Sorbitol buffer solution preparation

There are two basic procedures for enzyme immobilization by adsorption. Both share the same first steps preparation of the immobilization matrices (discussed above) and the aqueous enzyme solution. Typically, the aqueous solution is fairly concentrated in enzyme (approximately 5 to 40 mg/ml) and buffered (approximately 10 mM) to the optimal pH value of the enzyme. In addition, necessary co-factors, such as NAD(P)+ or NAD(P)H, should be included. Also, the presence of albumin or high-molecular-weight PEG at concentrations of 2 mg per g of matrix have been shown effective in protecting enzymes during the water removal stage of immobi-lization. °° Furthermore, the presence of sorbitol can increase activity and reduce side reactions, such as hydrolysis. -It is recommended that the aqueous solutions be centrifuged prior to use to remove any nonsolubilized matter. 

TABLE 2. PPase activity In supernatants and pellet after washing In buffers at pH 6.5 and 8.5. The washing solution contained 330 mM sorbitol, 30 mM tricine and 5 mM MgCl2. SO, SI, S2, S3-supernatants after osmotic shock, 1st, 2nd and 3rd washings respectively. Total PPase activity in Intact chloroplasts Is taken as 100%, corresponding to 7.7 pmoles/mg Ch1 m1n at pH 6.5 and 8.5. Only the last pellet was analyzed for PPase activity. Total Chi In preparation was 0.45 mg. 

Crude Solid. The simplest way to use enzymes in organic solvents is to suspend a precipitate or a lyophilisate. The enzyme does not need to be of high purity, but some care should be taken during the preparation. In aqueous solution, the enzyme has an optimal pH, dictated by the ionization state under which the amino acids involved in the catalysis must be to allow activity. The solid enzyme must be in the same ionization state when used in organic solvents (15). For this purpose, it is important to precipitate the enzyme or lyophilize it from a solution buffered at this pH. This applies to the other forms of solid enzyme preparations. The other important point is the drying of the preparation. It has been observed that the secondary structure of proteins can be affected by lyophilization (16). This can be avoided by the use of lyoprotectants such as sorbitol (17) or salts such as KCl (18). 

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