Research On The Activity Regulation Of N-Acetyl-β-D-Glucosaminidase From Litopenaeus Vannamei

The production per unit of Litopenaeus vannamei tops among three major prawns in the world.And it is known that N-Acetyl-β-D-glucosaminidase(NAGase EC.3.2.1.52) plays a pivotal role in molting and nutritious metabolism of prawns. Hence,our study of enzymatic changes of prawns when sick,the effects of environmental pollutants,forage addition and common drugs used for aquatic breeding on the activities of NAGase,along with the study of acute toxic assays on prawn living bodies and the effects of heavy metal ions on activity of NAGase of prawns will benefit the prawn breeding industry and provide a more sufficient and direct scientific basis for monitoring the pollution in aquatic environment for culturing the prawn.Meanwhile,our study fulfills the blank in this field both domestically and internationally.Our study focuses on the NAGase extracted from Litopenaeus vannamei and falls into three categories:(1) Study of differences of activities and properties of NAGase in prawns between health ones and the ones with "red body disease(RBD)".Our results show that the activities of NAGase in shell membrane and viscera of healthy prawns are 34.80 U/mg and 35.34 U/mg,respectively;while the activities of NAGase in shell membrane and viscera of prawns with RBD are 38.32 U /mg and 20.02 U/mg, respectively.This illustrate that there are differences between healthy prawns and the ones with RBD in activity and basic enzymatic properties of NAGase.Although catalytic reaction kinetic constant K_m and V_m value and activity energy of NAGase from the shell membrane and viscera of prawns with RBD are higher,the optimal temperature of NAGase is lower;also pH and thermal stabilities of NAGase are worse. At the same time,the sensitivity of NAGase changes between healthy prawns and prawns with RBD to outside factors,including heavy metal ions like Hg²⁺,Zn²⁺,Cu²⁺, Cd²⁺,Pb²⁺,Al³⁺;organic pollutants like ethanol,formaldehyde,phenol,methanol, dioxane,DMF and DMSO;common chemical dressing agent and denaturant like urea, NBS and SDS. (2) Study of different effects of common antidote for aquatic bodies,such as sodium hypochlorite,symclosene and quaternary ammonium on the activity of NAGase from shell membrane and viscera of Litopenaeus vannamei.Meanwhile,the inhibitions of sodium hypochlorite and symclosene to these two different enzymes are irreversible.Moreover,our results show that benzylpenicillin sotassium,streptomycin, gentamicin and kanamycin have no effect on the activities of these two kinds of NAGase.Our results also show that among common antibiotics,ciprofloxacin activates these two kinds of NAGase while norfloxacin and sulfalene inhibit them. Sulfalene shows mixed reversible inhibition on NAGase from shell membrane while noncompetitive inhibition on NAGase from viscera;norfloxacin shows mixed reversible inhibition on NAGase from viscera.We also study the effects of several common vitamins on the activity of NAGase extracted from viscera.Our data illustrate that VB₁₂,niacin and lactoflavin have no significant effect on the enzyme activity while VB₁,VB₆ and ascorbic acid inhibit it in distinct degrees.Further kinetic study shows that the inhibition of VB₆ on the enzyme is noncompetitive.(3) Study of culture conditions for Litopenaeus vannamei,and different effects of heavy metal ions on NAGase activities.We study how different metal ions such as Cu²⁺,Zn²⁺,Cd²⁺,Pb²⁺,Hg²⁺ affect the activity of NAGase extracted from shell membrane and viscera of Litopenaeus vannamei.Our results show that no significant change in the activity of NAGase extracted from shell membrane and viscera of Litopenaeus vannamei treated with these five metal ions in short time.Nevertheless, the activities of these two NAGase decrease after 48 hours treatment with metal ions, which indicates a significant dose-dependent inhibition.