Alkaline phosphatase encapsulated in gellan-chitosan hybrid capsules.

Alkaline phosphatase (ALP) was encapsulated in gellan-chitosan polyion complex (PIC) capsules using a convenient procedure. The recovery of ALP was about 50% when the capsules were prepared by dropping a solution of ALP and gellan mixture (ALP/gellan) into a chitosan solution. When p-nitrophenyl phosphate (p-NPP) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) were incubated with ALP/gellan-chitosan capsules as substrates for ALP, the transparent colorless capsules changed to yellow and blue, respectively. The encapsulation of ALP into the PIC capsules was also confirmed by SDS-PAGE and immunoblot analyses. The ALP and polypeptides of more than 30 kDa remained without release even after incubation at 4 degrees C for 14 d. The biochemical properties of the encapsulated ALP activity were similar to those of the intact enzyme. When the solution containing p-NPP was loaded on a column packed with ALP/gellan-chitosan capsules at 27 degrees C, approximately 75% of p-NPP was hydrolyzed by passing through the column. No significant leakage of ALP was observed during the procedure, indicating that the capsules were resistant to pressure in the chromatographic operation. Furthermore, 70% of the hydrolytic activity of the packed capsules remained after storage at 4 degrees C for one month. These results suggest that the polyion complex capsules could be useful materials for protein fixation without chemical modification. [Diagram: see text] Encapsulation of ALP into PIC capsules and the morphological changes seen in the absence of the ALP substrate and in the presence of p-NPP and BICP.

Convenient procedures for human hair protein films and properties of alkaline phosphatase incorporated in the film.

We have developed a novel method of extracting proteins from human hair in the absence of detergent called the "Shindai Method". Using the protein solution consisting of hard alpha-keratins and matrix proteins prepared by this method, we developed two procedures for preparing hair protein films. The protein solution was mixed with trichloroacetic acid (TCA), perchloric acid (PCA) or guanidine-HCl (GHA), and then exposed in distilled water. Light brown aggregates immediately formed (Pre-cast method). The other method is based on the same characteristics of the hair proteins to form protein aggregates. The protein was directly exposed to the solution containing TCA, PCA, GHA, HCl, H(2)SO(4) or acetate buffer (Post-cast method). The maximum yield was greater than 70%. These protein films were water-insoluble and mainly made up of alpha-keratins. Scanning electron micrographs showed that the fine surface of the protein films was composed of particles, filaments, and porous structures and the constitution was dependent on the preparation procedure used. When porcine intestine alkaline phosphatase (ALP) was mixed with the hair protein solution in a Post-cast method using acetate buffer (pH 5), ALP was incorporated into the alpha-keratin films. The activity retained in the protein film was approximately 8% of the original level. The biochemical properties of the ALP activity in the film were similar to those of the native enzyme.