固定化核酸酶的制备及其降解沼液中抗生素抗性基因sul 1的研究

Preparation of immobilized nuclease and its degradation on antibiotic-resistance genes sul 1 in biogas slurry

为了削减畜禽沼液中抗生素抗性基因,以壳聚糖微球为载体,戊二醛为交联剂,采用共价交联法将核酸酶固定到壳聚糖微球上,优化了固定化核酸酶的制备条件,并探究了固定化核酸酶对模拟废水和猪场沼液中磺胺类抗生素抗性基因sul 1的去除效应,为畜禽沼液中抗生素抗性基因进行生物酶法降解提供了新思路。结果表明:1)核酸酶成功固定化在壳聚糖微球表面。最佳制备条件为壳聚糖质量分数4%、戊二醛体积分数2%、交联时间8 h、固定化时间10 h。2)固定化核酸酶在模拟废水中的处理效果极佳,适用pH为5.5~8.5,适用温度为4~37℃,对模拟废水中sul 1的去除率可达100%。重复使用5次后,去除率仍保持在80%以上。3)对猪场原沼液中sul 1的去除效果不及模拟废水。稀释沼液可以有效缓解对核酸酶的抑制,稀释5倍后,在处理温度37℃、处理时间80 min条件下,对沼液中sul 1的去除率最高可达72.2%,重复使用5次后保持44.5%的酶活力。

In order to explore the method of reducing antibiotic-resistance genes(ARGs)in livestock and poultry biogas slurry,the nuclease was immobilized on chitosan microspheres by covalent cross-linking method with chitosan microspheres as the immobilization carrier and glutaraldehyde as the cross-linking agent.The preparation conditions of the immobilized nuclease were optimized,and the immobilized nuclease was used to remove the sulfonamides ARGs sul 1 from simulated wastewater and pig farm biogas slurry to provide a novel bio-enzymatic degradation approach for ARGs in livestock and poultry biogas slurry.Results showed that:1)nuclease was successfully immobilized on the surface of chitosan microspheres.The optimal preparation conditions were chitosan mass fraction of 4%,glutaraldehyde volume fraction of 2%,cross-linking time of 8 h,and immobilization time of 10 h.2)The immobilized nuclease exhibited outstanding treatment efficiency in simulated wastewater,within pH range of 5.5-8.5 and temperature range of 4-37℃.The removal efficiency of sul 1 in simulated wastewater approached 100%.Even after 5 repeated uses,the removal efficiency of sul 1 in simulated wastewater remained above 80%.3)The removal effect of sul 1 in raw pig farm biogas slurry was inferior to that in simulated wastewater.Dilution of the biogas slurry effectively mitigated the inhibitory effect on the nuclease.After a 5-fold dilution,at the treatment temperature of 37℃and treatment time of 80 min,the highest removal efficiency reached 72.2%.The enzyme activity remained at 44.5%after repeated use of 5 times in the biogas slurry.