Extraction and Characterization of Proteolytic Enzymes and Crude Aqueous and Acidic Antimicrobial Peptides from Skin of Oreochromis Sp. (Tilapia), and Hypophthalmichthys Nobilis (Bighead Carp)

Low, Jun Xian (2019) Extraction and Characterization of Proteolytic Enzymes and Crude Aqueous and Acidic Antimicrobial Peptides from Skin of Oreochromis Sp. (Tilapia), and Hypophthalmichthys Nobilis (Bighead Carp). Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Waste from fish processing industries have contributed to increase of environmental pollution globally. This crisis has forced various established industries to explore the potential of fish processing wastes into valuable bioactive compounds. This project aims to determine the antimicrobial activity of isolated freshwater fish‟s skin [skins from Oreochromis sp. (tilapia) and Hypophthalmichthys nobilis (bighead carp)] against Gram positive and Gram negative bacteria, to extract (crude aqueous AMPs extract, and crude acidic AMPs extract) and characterize proteolytic enzymes and antimicrobial peptide from Malaysia freshwater fish‟s skin that can be utilized in industries. The fish skin extracts were homogenized by 50 mmol/L Tris-HCl buffer, pH 7.8 and used for extraction of antimicrobial peptide. The lyophilized fish skins were extracted using distilled water and 0.1% acetic acid for antimicrobial assay. Protein content for crude antimicrobial peptides extracted was estimated by using Bradford assay. Partial purification of the antimicrobial peptides was conducted by ammonium sulfate precipitation, and the salt was removed by overnight dialysis. Molecular weight of the antimicrobial peptides was estimated by using SDS-PAGE. Screening of antimicrobial activity was performed by using disc diffusion method in triplicates. As for extraction of proteolytic enzymes, the lyophilized fish skins were homogenized with 0.02M Tris-HCl buffer, pH8.0. Lowry‟s assay was used to determine the protease activity and specific activity of filtrates collected by protease assay. Bradford‟s assay was conducted to estimate the protein content of the supernatant collected. The partial purification of protease was performed by using ammonium sulfate precipitation and dialysis. The effect of pH and temperature on enzyme activity was measured at pH ranging from 4 to 11 and temperature ranging from 20 ˚C to 90 ˚C. The protein content was measured for Bighead carp aqueous extract and acidic extract, Tilapia aqueous extract and acidic extract were 5.151mg, 3.971mg, 3.587mg and 2.654mg respectively. The molecular weight of bighead carp antimicrobial peptides was estimated at 27.0 kDa. Bighead carp extract in aqueous exhibited antimicrobial activity against Gram-positive bacteria, Staphylococcus aureus with 17 mm zone of inhibition. Bighead carp has significant higher proteolytic enzyme activity measured at 63.42U as compared to tilapia with protease activity recorded with 49.77U. One-way analysis of variance (ANOVA) was used to determine the significant differences between means of triplicates results in effect of pH and temperature. The optimum pH for tilapia, and bighead carp protease was 11 and 10 respectively. The protease extracted from tilapia, and bighead carp was stable at temperature from 20 ˚C to 50 ˚C. Protease enzymes also showed its capability as a blood destainer. In conclusion, crude aqueous bighead carp extract had the best antimicrobial potential while crude bighead carp extract had destaining activity in which could be applied in detergents industries.