Study On Improvement Of Laccase Production And Treatment Of Papermaking Wastewater By Inter-kingdom Protoplast Fusion

Papermaking industry is one of the most important industries in our country. With therapid development of papermaking industry, papermaking wastewater has become one of themain pollution sources of water environment. At present, many scholars study on treatment ofpapermaking wastewater by white rot fungi. But there are some shortcomings such as lowproduction of laccase, slow growth of the fungi and so on. It is not suitable for industrializedproduction. Protoplast fusion technology has broken the boundaries between microbialspecies, and it can realize the distant hybridization. Protoplast fusion makes gene transfermore perfect and increase the frequency of breeding and gene recombinstion rate. Protoplastfusion technology opens up a new way to build genetic engineering bacteria to treat industrialwastewater efficiently.Pleurotus ostreatus and Escherichia coli were the objects of study in this paper. Theconstruction of high-yield laccase-producing fusants was achieved by inter-kingdomprotoplast fusion. Fermentation efficiency, laccase activity and the rules of degradation ofpapermaking wastewater were studied. It is very important to solve the problems ofpapermaking wastewater treatment in China.The optimal conditions of protoplast preparation and regeneration of P. ostreatus B werestudied. Six factors including mycelial age of parents, enzymolysis time, enzymolysistemperature, the type and concentration of enzyme, osmotic stabilizer were investigated bysingle factor test and orthogonal test methods. The results showed that the optimal conditionsof protoplast preparation and regeneration are as follows: mycelial age6d,1.5%cellulase+1.0%snailase, osmotic stabilizer1M mannitol,30℃, enzymatic hydrolysis time3h.Identifying the fusant was studied from the characteristics of the colony, mycelialmorphology, growth curve and laccase enzyme activity. An ideal strain F was screened. Itsgrowth speed is faster than the growth rate of parent strain B, laccase enzyme activityincreased12.18%compared to the original strain B.The fermentation conditions of fusant F were studied. The results showed that theoptimal conditions of producing laccase are as follows: inoculum amount10%, carbon sourceglucose, nitrogen source peptone,0.5mmolL-1Cu2+and oscillation cultivation. The highestyield of enzyme activity reached190.5U ml-1.The papermaking wastewater was treated by fusant F. The results showed that theoptimal conditions of the papermaking wastewater degradation are as follows: initial pH6,initial concentration lower,30℃, mycelial age7d. The highest removal rate of COD andchromaticity can reach85%and94%respectively.