Researches On Fermentation Conditions For The Production Of Chloroperoxidase

Chloroperoxidase (CPO), which has high selectivity of isomers and universality, is a widely used heme-peroxidase. Its specific substrates are mainly chloride, bromide and iodide. When there are appropriate receptors, it can catalyze the formation of carbon-halogen bonds. With the application development of CPO in industry, agriculture and medicine in recent years, the demand of CPO is increasing. But there was no report on its fermentation and production in China.We bought Leptoxyphium fumago from UK. We studied the optimum growing conditions and biological characteristics, such as morphological, physiological and biochemical characteristics and anti-bacterial. On this basis, we studied the optimized composition of medium, environment and fermentation conditions in laboratory, and developed a simple, rapid, high yield isolation and purification procedure for Caldaroperoxidase isolation.The results show that Leptoxyphium fumago can grow on the medium containing different carbon and nitrogen sources. In neutral and acidic, it can grow well. In addition, we found that it has a good anti-bacterial, so that it can not easily be contaminated, except Bacillus Subtilis*. In order to optimize the fermentation conditions, the carbon, nitrogen, natural extracts and environmental conditions were varied according to the quadratic response surface common rotary combination design with the aim of assessing the effects of these variables and their interactions on the CPO of product. The predictive polynomial quadratic equations model was developed by Minitab software. The optimal fermentation conditions were fermented for 6-7 d at the temperature of 25℃, pH 7.0. The composition of the medium is Glucose 18.488 g / L, Sucrose 15 g/L, Starch 10 g/L, NaNO3 2 g/L, Yeast extract 7 g/L, the Potato Extract 1000 mL/L, KCl 3.25 g/L,KH2PO4 2.55 g/L, MgSO4·7H2O 2.39 g/L, FeSO4·7H2O 0.22 g/L. the liquid volume of medium is 50mL/250mL shake flask, and the inoculum is 2 cm2 in a flask. In this condition, the enzyme activity can reach 770.244 IU/mL. In verification test, the actual activity is 755.366 IU/mL, and the error is less than 1% We also developed the best isolation and purification procedure. Firstly, separate mycelium from broth to obtain crude enzyme solution for ultrafiltration. Then, after salting out protein by ammonium sulfate, the protein was done with DEAE-cellulose 53 ion-exchange chromatography and Sephadex G-100 gel chromatography. In this way, the activity of CPO reached to 6146.24 U/mL, which was 17.01 times than the crude enzyme solution. With the studies on enzymatic characterizations of CPO, we found that its Km is 12μmol/(L·min), the optimum reaction pH is 2.75 and the optimum reaction tempreture is 25℃. The reaction rate was influenced by temperature obviously.