阐明磺胺类抗生素生物降解机理对加强环境中磺胺类抗生素的生物去除具有重要的意义。本研究筛选出可以高效降解磺胺类抗生素的降解菌株，以4类（共11个）磺胺为降解对象，利用基因组学和量子化学计算手段对磺胺类抗生素的微生物降解机理进行研究。16S rRNA基因测序分析结果表明分离出来的降解菌株属于Paenarthrobacter属，为革兰氏阳性菌。降解动力学试验表明，该菌株对磺胺二甲基嘧啶的降解速率最大，为0.058 mmol?L-1∙h-1，对磺胺甲氧哒嗪的降解速率最小，为0.013 mmol?L-1∙h-1。虽然降解菌株对不同类型磺胺的降解速率差异大，但基本符合对磺胺的适应时间越长，降解速率就会相对较慢的规律。代谢产物分析结果表明磺胺的胺化杂环侧基结构会成为稳定的终产物。通过基因组学分析，发现该降解菌株里含有负责降解磺胺的功能基因sad基因簇。该降解功能基因簇编码的双组份黄素单加氧酶会催化过氧态的黄素作为亲电试剂攻击磺胺，发生亲电反应，导致磺胺的S-N键发生断裂，并生成胺化杂环侧基结构作为磺胺生物降解的最终稳定产物。量子化学计算结果显示：在磺胺的生物降解过程中，中性分子是磺胺发生亲电反应的主要形态，C7是反应的活性位点。由于不同磺胺的磺酰胺基端取代基结构会影响磺胺分子的电子结构，从而导致C7的反应活性不同。此外，磺胺的存在形态也会导致表观反应速率上的差别。由量子化学计算得到的理论指标从分子层面解释了导致不同磺胺的降解反应差异的原因。本研究可以为预测实际应用中微生物降解抗生素的行为规律提供一定的理论参考与指导价值。
Elucidating the biodegradation mechanisms is of great significance to enhance the biological removal of sulfonamide antibiotics in the environment. In this study, one degrading strain that could efficiently degrade sulfonamide antibiotics was isolated. Eleven sulfonamide antibiotics belonging to four types were selected as biodegradation targets. The biodegradation mechanisms of sulfonamide antibiotics were analyzed by means of genomics and quantum chemistry calculation.According to 16S rRNA genes sequencing result, the highly efficient degrading strain was identified as genus Paenarthrobacter, which is gram-positive. The kinetics experiment showed that the biodegradation rate of sulfamethazine was the highest (0.058 mmol?L-1∙h-1) and that of sulfamethoxypridazine was the lowest (0.013 mmol?L-1∙h-1). Although the biodegradation rates of different types of sulfonamide antibiotics varied greatly, it basically conformed to the pattern that the longer the adaptation time to antibiotics was, the slower the degradation rate would be. The aminated heterocyclic side group of each sulfonamide was detected as a stable product according to the result of metabolites identified by mass spectrometry.It was found that there was a functional sad gene cluster responsible for the biodegradation of sulfanilamide antibiotics through genomics analysis. A two-component flavopretein monooxygenase encoded by the gene cluster catalyzed the electrophilic reaction of hydroperoxyflavin against sulfanilamide antibiotics, resulting in the breaking of S-N bond of sulfonamide antibiotics and the formation of aminated heterocyclic side moiety as the final stable product of sulfanilamide biodegradation.The calculation results of quantum chemistry showed that neutral molecule was the main form of electrophilic reaction, and C7 was the active site. Sulfonamide antibiotics with different sulfo-substituents had different electronic structures, as a result, the activity of C7 was different. In addition, the existing forms of sulfonamides also led to the difference of apparent reaction rate. The theoretical indexes calculated by quantum chemistry explained the reasons for the difference of biodegradation reactions of sulfonamide antibiotics from the molecular level, which provided theoretical reference and guidance value for predicting the biodegradation behavior and pattern of antibiotics in practical application.
