Purification Of Endogenous Proteases From (Litopenaeus Vannamei) Shrimp Head And Their Enzymology Properties

Shrimp head is the main by-product in headless and peeled prawns processing. It is estimated that the output of shrimp head in China was up to 200,000 tons annually, seventy percent of which is from Litopenaeus vannamei. Shrimp head contains not only abundant protein but also rich endogenous protease, which can easily lead to autolysis on certain conditions. Shrimp head is perishable and its quality is so difficult to control that it is often made into feed and not fully used. All above bring out not only waste of resource but also environmental pollution.Results of our study will provide a theoretical basis to ensure the quality of shrimp head, makeing full usage of endogenous protease to recover nutrients in shrimp head such as protein with low cost. In addition, the characteristics of endogenous proteases from shrimp head could be recognized deeply. Firstly this paper studied the change rule of the activity of endogenous protease from shrimp head with pH, the results indicated there were two peaks of enzyme activity, the optimum were pH 2.0 and 8.0, respectively. This paper systematically studied the purification and characterization of endogenous proteases on this basis. The homology of these two kinds of endogenous protease was preliminary analyzed by LC-ESI-MS/MS.The main results were as following:(1) The research used Litopenaeus Vannamei heads as materials and optimized the extraction conditions. The impact of pH, temperature and solid-liquid ratio on the activity of endogenous proteases was studied. And then the optimum conditions to extract enzyme was optimized through orthogonal experiment. The optimum conditions to extract acidity protease by orthogonal experiment were: pH 3.0, the ratio of solid-liquid of 1:2, temperature 30℃. For the alkaline protease, experiment results showed that the optimum conditions were: pH 8.0, the ratio of solid-liquid of 1:3, temperature 50℃. For the acid protease, preliminary separation results showed that the saturation ammonium sulfate in first precipitation fraction was 10% and 50% in second. For the alkaline protease, preliminary separation results showed that the saturation ammonium sulfate in first precipitation fraction was 20% and 80% in second.(2) Endogenous alkaline protease from Litopenaeus vannamei shrimp head was purified to homogeneity by 20%-80% of ammonium sulfate fractions, Q-Sepharose F.F anion chromatography and SephadexG-150 gel chromatography with a 26.5-fold increase in specific activity and 32.8% recovery. The purified alkaline protease specific activity was 1386.16 U/mg. The result of SDS-PAGE showed a single band and the molecular weight of alkaline protease was 79.95 kDa.(3) Endogenous acidity protease from Litopenaeus vannamei shrimp head was purified to homogeneity by 10%-50% of ammonium sulfate fractions, SephadexG-100 gel chromatography and DEAE-Sepharose F.F anion chromatography with a 15.7-fold increase in specific activity and 22.2% recovery. The purified acidity protease from Litopenaeus vannamei shrimp head was obtained. The purified acidity protease specific activity was 698.09 U/mg. The result of SDS-PAGE showed a single band and the molecular weight of acidity protease was 27.45 kDa.(4) The enzymatic characteristics of alkaline protease: The alkaline protease optimum pH was 8.0 with high stability in alkaline range. The optimum temperature was 50℃and thermal stability is well. Enzyme Km value and Vmax value are 2.54g/L and 7.11μg/min. Fe³⁺,Mn²⁺ and Hg⁺ can inhibit the enzyme activity completely. TryPsin inhibitor can strongly inhibit the activity of the enzyme. DTT can activate the enzyme activity. Inferred the protease from Litopenaeus vannamei shrimp head was a kind of trypsin.(5) The enzymatic characteristics of acidity protease: The optimum pH and temperature for the enzyme activity were pH 3.0 and 30℃, respectively. Thermal and pH stability were not good. Enzyme Km value and Vmax value are 2.01g/L and 26.39μg/min. The enzyme was strongly inhibited by Hg⁺, but Ca²⁺ could activate the enzyme activity. Activity was completely inhibited by PepstatinA inhibitor and ethylenediamin etetraacetic acid (EDTA). Inferred the protease was a metalloprotease and the character of that was similar to the pepsin.(6) At the excitation wavelength ofλex=278nm and 295nm, the fluorescence spectra of pH and ionic strength to shrimp head endogenous protease was determined by Shimadzu RF-5301 PC fluorescence spectrophotometer. Among them, the fluorescence intensity of endogenous alkaline protease as pH and ionic strength enhancement enhanced, but the position of fluorescence emission peak does not move, and under the same conditions, the secondary structure of acid protease is changed. By Spectrum-100 IR Spectrometer FTIR, with protease solution daubed on KBR chip, scanning scope 900-1700cm-1 and resolution 4cm-1, the result shows that different types of proteinase possesses different spectral bands data, but both kinds of endogenous protease containα- helix andβ- folding.(7) Endogenous alkaline and acid protease were purified from Litopenaeus vannamei Shrimp Head, protein sequencing was detected by reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The trypsin and pepsin were identified through matching in the website of NCBI. By comparing parts of the detected amino acid sequences of this two kinds of endogenous protease with the amino acid sequence of trypsin and pepsin from other different species with the Vector NTI suite 8.0 software, respectively. The results indicated that the terminal amino acid sequence of the activation segment of the endogenous alkaline protease was LSSPATLVSRVATVSLPR, which showed high homology to trypsin from Pig. The terminal amino acid sequence of the activation segment of the endogenous acidity protease was EFGLSETEPGTNF, which showed high homology to gastricsin from Xenopus laevis.