Evaluation And Regulation For Inhibitory Effects Of Tetracycline Antibiotics On Functional Enzymes

In recent years,antibiotics have been widely used in medicine,livestock and aquaculture industries.Due to their low body metabolic rate,most antibiotics are excreted into the environment through urine and feces in the form of original drugs or metabolites.It will cause serious pollution to water and soil,which will affect the activity of environmental microorganisms and functional enzymes.Tetracycline antibiotics?TCs?,as one of the most widely used antibiotics,have received widespread attention worldwide for their toxicity effects and regulatory strategies.At present,TCs toxicity pollution has been widely discussed,but there is no systematic research on the problem of TCs inhibition on functional enzyme activity in water,soil and sewage treatment:most studies only focus on the inhibition effect of antibiotics on single-functional enzyme activity,and do not involve the specific molecular mechanism of toxicity inhibition.Therefore,to clarify the differential inhibitory effect of antibiotics on the activity of functional enzymes and molecular mechanism will not only provide a theoretical basis for the systematic evaluation of the antibiotics toxicity effects and environmental risks,but also provide a technical basis for regulating the engineering application of antibiotic wastewater.With TCs served as the basic research substance,their inhibitory effect and molecular mechanism on catalase,urease,alkaline phosphatase and sucrase were evaluated by toxicity test,fluorescence spectrum experiment and molecular docking simulation technology;Furthermore,the chemical oxidation technology UV/H₂O₂ was used to control potential toxicity of TCs,and the inhibitory effects and structural characteristics of antibiotics/regulated by-products on catalase were analyzed through toxicity tests and molecular docking simulation,then the differential molecular mechanism was elucidated.The theoretical simulation data were compared with toxicity test results to fully explore the toxicity mechanism of TCs inhibiting enzyme activity.This study provides valid theoretical support to control their potential risk.The main research is as follows:?1?Study on the toxicity effects and mechanisms between TCs and functional enzymes.Toxicity experiments showed that TCs had significant inhibition on catalase activity.After linearly fitting the inhibition rate to the antibiotic concentration,the order of inhibition was tetracycline>oxytetracycline>chlortetracycline>doxycycline;TCs had significant promotion on urease activity.After linearly fitting the promotion rate to the antibiotic concentration,the order of promotion was tetracycline>oxytetracycline>doxycycline>chlortetracycline;TCs had an promoting effect on alkaline phosphatase activity.After linearly fitting the promotion rate to the antibiotic concentration,the order of promotion was oxytetracycline>tetracycline>doxycycline>chlortetracycline;TCs had a weak effect on sucrase activity.The interaction mechanism between TCs and functional enzymes were elucidated by fluorescence spectroscopy experiments and molecular simulation techniques.The results of fluorescence analysis showed that the fluorescence intensity had a significant downward trend,TCs and the functional enzymes have quenched.Combined with toxicity experimental data,the fluorescence quenching types of TCs on catalase,urease,alkaline phosphatase and sucrase could be inferred:TCs were statically quenched at the catalytic active center of catalase,the fluorescence intensity had a positive correlation with toxicity at 344.4 nm;TCs and urease were statically quenched,the fluorescence intensity was strongly correlated with toxicity.It is inferred that the reaction was caused by allosteric effects,and the interaction effect center was the non-catalytic active center;TCs and alkaline phosphatase were statically quenched.There was a weak positive correlation between fluorescence intensity and toxicity,and it is inferred that the reaction was caused by allosteric effects,and the interaction effect center was the non-catalytic active center.TCs were statically quenched at the non-catalytic active center of sucrase.Combined with molecular simulation technology research found:1)TCs form complexes with catalase in the catalytic active center,combination areas/energy changes and TCs toxicity were positively correlated,which could be used as an important factor in evaluating the mechanism of TCs inhibiting catalase activity.In the interaction between TCs and catalase,hydrogen bonding such as O^1C?Glu₂₅₂,O¹?Arg₁₉₅,O⁶?Asp₂₄₉49 and ion bonds for N⁴ with Glu₂₅₂52 were positively related to toxicity;But hydrogen bonding such as O¹⁰?Pro₃₆₃,O¹⁰?Lys₄₅₅,O¹²?Asn₁₂₇27 and ion bonds for N⁴ with Asp₃₇₉79 were negatively related to toxicity,the stronger the above effects,the less the inhibitory effect of TCs on catalase;Hydrogen bonding such as O¹?Arg₃₃₄,O⁶?Val₃₅₁,O¹⁰?Ser₃₁₆16 had little correlation with toxicity.2)TCs form complexes with urease in the non-catalytic active center,combination area has little correlation with toxicity;energy changes was negatively correlated with toxicity,which could be used as an important indicator to evaluate the mechanism of TCs on urease activity;In the interaction between TCs and urease,hydrogen bonding such as C⁴?Glu₂₅₇,N⁴?Asp₂₈₃,C⁴³?Glu₂₅₇57 and ionic bonding of N⁴-Glu₂₅₇,N⁴-Asp₂₈₃,N⁴-Asp₂₂₁,N⁴-Glu₃₆₉69 were positively related to toxicity;But hydrogen bonding such as O⁶?Glu₁₄₅,N⁴?Asp₁₁,C^4'?Glu₁₄₅45 and ionic bonding such as N⁴-Asp₁₁1 were negatively related to toxicity,the stronger the above effects,the less the inhibitory effect of TCs on urease;hydrogen bonding such as N²¹?Glu₂₂₀,N²¹?Asp₂₈₃,O¹¹?Ile₅₃₄34 and ion bond for N⁴ with Phe₅₆₇67 had less correlation with toxicity.3)TCs form complexes with alkaline phosphatase in the non-catalytic active center,combination areas/energy changes were positively correlated with toxicity,which could be used as an important indicator to evaluate the mechanism of TCs on alkaline phosphatase activity;In the interaction between TCs and alkaline phosphatase,hydrogen bonding such as C⁴²?Asp₃₈₀,C⁴¹?Asp₃₈₀,O¹³?Glu₄₁₁and the ionic bonding of N⁴-Asp₃₈₀,N⁴-Glu₄₀₇07 and N⁴-Glu₄₁₁11 were positively related to toxicity;But hydrogen bonding such as N²¹?Asp₁₅₃,O²¹?Ser₁₀₂,O²¹?Arg₁₆₆66 and ion bond for C⁶ with His₃₃₁31 were negatively related to toxicity,the stronger the above effects,the less the inhibitory effect of TCs on alkaline phosphatase;Hydrogen bonding such as O⁶?Asp₁₀₁,N²¹?Val₉₉,N²¹?Asp₃₂₇,O⁶?Glu₁₅₀50 had less correlation with toxicity.4)TCs form complexes with sucrase in the non-catalytic active center.The hydrogen bonds involved in the interaction between tetracycline and sucrase include N⁴?Glu₂₀₃,C⁴¹?Glu₂₀₃,N²¹?Leu₂₀₇,etc.,and ion bonds include N⁴-Glu₂₀₃,N⁴-Glu₂₀₉;The hydrogen bonds involved in the interaction of chlortetracycline and sucrase include N⁴?Glu₂₀₃,C^4'?Asp₂₂,C^4A?Glu₂₀₃,etc.,and ion bond include N⁴-Glu₂₀₃;The hydrogen bonds involved in the interaction of oxytetracycline and sucrase include O¹¹?Asn₁₁₄,N²¹?Glu₂₀₃,N²¹?Asn₂₂₈,etc.,and ion bonds include N⁴-Glu₂₀₃,N⁴-Asp₂₂,N⁴-Glu₂₅₁;the hydrogen bonds involved in the interaction of doxycycline and sucrase include N⁴?Glu₂₀₃,O¹¹?Asn₁₁₄,N²¹?Glu₂₀₃,etc.,and ion bond include N⁴-Glu₂₀₃,so the above hydrogen bonds and ion bonds were the key indicators for evaluating the interaction mechanism of TCs and sucrase.?2?Effect and mechanism for UV/H₂O₂ regulation of TCs inhibiting catalase activity.TCs were pretreated by UV/H₂O₂ oxidation,and the order of degradation rate was as follows:k_tetracycline>k_{oxytetracycline}>k_{chlortetracycline}.Then the enzyme activity inhibition experiment was carried out on catalase.By comparing the inhibition rates of the samples before and after oxidation,it was found that the inhibition of catalase activity by the oxidized antibiotic samples was greater than that of the corresponding concentration standard.This indicates that the by-products contained in the oxidized antibiotic samples were also somewhat toxic to catalase.Subsequently,the typical by-product structures were obtained through mass spectrometry separation and molecular docking simulation experiments between DBPs and catalase were performed to obtain hydrogen bonding interactions.The study found that the order of TC-DBPs total hydrogen bonding interactions was C₂₀H₁₈NO₁₀>C₂₀H₁₈NO₉>C₂₀H₁₈NO₈;the order of CTC-DBPs total hydrogen bonding interactions was C₂₀H₁₇ClNO₈>C₂₁H₁₉ClNO₉>C₂₁H₁₉ClNO₇;the order of OTC-DBPs total hydrogen bonding interactions was C₂₂H₂₅N₂O₁₁>C₂₂H₂₃N₂O₁₀>C₂₁H₂₃N₂O₈>C₂₁H₂₅N₂O₈>C₂₂H₂₅N₂O₁₀>C₂₂H₂₃N₂O₉.Compared with the hydrogen bonds between TCs and catalase,the tetracycline by-product C₂₀H₁₈NO₉ retained the same hydrogen bonding effects as the protoxin such as O¹¹?Asn₁₂₇,O¹?Lys₂₅₆,etc.;C₂₀H₁₈NO₁₀0 retained the same hydrogen bonding effects as the protoxin such as O¹¹?Asn₁₂₇,N²¹?His₃₆₈,etc.;C₂₀H₁₈NO₈ retained the same hydrogen bonding effects as the protoxin such as O¹¹?Asn₁₂₇,N²¹?His₃₆₈68 and so on.The chlortetracycline by-product C₂₁H₁₉ClNO₉ didn't have the same hydrogen bond;C₂₀H₁₇ClNO₈ retained the same hydrogen bonding effects as the protoxin N^2'?Asn₃₂₃,O¹²?SO4₅₀₅;C₂₁H₁₉ClNO₇ retained the same hydrogen bonding effect as the protoxin O¹²?SO4₅₀₅.Oxytetracycline by-product C₂₂H₂₅N₂O₁₀0 retained the same hydrogen bonding effects as the protoxin N⁴?Glu₁₂₅,C⁵?Asp₃₇₉,O⁶?His₃₆₈;C₂₂H₂₅N₂O₁₁1 retained the same hydrogen bonding effects as the protoxin C⁵?Asp₃₇₉,O⁶?His₃₆₈;C₂₂H₂₃N₂O₁₀0 retained the same hydrogen bonding effect as the protoxin O⁶?Asn₃₆₉;C₂₁H₂₅N₂O₈ retained the same hydrogen bonding effects as the protoxin O¹³?His₃₅₄,O⁶?Asn₃₆₉;C₂₁H₂₃N₂O₈ retained the same hydrogen bonding effects as the protoxin O¹³?His₃₅₄,O⁶?Asn₃₆₉;C₂₂H₂₃N₂O₉ retained the same hydrogen bonding effect as the protoxin O¹³?His₃₅₄.Therefore,the above hydrogen bonding could be used as important indexes for evaluating the inhibitory effects of TCs and their by-products on catalase activity.In summary,In summary,this study found that TCs had significant differences in the toxicity effects and mechanisms of catalase,urease,alkaline phosphatase and sucrase activities;In view of the significant inhibitory effect of TCs on catalase,it could be regulated by the UV/H₂O₂ combined oxidation method in advanced oxidation.However,the inhibition effect and mechanism evaluation showed that the toxicity of by-products could not be ignored;Through molecular simulation,it was found that some key sites and hydrogen bonding that were closely related to toxicity effects could be used as an important indicator to fully regulate the mechanism of TCs inhibition on catalase activity.