The Effects And Mechanisms Of Typical Veterinary Antibiotics On Urease And ?-glucosidase

Antibiotics were often used in the prevention and treatment of animal diseases Because they played an extremely important role in controlling infectious diseases of livestock and poultry.they,The application amount of antibiotics increased gradually with the development of intensive livestock and poultry breeding,w-hich led to environmental pollution.After entering into water and soil environment,they would affect the activities of environmental microorganisms and enzymes,and then affected the process of carbon and nitrogen conversion.Urease(EC3.1.1.5)was one of the most important nitrogen cycle indicator enzymes in the environment and could rapidly catalyze the hydrolysis of urea to ammonia and carbon dioxide.?-glucosidase(EC3.2.1.21)was one of the most important carbon cycle indicator enzymes in the environment,which hydrolyzed and bound to terminal non-reducing ?-D-glucose bonds while releasing P-D-glucose and corresponding ligands.The effect of small molecular environmental pollutants on enzyme activity in soil had been reported,but the effect on enzyme activity mechanism,especially on enzyme structure,was rarely reported.To reveal its effect on enzyme activity,this experiment learned the interaction mechanism between three typical antibiotics(sulfamethazine,enrofloxacin and tetracycline)and two important enzymes(urease and ?-glucosidase)in carbon-nitrogen conversion from the molecular structure level,and then provided the theory for the mechanism of antibiotic promoting or inhibiting enzyme activity.The effects of sulfamethazine,enrofloxacin and tetracycline on urease and ?glucosidase activity were determined by pure chemical experiments using phenol-sodium hypochlorite colorimetric method and p-nitrobenzene-?-D-glucoside(pNPG)method.Then the interaction of three antibiotics with urease and ?-glucosidase in water environment was studied by fluorescence spectroscopy combined with UV-Vis absorption spectroscopy.The results of urease experiment showed that both sulfamethazine and enrofloxacin promoted urease activity,while tetracycline inhibited urease activity with low concentration,but promoted with high concentration.The order of action of three antibiotics on urease activity was as follows:tetracycline>enrofloxacin>sulfamethazine,which gradually enhanced with the increase of benzene ring quantity.The fluorescence spectra showed that sulfamethazine,enrofloxacin and tetracycline all formed 1:1 complex with urease and quenched the endogenous fluorescence of urease by static quenching,and their binding constants were 1.66×103,2.82×104,and 1.27×105 KA·L-1·moL-1,respectively.The combined UV-Vis absorption spectra indicated that sulfamethazine,enrofloxacin and tetracycline all led to changes in urease conformation.The results of molecular docking showed that all three antibiotics formed hydrogen bonds with urease.Sulfadiazine formed two hydrogen bonds with ARG439(Arginine)and VAL591(Valine),enrofloxacin formed three hydrogen bonds with ARG439 and CYS592(Cysteine)and tetracycline formed two hydrogen bonds with ASP494(Aspartic acid).Ttheir binding energies were-5.55,-5.82,and-6.13 kJ·mol-1 respectively,indicating that the greater the enzyme activity,the lower the binding energy.The molecular docking parameter information indicated that the hydrogen bonding force,van der waals force and electrostatic force were all the main forces of the interaction between the three antibiotics and urease.The results of(3-glucosidase study showed that all three antibiotics promoted?-glucosidase activity,and the order of extents was:tetracycline>enrofloxacin>sulfamethazine,which gradually enhanced with the increase of benzene ring quantity.The fluorescence spectra showed that sulfamethazine,enrofloxacin and tetracycline all formed 1:1 complex with ?-glucosidase and quenched the endogenous fluorescence of?-glucosidase by static quenching.Their binding constants were 1.66× 103,2.82×104,and 1.27 ×105 KA·L-1·moL-1 espectively.Combined with UV-Vis absorption spectra,it was shown that sulfamethazine,enrofloxacin and tetracycline all lead to ?-glucosidase conformation changes.The molecular docking results showed that all three antibiotics formed hydrogen bonds with ?-glucosidase,among which sulfadiazine and enrofloxacin formed two hydrogen bonds with SER304(Serine)and ARG280(Arginine),tetracycline formed seven with LYS396(Lysine),GLN276(Glutamine),SER301(Serine),ASP283(Aspartic acid),and GLU279(Glutamine acid),and the above amino acids were all hydrophilic amino acids.The binding energies of the three antibiotics were-5.72,-6.06,and-7.70 kJ·mol-1 respectively,indicating that the larger the enzyme activity,the lower the binding energy.The molecular docking parameter information indicated that hydrogen bonding,van der Waals force,and electrostatic force were the main forces that interacted with the three antibiotics and ?-glucosidase.