Effect Of Citrate On Catalase Production Of Serratia Marcescens And Its Mechanism

Catalases (CAT) play an important role in food industry, textile and pulp field by degrading H₂O₂ to H₂O and O₂. In an experiment to optimize the culture conditions for catalase production by S. marcescens SYBCT02, an interesting phenomenon occurred that the addition of citrate to the culture medium significantly stimulated the accumulation of CAT. To explore the mechanism of citrate inducing CAT production, experiments were designed using Serratia marcescens SYBCT02 as material, the connection of CAT production with antioxidant or oxidative stress was investigated. The main results are as follows:(1) The effect of citrate concentration on CAT production by S. marcescens was investigated. After adding 20 g/L citrate to the culture medium, CAT production by S. marcescens SYBCT02 was four times higher than in the citrate-free medium. A stimulating effect of citrate on the CAT activity was also observed in both S. marcescens SYBC08 and S. marcescens SYBCYH in this study, which indicated the universality of this phenomenon in Serratia marcescens.(2) Three kinds of carbon sources, i.e. glucose, citrate and a mixture constituted by glucose and citrate, with the same amount of carbon element (0.72 mol/L), were used in the culture medium for CAT production by S. marcescens SYBCT02. After determining the contents of cellular antioxidant substrates and the activities of cellular antioxidant enzymes, an occurrence of oxidative stress was found in the cells of S. marcescens SYBCT02 cultivated in the media with citrate. Compared with the control, the increased amounts of hydrogen peroxide (H₂O₂) and hydroxyl radical (·OH) induced the synthesis of CAT after an addition of citrate to the culture media. This conclusion was further confirmed by the addition of exogenous reactive oxygen (produced by menadione and paraquat) to the culture media.(3) The reason for more CAT production in citrate plus glucose medium was investigated by determining the absorption rates of citrate and glucose in three different kinds of media. It was found that the absorption rate of citrate in the composite carbon sources was higher than that in the citrate-only medium, suggesting citrate absorption by S. marcescens SYBCT02 improved when glucose existed in the medium. As a crucial intermediary metabolism substance, citrate could join in the metabolism and regulate the enzymes in TCA cycle, affecting EMP and HMP pathways indirectly. EMP pathway was downregulated whereas TCA cycle and HMP were both improved by citrate in the end. During the whole metabolism, the two carbon sources made a contribution to CAT production through different pathways.(4) Catalase gene of S. marcescens SYBCT02 was cloned through PCR and expressed in E. coli. The acquired CAT gene sequence shared 98% identity with that of S. marcescens SYBC08. Citrate addition could increase the CAT production by S. marcescens SYBCT02 evidently. However, the same result was not attainable for the recombinant bacterium.