| Catalase is widely distributed in nature and found in all aerobic microorganisms, plant andanimal cells. Physiologically it acts as a regulator of H2O2 levels in organelles and as a specificperoxidase. Catalases from various sources are utilized in numerous industrial applications, suchas diseases diagnosis and food sterilization. Recently, catalase is used in removal of hydrogenperoxide of bleaching wastewater as a substitute for washing with huge amount of wateror usingchemical reducing agents. Compared with extract from organelles, biosynthesis of catalase bymicroorganisms has much more advantages.In this paper, the strain of Thermoascus aurantiacus WSH 03-01 was studied as a producerof catalase. The effect of revulsant on catalse fermentation was investigated in the beginning.Proper inoculum size and agitation speed were found by experiment in a 7 L fermentor. Thekey factor for producing catalse was determined by detecting ethanol's consuming processduring the catalse fermentation. Based on the result, catalse was high synthesized with themethod of adding ethanol continuously into the bioreactor. Thus, the scale-up of catalseproduction from 7 L fermentor to 30 L fermentor was also achieved. Furthermore, the effects ofvarious stabilizers on storage stabilities of catalase were observed, and the removal efficiency ofhydrogen peroxide by the obtained catalase was verified initially. The main content of thisdissertation is as follows:(1) The catalase activity increased to 26.0% when 88 mmol/L hydrogen peroxide was addedat 0 h. When 1 μmol/L menadione was added at 24 h or 48 h, the catalse activity increased 11.0%and 23.1%, respectively, compared with that of control. The catalase activity increased 32.5%when menadione was added at 36h and 48h. According to the result of menadione additionoptimization, the catalse activity reached 1725 U/mL when 88 mmol/L hydrogen peroxide wasadded at 0 h and 1 μmol/L menadione was added at 48 h,which was 60.6% higher than that ofcontrol (1074 U/mL). It indicates that catalse can be induced by reactive oxygen speciesgenerated by menadione and hydrogen peroxide.(2) The effects of inoculum size and agitation speed on catalse fermentation were optimizedin a 7 L fermentor. The proper conditions for catalse fermentation were: inoculum size 7%,agitation speed 200 rpm. The catalase activity reached 1851 U/mL in a 7 L fermentor at 96hunder the optimized conditions.(3) The speed of adding ethanol was confirmed as 0.14 mL/(h·L) by determine ethanol'sconsuming curve in the course of catalse fermentation. The catalse activity reached 2453 U/mLwhen ethanol was added continuously into the fermentor. The result showed that the catalseactivity reached 2287 U/mL in a 30 L fermentor. A two-stage agitation speed control mode wasalso developed to solve the low dissolved oxygen at the late phase of the fermentation. By thisway, the dry cell weight reached 9.56 g/L and the catalase activity reached 3652 U/mL.(4) The result of reserving condition study showed that the reservation efficiency of thecatalase activity exceeded 90% even it was conserved 60 days at 40℃ when 20 mmol/Lsodium chloride or 90g/L glycerol or 0.5g/L glutin was added into the concentrated catalase byultrafiltration.(5) The removal efficiency of hydrogen peroxide by the catalase obtained in this study washigher under the following conditions: pH range as 5.5~8.0, temperature between 15℃~40℃.Hydrogen peroxide at 100 ppm was removed completely by the enzyme after 15 minutesreaction. |
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