| Glucose oxidase (GOD) and horseradish peroxidase (HRP) have a wide range of applications in food, medicine, chemical testing and many other fields, but HRP through plant extraction, separation and purification technology is complex and the cost is high. Trichoderma reesei and Pichia pastoris are excellent eukaryotic host for heterologous expression and suitable for large scale industrial production application.We expressed GOD and HRP in T. reesei and P. pastoris, meanwhile, developed degradation methods for Sudan dyes by the two enzymes system.In order to obtain GOD and HRP and develop other functions, we expressed two enzyme genes in T. reesei and P. pastoris, respectively. However, the two recombinant T. reesei strains plcs-ClGo and plsl-Prx have low enzyme activities. The GOD and HRP activities from the two recombinant P. pastoris strains pPIC9k-Go and pPIC9k-Prx are 74.3 IU/mL and 267.3 IU/L, respectivery. And we obtained the recombinant strains pPIC9k-GH4B which could expressed a fusion protein which is ligating a GOD and a β-glycosidase to amplify GOD substrate spectrum, The activities of GOD and P-glycosidase are 16.4 IU/mL and 0.77 IU/mL, respectively. In addition, we obtained another recombinant strain pPIC9k-PEVG which could co-expressed the GOD and HRP and the activities of enzymies are 20.9 IU/mL and 254 IU/L, respectively. This study found GOD and HRP play a cooperative function in the degradation of Sudan dyes under the condition of pH 6.0. And adding suitable amount of phenolic substance can further improve the degradation rate of Sudan. The degradation rate of Sudan Ⅰ increased from 21.93% to 87.5% with 20 μM bisphenol A, and the degradation rates increased from 0 to 58.3% and 68.75% for Sudan II and Sudan III, respectively. The GOD slow release hydrogen peroxide could reduce the harm of HRP, and phenol promote the efficiency of Sudan dyes degradation at non basic condition, which provides a new thought for toxic substances degradation. |
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