BIOCHEMICAL ANALYSIS AND PEPTIDE MODELING OF LYSOZYME IN INDIAN FENNEROPENAEUS INDICUS SHRIMP SPECIES.

of activator and inhibitor on lysozyme activity is obtained.The final results of our project clearly explain that the identified motif peptide sequence and the 3D peptide structure are potential candidates for drug docking studies and also act as novel molecular markers useful for pharmacoinformitics and clinical endocrinology studies. the sample is estimated by plotting OD value at 640nm against the standard protein using Lowry’s method. The biochemical analysis of the enzyme is done by using Sodium Dodecyl Sulphate - Poly Acrylamide Gel Electrophoresis and High Performance Liquid Chromatography. The proteins get separated on the basis of molecular weights. When the electrophoretic run is completed the gel is immersed in 0.25% Coomassie blue solution for 30 minutes for staining. The gel is destained with 40% methanol and 10% acetic acid. The protein bands are observed clearly under UV- Trans illuminator. The mobile phase used in HPLC is 0.1% phosphoric acid and acetonitrile in the ratio 80:20. Sample is precipitated by 60% ammonium sulphate and the precipitate is recovered and purified further on G 200 sephadex. The sample is placed in auto sampler tray and run the HPLC. High pressure liquid chromatogram is obtained with the help of computer. Bioinformatics analysis were carried out for lysozyme protein , motif were identified and identified motif are modeled using pepfold server.

FAO defines aquaculture as "the farming of aquatic organisms, including fish, mollusks, crustaceans, and aquatic plants". Fanning here implies some form of intervention in the rearing process to enhance production, such as regular stocking, feeding and protection front predators. Aquaculture is the only alternative to mitigate the threats to the world's fisheries by taking the pressure off from wild fish stocks while supporting livelihoods and food production. Aquaculture production has increased tremendously over the past few decades and today accounts for almost a half of global fish production by weight, while production from wild fisheries has largely slowed or stagnated. The enzyme lysozyme is extracted from Fenneropenaeus indicus and it is estimated by using Lowry's method. Lysozyme is biochemically analysed with SDS-PAGE and HPLC. The activity of the enzyme is assayed and characterized by determining the optimum pH and temperature. The effect of activator and inhibitor on lysozyme activity is obtained.The final results of our project clearly explain that the identified motif peptide sequence and the 3D peptide structure are potential candidates for drug docking studies and also act as novel molecular markers useful for pharmacoinformitics and clinical endocrinology studies. F. indicus is a marine shrimp (with estuarine juveniles) which likes mud or sandy mud habitats at depths of 2-90 metres (6-300 feet). It is one of the major wild caught commercial species of the world. Adults are marine and the juveniles are estuarine.

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F. indicus tolerates low water quality conditions and tolerates low dissolved oxygen better than Penaeus monodon (Rosenberry, 2004). Ideally, oxygen levels should be between 5 and 10 ppm, though higher levels (up to 20 ppm) do not seem to cause any problem. At night, the minimum oxygen levels should be 3.6 ppm. Similar to P. monodon (Groth et al., 2001), it still survives when dissolved oxygen (DO) levels are around 1.5 ppm (mg/L). It tolerates salinities from 4 ppt (L Evans, Ecotao Enterprises, South Africa, personal communication, 2004) to 50 ppt (A Abedian, Tarbiat Modares University, Iran, personal communication, 2004), high temperatures and high densities, and it is readily available in the wild. F. indicus also reaches sexual maturity and spawns in ponds. It tolerates 45 ppt salinity in the Red Sea (De Bruin et al., 1994). The ideal growout water temperatures are from 25 to 32°C. Between 20 and 23°C, F. indicus grows too slowly for commercial production, although it will survive quite well at these temperatures. Between 18 and 20°C, the growth rate is roughly a seventh of that at 28°C. The animal can live at temperatures down to 15°C, but shows stress, such as a slow or lack of recovery from moulting at these low temperatures. In some areas, such as Thailand (Phongdara et al., 1999) and Australia, banana prawns comprise two species, F. merguiensis and F. indicus, which are very similar in morphology. They can be identified by a distinct difference in the rostrum.
F. indicus is a marine shrimp (with estuarine juveniles) which likes mud or sandy mud habitats at depths of 2-90 metres (6-300 feet). It is one of the major wild caught commercial species of the world. Adults are marine and the juveniles are estuarine.
F. indicus tolerates low water quality conditions and tolerates low dissolved oxygen better than Penaeus monodon (Rosenberry, 2004). Ideally, oxygen levels should be between 5 and 10 ppm, though higher levels (up to 20 ppm) do not seem to cause any problem. At night, the minimum oxygen levels should be 3. . The ideal growout water temperatures are from 25 to 32°C. Between 20 and 23°C, F. indicus grows too slowly for commercial production, although it will survive quite well at these temperatures. Between 18 and 20°C, the growth rate is roughly a seventh of that at 28°C. The animal can live at temperatures down to 15°C, but shows stress, such as a slow or lack of recovery from moulting at these low temperatures.

Methods:-
The enzyme lysozyme is extracted from the F. indicus. The amount of protein present in the sample is estimated by plotting OD value at 640nm against the standard protein using Lowry's method. The biochemical analysis of the enzyme is done by using Sodium Dodecyl Sulphate -Poly Acrylamide Gel Electrophoresis and High Performance Liquid Chromatography. The proteins get separated on the basis of molecular weights. When the electrophoretic run is completed the gel is immersed in 0.25% Coomassie blue solution for 30 minutes for staining. The gel is destained with 40% methanol and 10% acetic acid. The protein bands are observed clearly under UV-Trans illuminator. The mobile phase used in HPLC is 0.1% phosphoric acid and acetonitrile in the ratio 80:20. Sample is precipitated by 60% ammonium sulphate and the precipitate is recovered and purified further on G 200 sephadex. The sample is placed in auto sampler tray and run the HPLC. High pressure liquid chromatogram is obtained with the help of computer. Bioinformatics analysis were carried out for lysozyme protein , motif were identified and identified motif are modeled using pepfold server.

Results And Discussion:-
As with all forms of gel electrophoresis, molecules may be run in their native state, preserving the molecules' higher-order structure or a chemical denaturant may be added to remove this structure and turn the molecule into an unstructured linear chain whose mobility depends only on its length and mass-to-charge ratio. In most proteins, the binding of SDS to the polypeptide chain imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by approximate size during electrophoresis. The result of SDS-PAGE is shown in Fig 1. Highperformance liquid chromatography is a technique in analytical chemistry used to separate, identify and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column. The chromatogram obtained for standard and sample collagenase is given in Fig 2 and 3.
The target sequence is retrieved from NCBI in FASTA format, presence of motif in the lysozyme were identified using scan prosite motif server. The motif region of target sequence was modeled using pep fold server (Fig 4,5).  . Further very little information is available on the wide spectrum activity of shrimp lysozyme, particularly from F. indicus. This forms the basis of the present study where in lysozyme has been identified from Indian shrimp F. indicus and the functional characterization has been carried out. The future studies on this enzyme may lead to find out more insights.