Methanol → Lysine dihydrochloride

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In 1N NaOH, 2 g of Sephadex G-25 (dextranepichlorohydrin copolymer made by Pharmacia Co.) was immersed. Then the copolymer was separated by filtration. It was allowed to react with cyanuric chloride for 15 seconds in dioxane. The reaction mixture was washed with cold acetone and ice water and then 200 mg of the enzyme Pronase E was dissolved therein. In a borate buffer solution, the s-triazine derivative of Sephadex G-25 obtained as described above was immersed and gently agitated for 5 hours at pH 8.0 and 4° C to have the enzyme Pronase immobilized. The amount of the enzyme immobilized was 67 mg per g of carrier and the specific activity of the immobilized enzyme at 40° C and pH 6.0 was found to be 2.53 μ.moles/mg.min. The optimum pH of this immobilized enzyme on DL-lysine methyl ester dihydrochloride as the substrate was found to be around 6.1. The immobilized enzyme was packed in a jacketed column. With the column temperature kept at 40° C, 50 ml of a DL-lysine methyl ester dihydrochloride solution having a concentration of 10% and kept at pH 6.8 was fed downflow from the column head at space velocity of 13 hr-1. pH of the effluent was lowered to 5.75 as the reaction proceeded. When the effluent from the column was tested for conversion by the ninhydrin process under conditions selected so as to permit coloration with amino acids and no coloration with amino acid esters, it was revealed that 40% of the DL-form had undergone the reaction. The effluent was mixed with a volume of methanol about four times as large, and the salt consequently precipitated therein was removed. Thereafter, the mixture was concentrated to about 20 ml by evaporation of the solvent. To the concentrate was added about 100 ml of acetone to produce 2.02 g of L-lysine dihydrochloride in the form of precipitate. This salt was found to have specific optical rotation, [α]54620°C, of +15.8 (C = 2, 6N-HCl). When it was recrystallized from watermethanol, the purified product showed a specific optical rotation of +19.2. This operation of the resolution by use of the column described above was performed a total of ten cycles over a period of 15 days with the same space velocity. During the period, the specific optical rotation of the precipitate remained unchanged, although the conversion tested by the ninhydrin process fell to eventually 30% of the DL-form.

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