Activation Mechanism Of Endogenous Protease In Shrimp Head Of Litopenaeus Vannamei Induced By UV-C Irradiation Stress

A large amount of shrimp head by-products is produced during the processing of prawn.Shrimp head is rich in endogenous enzymes,with high enzyme activity,it is is prone to autolysis.Protein and other useful components in shrimp head can be recovered by autolysis of endogenous enzymes,and biological enzyme preparation can also be prepared by extracting the endogenous enzymes.Previous studies found that UV-C irradiation stress can promote the autolysis of shrimp head,mainly due to its activation of the endoproteinase,but the activation mechanism of endoproteinase and whether it has activation effect on other endoproteinases are still unclear.On the basis of investigating the effect of UV-C irradiation stress on the activation of the main endogenous enzymes in the head of Litopenaeus vannamei,taking the endogenous protease of Litopenaeus vannamei as the research object,by comparing and analyzing the differences in the factors affecting the enzyme activity,the enzymatic quality,the molecular weight and the molecular conformation of the purified endogenous acid and alkaline protease before and after UV-C irradiation stress,this paper explored the molecular mechanism of its inducing the activation of endogenous protease in the head of Litopenaeus vannamei.The main results are as follows:(1)The effects of temperature and p H on the main endogenous enzymes in shrimp head of Litopenaeus vannamei before and after UV-C irradiation stress were compared.The results showed that the optimum p H and temperature of acidic proteinase,alkaline proteinase and polyphenol oxidase were not significantly changed,but the optimum p H and temperature of lipase and chitinase were changed significantly.The optimum p H of the endogenous acidic protease of shrimp head is 3 and the temperature is about 60 ?.The optimum p H of the alkaline protease was 8 and the temperature was about 50 ?.The optimum p H of the endogenous chitinase was 5?6 and the temperature was about 40 ?.The optimum p H of the endogenous lipase was p H 10 and the temperature was about 40 ?.The optimum p H of the endogenous polyphenol oxidase was 8 and the temperature was about 25 ?.Under UV-C irradiation stress,the main endogenous enzymes including acid protease,alkaline protease,lipase and chitinase in the head of Litopenaeus vannamei were significantly activated.(2)The changes of the endogenous protease activity before and after UV-C irradiation stress were compared by specific inhibitors and inorganic ions.The results showed that the enzyme activity could be increased by adding appropriate Na Cl,and the changes of enzyme activity before and after UV-C irradiation stress were similar.The optimum ratio of material to liquid is 1:3.After UV-C irradiation stress,the endogenous alkaline protease activity of shrimp head increased by 42 %,while that of shrimp head with specific trypsin inhibitor increased by 5 %.After UV-C irradiation stress,the endogenous acidic protease activity of shrimp head increased by 59 %,while that of shrimp head with specific pepsin inhibitor increased by 36 %.The above results indicated that the endogenous acidic protease of shrimp head had pepsin-like activity,while the endogenous basic protease of shrimp head had a highe trypsin-like activity.(3)By means of ammonium sulfate fractional precipitation,gel chromatography,ion-exchange chromatography,electrophoresis and other means,the endogenous proteases of shrimp head before and after UV-C stress were isolated and purified.The molecular weight of the highly active endogenous proteases was determined and its homology was analyzed and identified.The results showed that the concentration of ammonium sulfate was 70% for the best precipitation of acidic protease and 60 % for the best precipitation of alkaline protease.After purification,the molecular weight of the endogenous alkaline protease in shrimp head is about 23 k Da,and the endogenous acidic protease in shrimp head is about 32 k Da.The endogenous basic protease of shrimp head has high homology with trypsin molecular structure of Litopenaeus vannamei and has trypsin-like properties.The endogenous acidic protease of shrimp head has high homology with aspartic acid protease,which has the properties of pepsin.(4)The effects of UV-C irradiation stress on the spatial conformation of endogenous protease in Litopenaeus vannamei head were investigated by spectral analysis.The results showed that UV-C irradiation stress treatment had little change in the secondary structure of the endogenous alkaline protease of shrimp head,but UV-C irradiation stress treatment may make the spatial conformation of the endogenous alkaline protease of shrimp head expand moderately,making it easier for the active center to bind to the protein substrate and give full play to its catalytic ability.After UV-C irradiation stress treatment,the endogenous acidic proteinase of shrimp head moved to the low-frequency region of-folding,and after UV-C irradiation stress treatment,the peak of the maximum emission wavelength showed a red shift with a span of 10?25 nm.It indicates that UV-C irradiation stress may destroy hydrogen bonds and other secondary bonds in protein molecules,thus damaging and changing the spatial structure of proteins.In the process of proteindenaturation,the molecular side chain groups of aromatic amino acids are gradually exposed to aqueous solution,and their environmental polarity increases,thus increasing their absorption peak.Changes in the spatial conformation of endogenous proteases induced by UV-C irradiation stress may be the main reason for their activation.UV-C irradiation stress may cause the spatial conformation of endogenous protease to unfold moderately,making it easier for the active center to bind to the protein substrate and give play to its catalytic ability.