Construction Of Recombinant Catalase Engineering Strain And Its Fermentation Optimization

Catalase (CAT), one of the derivatives of erythrocyte, can catalyze H₂O₂ into H₂O and O2, preventing the chelation of H₂O₂ and O2 into OH. Due to the unique characteristic and application, CAT is becoming more and more attractive in textile areas and so on. Currently, several brands of CAT are being sold on market and their applications are of great prospect. The previous study in our lab has successfully screened a high CAT producing strain (Bacillus subtilis WSHDZ-01) with resistance to high heat and high alkalinity from textile waste. However, the major problem was that the total enzyme activity was not very high and the extracellular CAT production was low, requiring great cost for cell rupture in order to obtain the intracellular enzyme. In this research, Escherichia coli has been used as host for expression of CAT gene from Bacillus subtilis WSHDZ-01. Then, we optimized the fermentation process for great extracellular expression of recombinant enzyme. The main contents of this study are as follows:(1) The recombinant CAT producing strain E. coli BL21(DE3)(pET-20b(+)/katA) was constructed by inserting katA gene from Bacillus subtilis WSHDZ-01 into the down stream of signal peptide in pET-20b(+) and expressing the katA gene in E. coli BL21(DE3). However, the enzyme production was not high in shaken flask fermentation. As we know, E. coli had great difficulty in secrete CAT, which is of high molecular weight. Therefore, we planed to enhance CAT secretion by Gly addition and preliminarily optimized Gly adding strategy: the beginning fermentation medium was TB, temperature was 30°C, pH was 7.0, inoculation volume was 3%, and 200 mM Gly was added at 10 h, achieving enzyme activity up to 19211.7 U/mL.(2) We optimized the fermentation process for CAT production by E. coli BL21(DE3)(pET-20b(+)/katA) in shaken flasks. First, the single factor tests optimized carbon source, nitrogen source, temperature and pH, finding that the optimal carbon source was glycerol (5.0 g/L), nitrogen source was Angela yeast powder (40 g/L), the optimal temperature was 30°C, and optimal pH was 7.0. Based on the above results, we conducted orthogonal tests, studying the effects of the four factors on cell growth and CAT production, and the optimal fermentation conditions were determined as follows: glycerol 5.0 g/L; Angela yeast powder 35 g/L, KH₂PO₄ 2.32 g/L,K2HPO4 16.43 g/L, initial pH 7.5, temperature was firstly set as 37°C and then changed to 30°C. Under the optimal conditions, the extracellular CAT activity was up to 24152.7 U/mL in shaken flasks.(3) The fermentation process of E. coli BL21(DE3)(pET-20b(+)/katA) for CAT production has been preliminarily studied in 3 L reactor. Different glycerol concentrations had great impact on cell growth and extracellular CAT production: the high initial glycerol concentration was beneficial to cell growth and CAT production. However it could also resulted in extended fermentation cycle. The fermentation processes with different agitation rates were studied: high agitation rates could enhance cell growth, but would inhibit CAT secretion; low agitation rate was adverse to cell growth, but could achieve higher extracellular enzyme activity. Finally, we chose agitation rate of 400 r/min combine with 300 r/min for fermentation.(4) Based on the above study, we investigated the effects of fermentation with glycerol feeding on cell growth and extracellular enzyme production. The results indicated during the fermentation process with constant glycerol, CAT synthesis was greatly inhibited, and the enzyme would begin to secrete after the glycerol feeding; however the fed-batch fermentation with 15 g/L glycerol feeding greatly enhanced extracellular enzyme production, achieving final extracellular CAT up to 50369.5 U/mL.