Crystallization of alkaline protease as a means of purification process

Abstract

A purification protocol of alkaline protease purification using crystallization was developed by investigating the effects of pH, temperature, initial enzyme concentration, salt (as crystal inducer) concentration, and the presence of impurity proteins. A commercial alkaline protease solution was used as a starting material and NaCl was used as a crystal inducing salt. The crude enzyme solution was first diafiltered against deionized water and then concentrated by ultrafiltration. To the enzyme concentrate appropriate amount of NaCl was added to induce the crystallization which was lasted for 24 hours, and the enzyme crystals formed were filtered and washed with deionized water before being resolubilized. Crystal habit was typical needle shape, and the reaction order of its formation was estimated to be 1.53. The crystallization was strongly influenced by initial enzyme concentration. Solubility of alkaline protease at 25°C was 24.8 mg/ml, which was about one half of that of 4°C. Enzyme recovery yield of the purification process including the crystallization step ranged 50 to 60 %. The crystallization step was shown to successfully exclude impurity proteins from their habits as evidenced by gel permeation chromatography. The optimum condition for the crystallization was: pH 9.0, 25°C temperature, ca. 53 mg/ml or higher enzyme concentration, and minimum 5 % (w/w) NaCl concentration. In summary, an enzyme purification protocol based on crystallization was established, which can be applied to obtain a higher-purity alkaline protease solution on a large scale.