استخلاص و تنقية إنزيم الكولاجينيز من مخلفات أمعاء بعض الأسماك

الملخص EN

Five marine and freshwater fish species bighead carp Aristichthys nobilis, silver carp Hypophthalmichthys molitrix , shad Tenualosa ilisha, mullet Liza carinata and spotted leatherskin Scomberoides commersonianus, were used applying four solutions included distilled water, sodium chloride (at concentrations of 2, 5 and 7%), Tris-HCl buffer 0.05 M with pH 7, 7.5 and 8.5 sodium phosphate buffer 0.05 M , pH 5, 6.5 and 7 to limitation the best enzyme source and extraction solution.

It was found that bighead carp was the better source for obtaining enzyme in comparison with other sources and sodium phosphate buffer 0.05 M with pH 6.5 was the best extraction solution where it gave the highest enzyme activity of 220.45 U/ mg protein.

Ammonium sulphate added to enzyme extract with saturation rate 20-80% as a primary step for purification where the enzymatic and specific activities reached 718.67 U/ ml and 659.33 U/ mg protein, respectively, and an enzyme yield of 22.56% with 4.44 purification times.

Then, concentrated enzyme extract passed through ion exchange chromatographic column DEAE Sephadex A- 50.

Ion exchange process produced two enzymes W and C with specific activity of 140.22 and 359.40 U/ mg , enzyme yield 10.56 and 36.56% and purification times 1.57 and 4.75 , respectively.

The two enzymes subjected to and additional purification step of gel filtration using Sephadex G-100 gel filtration column where the specific activity for both enzymes W and C reached 139.19 and 350.21 U/ mg protein, 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 enzymes revealed the appearance of one protein band for each using Poly Acryl Amide gel electrophoresis without denaturizing agents