Optimum culture conditions of Brevibacterium sp. CH2 and stabilizing effect of various organic acids on nitrile hydratase were investigated.
The best carbon and nitrogen sources of Brevibacterium sp. CH2 were glucose and a mixture of bacto peptone, yeast extract and malt extract, respectively. The addition of ferric and ferrous ions greatly enhanced nitrile hydratase formation. The effects of nitriles, amides, and acids as an inducer on the formation of nitrile hydratase were investigated. Isobutyramide was the best inducer among those compounds tested. The maximum total activity was 630 units/ml of culture broth and the specific activity was 70 units/mg of dry cells, when Brevibacterium sp. CH2 was cultivated for 21 hours at 30℃ in the following optimized medium; 15g of glucose, 5g of bacto peptone, 3g of yeast extract, 3g of malt extract, 1g of $KH_2PO_4$, 1g of $K_2HPO_4$, 1g of NaCl, 0.5g of isobutyramide, 0.2g of $MgSO_4·7H_2O$, and 0.02g of $FeSO_4·7H_2O$ per liter of distilled water with pH controlled at 7.1.
As a result of the medium optimization, the specific activity of nitrile hydratase was 2.2 times increased and the substrate inhibition effect of nitril hydratase was considerably reduced. The new values of kinetic constants are as follows; $V_{max}$:6.31mol/h/g, $K_m$:0.015mol/L, $K_s$:0.55mol/L and $K_p$:0.065 mol/L.
The best stabilizing agent among the organic acids tested was n-butyric acid. When 20 mM of n-butyric acid was added in the buffer solution containing suspended cells, the initial enzyme activity was perfectly maintained for 20 days at 5℃. Consequently, we could obtain 400g of acrylamide per 1g of dry cell.
However, the strain Brevibacterium sp. CH2 is susceptible to a serious product inhibition. Therefore there exists a room for the improvement of Brevibacterium sp. CH2 by the study of product adaptation method or mutagenetic research, etc.
