Screening And Identification Of Antibiotic Resistant Bacteria And The Application Of The Key Enzyme β-lactamase

With the rapid development of pharmaceutical industry, a large amount of pharmaceutical wastewater, especially antibiotic production wastewater is generated by pharmaceutical industries every year, which will cause a series of environmental problems. Such wastewater normally contains complicated components with high toxicity, high chemical oxygen demand(COD) and low biodegradability, which would do great harm to the ecological environment and people’s health if they are discharged without any proper pretreatments. Although several physical and chemical treatment processes have been developed to remove the pharmaceuticals from wastewater, they usually exhibit low degradation efficiency toward some organic compounds in the wastewater. Moreover, some chemical methods can even cause secondary pollution. Therefore, it is of more importance to find an efficient and environmental friendly way to treat antibiotics polluted wastewater. Biological treatments have emerged as a powerful technology for treating pharmaceutical waste because bacteria can use the toxic pollutants in waste water as the only carbon source or main carbon source for their growth. These methods are characterized as high efficient, simple operation management, low cost of the processing equipment and environment friendly processes. Thus, the enzymatic treatment of antibiotic wastewater is of great significance.In this study, our goal is to develop an efficient biodegradation system to treat antibiotic production wastewater. For this, several β-lactamase resistant strains were selected from antibiotic production wastewater without adding any extra carbon sources. Those obtained strains were subsequently subjected for further characterization. The genes encoding β-lactamase were cloned, sequenced and expressed. For further application, the expressed β-lactamase was immobilized by using CLEAs method. The main results are summarized as follows: 1) two superiority strains were selected from wastewater by using wastewater organic compounds as the sole carbon source. They were identified as Acinetobacter baumannii and proteus, respectively. 2) Antibiotic susceptibility test indicated that the two strains showed the resistance against high levels of ampicillin, and contain the extended-spectrum β-lactamase. 3) We cloned and synthesized the β-lactamase gene. The expression conditions were optimized: 2% of the microbial quantity, 100 μg/mL Amp, then cultivated bacteria at 37 ℃ to reach OD600 at 0.6-0.8 with shaking speed of 180 rpm. The β-lactamase was then expressed by adding 0.05 mg/m L arabinose at 16 ℃ stand for 1.5 h. 4) We used the cross-linked enzyme aggregates method to immobilize the beta-lactamase. The optimal immobilized conditions are as follows: 1 ml 25 mg/m L enzyme fluid, add BSA of a third of enzyme levels(W/W) as a protective agent, 5 ml 75 % saturated ammonium sulfate solution as a precipitation agent and the concentration of 0.5 % glutaraldehyde as a crosslinking agent. The immobilized enzyme retained about 94.5 % activity of the free enzyme; retained 70 % activity after storage at 70 ℃ for 6 hours; retained 90 % activity after storage at 4 ℃ for 2 weeks.In contrast, the free enzyme totally lost activity after storage at 60 ℃ for 2 hours and at 4 ℃ for 30 hours. Thus, the obtained immobilized enzyme is more stable than the free enzyme and can be recycled. 5) We added the immobilized beta-lactamase into the antibiotic wastewater, and put them in the shaker for 35 hours at 30 ℃ with 180 rpm shaking rate. Finally we found that chemical oxygen demand of the pharmaceutical wastewater was removed 72.9% after 24 hours. However, the free enzyme had no such an effect on the pharmaceutical wastewater because the free enzyme activity was lost in the pharmaceutical wastewater. The results showed that the enzymatic method of treating antibiotic wastewater is efficient and is potencial in the industrial wastewater treatment.