded efficiently by laccase from Paecilomyces sp. S152. The increasing laccase activity under the complex-induction condition may be due to the defensive effect of microorganism, which inducing the secretion of laccase to reduce the position of methylene blue. The intra- and extra- cellular location of laccases were detected in the cells of Paecilomyces sp. S152. To improve cell permeability, Tween 80 (1.5 g/l) was added into fermentation broth and an enhanced extracellular laccase activity was detected with the shortened fermentation period (6 days) and improved (946 U/l/d).4. Based on the kinetics of laccase production under different culture temperature, a two-stage temperature-shifted strategy was developed as follows: temperature was controlled at 35oC during the first 92 h of cultivation, and then switched to 30oC. By applying the strategy, a combination of the optimum laccase-excretion periods was achieved, and a succession of qp (specific laccase formation rate) peak time was observed, and thus, the laccase production was increased by 45% and 117% compared with that of 30oC and 35oC cultivation, respectively. Also, the increased laccase productivity and laccase yield to glucose were obtained by applying the temperature-shifted strategy. The possible mechanisms for enhanced laccase activity by applying the strategy were explained as: higher temperature was more favorable to improve cell growth and cell permeability, and avoid glucose repression in early period of cultivation; while a temperature-stress by reducing incubation temperature at later period of cultivation would induce the secretion of protease, which may be involved into the activation of pro-laccase, resulted in the enhancement of laccase activity.5. Laccase isozyme Lac 1 and Lac 3 were purified from the fermentation broth of Paecilomyces sp. S152 by ethanol precipitation, anion exchange (Q-Sepharose HP) and gel filtration chromatography (Superdex 75). The purified Lac 1 and Lac 3 exhibited a high specific activity of 196.75 and 327.43 U/mg for ABTS. The purification folds of Lac 1 and Lac 3 are 19.95 and 33.21, with the recovery yield of 19 and 27.66%, respectively. The molecular weight of Lac 1 and Lac 3 are both 79 kDa. Both enzymes were absence of blue copper absorption spectrum and exhibited yellow color, which were different from the wildly existed blue laccases and similar to several yellow laccases from other fungi. The value of Km and Kcat/Km on ABTS and guaiacol revealed that the substrate affinity and catalytic rate of Lac 3 were superior to that of Lac 1. Furthermore, a protease was purified from the fermentation broth of Paecilomyces sp. S152 and identified as serine protease which could be strongly inhibited by serine protease inhibitor PMSF. In the cell-free system, the activation of pro-laccase was inhibited in the presence of PMSF; addition of 1 mmol/l PMSF during fermentation resulted in a significant decrease of both protease and laccase activities. The results above suggested that the presence of protease could promote the activity of laccase and, while inhibition of protease activity would not favor for activation of pro-laccase.