Improvement of nitrile hydratase activity and enhanced production of acrylamide using brevibacterium CH2

Abstract

A number of microorganisms are known to possess a capability of converting nitrile compounds to the corresponding amides. Chapter 1 summarizes the commercial use of acrylamide, its current production methods, the applications of nitrile converting enzymes, the enzyme stabilization by additives, and the immobilization methods for cells. The acrylonitrile adaptation of Brevibacterium sp. CHI for the increased production of acrylamide was described. A bacterial strain having higher nitrile hydratase activity and acrylonitrile concentration tolerance, numbered Brevibacterium sp. CH2, was developed by repeated subculturing of CH1 strain in the broth containing acrylonitrile with slightly increased acrylonitrile concentration. The specific nitrile hydratase activity of the strain increased up to 32 units/mg cells, 3.2 times higher than that of CH1 strain and the enzyme was not inhibited by the substrate, acrylonitrile concentration of 6\%(v/v). The effects of temperature and pH on the activity of the cells were investigated and the substrate specificity of the strain was also studied. The fed-batch culture of CH2 strain with regulatory substrate feeding gave a cell density of up to 80 g/L and nitrile hydratase activity was maintained at above 22 units/mg cells. The cell growth yield was ca. 0.5g dry wt per g glucose consumed. The total nitrile hydratase activity in the fedbatch culture with the regulatory feeding increased up to 1784 units/ml, which amounted to four times that of the batch culture. The productivity of the enzyme formation and the cell mass in regulatory fed-batch mode greatly increased as compared with batch culture. The effects of acrylonitrile and acrylamide on the enzyme action of nitrile hydratase of CH1 and CH2 strain used for the biotransformations of nitriles were described. The excessive substrate (acrylonitrile) and product (acrylamide) inhibited the activity of the enzyme competitively. In comparison with 0.2 mol/L of CH1 strain, the substr...