Process development for microbial transglutaminase production

Transglutaminase (EC 2.3.2.13) catalyses in vitro cross-linking reactions in various proteins. This enzyme has been used in attempts to improve the functional properties of protein foods. Up to now, commercial transglutaminase has been obtained from animal tissues. The complicated separation and purification procedure results in an extremely high price of the enzyme, which hampers a wide application in food processing. Recently, studies on the production of microbial transglutaminase (MTG) have been launched. The enzyme obtained from microbial fermentation has been applied in the treatment of various food proteins. Food treated with MTG appeared to have an improved flavour, appearance and texture. In addition, MTG can increase shelf-life and reduce allergenicity of certain foods. However, much work has to be done before this interesting enzyme can be produced on an industrial scale in an economic manner. This thesis focuses on the development of an economic and practical process for MTG production.

The study is directed mainly to process development and optimization with respect to MTG production in order to improve the productivity and to minimize the cost.

First, a stoichiometric model was developed for the application of medium design in MTG production by Streptoverticillium mobaraense. The model avoids dealing with all the metabolic reactions involved by simply lumping them into a single reaction. With the help of the analysis of the nutrients' roles and biochemical knowledge of the micro-organism, all stoichiometric coefficients in the model were calculated. Together with the experimental measurements, these coefficients were used for medium design. With this designed medium, MTG activity and specific productivity were increased significantly.

Second, analysis of amino-acid metabolism was done using mass balances. Depletion of free amino acids in a batch fermentation is probably the limiting factor for further cell growth and MTG production. The cause of this limitation is probably the cross-linking of amino acids by the MTG produced.

Third, an integrated fermentation technique was applied for further optimization of MTG production. A strategy was developed on the basis of the stoichiometric requirements for cell synthesis and MTG production and of the consideration that the product itself causes crosslinking of the substrates. By applying fed-batch techniques using an inorganic nitrogen source in the feed that cannot be cross-linked, MTG production was further increased significantly.

Finally, on the basis of our studies and others' work, trends with respect to the production and application of transglutaminase are signalled in the last chapter.