Microbial Synthesis And Properties Of Novel Microplastic Degrading Enzymes

The pollution of microplastics is becoming more and more serious.One of the solutions is to biodegrade plastics to save resources and alleviate the environmental threats brought by plastics.Polyethylene terephthalate（PET）,a petroleum-based plastic in huge quantities,is often used as packaging material or textiles,and it is not recycled much and is thrown away in the environment.Improving the biodegradation efficiency of PET and recovering the monomers of PET can realize the recycling of PET,alleviate the current situation of resource scarcity and reduce environmental pressure.In this paper,the cutinase ICCG,which has the highest PET degradation efficiency,was used as the research object,and a high-performance liquid chromatography method for the detection of terephthalic acid,a PET degradation product,was established.The enzymatic properties of ICCG were explored,and the results were as follows:（1）An ion suppression chromatography detection method was established for terephthalic acid（PTA）,a degradation product of PET.The optimized chromatography conditions were:acetonitrile-water=13%-87%（v:v）;acetic acid adjusted mobile phase p H=3.00;flow rate:1.0m L/min;injection volume:5μL;detection wavelength:242 nm;temperature:room temperature.（2）Using genetic engineering technology,a new genetically engineered bacterium E.coli BL21-p ET22b-ICCG was constructed,which can normally express the active PET-degrading enzyme ICCG,and this engineered bacterium is not suitable for LB medium induced by IPTG,and suitable for lactose-induced LB medium.Autoinduction medium.（3）The main carbon source components（glucose,glycerol,and lactose）of ZYM autoinduction medium were optimized by single factor and surface response experiments.,the activity of recombinant E.coli supernatant at this concentration increased by about 56.53%compared with the original ZYM self-induction medium.（4）The enzymatic properties of ICCG were studied,the temperature activity range was 40℃-90℃,and the optimum reaction temperature was 72℃.The optimum reaction p H is 8.00.When the p H is lower than 5,almost no reaction occurs,and the reaction conditions are alkaline.In the50m L reaction system,when the ICCG concentration was constant,the substrate PET addition amount of 0.1g had higher degradation efficiency and PET degradation rate（ICCG concentration was 0.46mg/L,and the 8h PET complete degradation rate was 5.36%）.When investigating the effects of 7 kinds of metal ions(Ca²⁺,Mn²⁺,Mg²⁺,Fe²⁺,Co²⁺,Cu²⁺,Zn²⁺)on the catalytic activity of ICCG,it was found that 5m M Ca²⁺contributed to the catalytic effect of ICCG,and high concentrations of Mn²⁺,Mg²⁺,Co²⁺,Cu²⁺It may have an activating effect on ICCG activity,low concentration of Fe²⁺may have an activating effect on ICCG activity,and Zn²⁺has an overall inhibitory effect on ICCG catalytic activity.Organic reagents（methanol,ethanol,acetonitrile）have little effect on the catalytic activity of ICCG.5%-10%methanol and 5%ethanol can activate the activity of ICCG,but acetonitrile has a weak inhibitory effect on the activity of ICCG on the whole.The presence of a certain concentration has an activating effect on ICCG activity.The surfactant glycerol can activate the activity of ICCG to a certain extent at5%-10%.The protein deformer urea can inhibit the activity of ICCG to different degrees.EDTA has a significant activation effect on ICCG at 5m M.In addition,ICCG has high thermal stability.The activity of ICCG is higher than that in the unincubated state when incubated at the optimum temperature,and the ICCG activity remains high after incubation for 20 h.In this paper,a test method for terephthalic acid was established,which can provide a reference for the subsequent characterization of PET degradation efficiency;a new ICCG genetically engineered bacteria was constructed.The chemical properties were explored in detail to provide some theoretical support for optimizing the degradation efficiency of PET in the future.