Efficient production of chitinase by immobilized Wasabia japonica cells in double-layered gel fibers

Abstract

Cell leakage during the cultivation of Wasabia japonica cells was prevented by immobilizing the cells in the inner layer of double-layered calcium alginate gel fibers. The double-layered gel fibers were used for chitinase production in Erlenmeyer flasks. Cell leakage from the double-layered gel fibers was not observed during the cultivation. The specific productivity by the immobilized cells was higher than that of freely suspended cells under the same culture conditions. When the dissolved oxygen concentration was raised by aeration with pure oxygen, the chitinase specific productivity by the immobilized cells increased to a level about 5 times higher than that of the freely suspended cells. Similar results were obtained in a bubble column reactor. At high chitinase concentrations, product inhibition was observed. The rate of chitinase production in the batch culture could be represented by the equation dP dt =η P ·(1− P P m ) 0.24 , where P=chitinase concentration (U-enzyme/ l-broth), P m= limit chitinase concentration (U-enzyme/ l-broth) and η P=maximum chitinase production rate (U-enzyme/ l-broth·d). From this equation, it is clear that during chitinase production, product inhibition occurs from the start of the culture. In order to maintain the chitinase production rate above 80% of the maximum production rate, it is necessary to keep the chitinase concentration in the broth below 2.0 U/ml. A system for continuous production with simultaneous recovery of chitinase was developed. A production column containing W. japonica cells immobilized in double-layered gel fibers was coupled to a chitin column, and by circulating the fermentation broth between the two columns, continuous production with simultaneous recovery of chitinase was achieved. With this system, it was possible to maintain high and stable chitinase production for 40 d, and a large amount of chitinases (3,211 U) was obtained.