Extraction and Purification of Collagenase from Some Fish Wastes


Five marine and freshwater fish species bighead carp Aristichthysnobilis, silver carp Hypophthalmichthys molitrix , shad Tenualosa ilisha, mulletLiza carinata and spotted leatherskin Scomberoides commersonianus, were usedapplying four solutions included distilled water, sodium chloride (at concentrationsof 2, 5 and 7%), Tris-HCl buffer 0.05 M with pH 7, 7.5 and 8.5 sodium phosphatebuffer 0.05 M , pH 5, 6.5 and 7 to limitation the best enzyme source and extractionsolution. It was found that bighead carp was the better source for obtaining enzymein comparison with other sources and sodium phosphate buffer 0.05 M with pH 6.5was the best extraction solution where it gave the highest enzyme activity of 220.45U/ mg protein. Ammonium sulphate added to enzyme extract with saturation rate20-80% as a primary step for purification where the enzymatic and specificactivities reached 718.67 U/ ml and 659.33 U/ mg protein, respectively, and anenzyme yield of 22.56% with 4.44 purification times. Then, concentrated enzymeextract passed through ion exchange chromatographic column DEAE Sephadex A-50. Ion exchange process produced two enzymes W and C with specific activity of140.22 and 359.40 U/ mg , enzyme yield 10.56 and 36.56% and purification times1.57 and 4.75 , respectively. The two enzymes subjected to and additionalpurification step of gel filtration using Sephadex G-100 gel filtration column wherethe specific activity for both enzymes W and C reached 139.19 and 350.21 U/ mgprotein, enzyme yield of 5.77 and 22.43% and purification times 4.26 and 19.68,respectively. Results for determination the enzyme purity for the two enzymesrevealed the appearance of one protein band for each using Poly Acryl Amide gelelectrophoresis without denaturizing agents .