Expression and biochemical characterization of a Bacillus subtilis catalase in Pichia pastoris X-33
Protein expression and purification(2023)
摘要
Catalase, which catalyzes the decomposition of H2O2 to H2O and O2, is widely used to reduce H2O2 in industrial applications, such as in food processing, textile dyeing and wastewater treatment. In this study, the catalase (KatA) from Bacillus subtilis was cloned and expressed in the yeast Pichia pastoris X-33. The effect of the promoter in the expression plasmid on the activity level of the secreted KatA protein was also studied. First, the gene encoding KatA was cloned and inserted into a plasmid containing an inducible alcohol oxidase 1 promoter (pAOX1) or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). The recombinant plasmids were validated by colony PCR and sequencing and then linearized and transformed into the yeast P. pastoris X-33 for expression. With the promoter pAOX1, the maximum yield of KatA in the culture medium reached 338.8 +/- 9.6 U/mL in 2 days of shake flask cultivation, which was approximately 2.1-fold greater than the maximum yield obtained with the promoter pGAP. The expressed KatA was then purified from the culture medium by anion exchange chromatography, and its specific activity was determined to be 14826.58 U/mg. Finally, the purified KatA exhibited optimum activity at 25 degrees C and pH 11.0. Its Km for hydrogen peroxide was 10.9 +/- 0.5 mM, and its kcat/Km was 5788.1 +/- 25.6 s-1 mM-1. Through the work presented in this article, we have therefore demonstrated efficient expression and purification of KatA in P. pastoris, which might be advantageous for scaling up the production of KatA for use in a variety of biotechnological applications.
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关键词
Expression,Catalase,Pichia pastoris,Promoter,Characterization
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