| Polychlorinated biphenyls(PCBs)are a kind of persistent organic pollutant widely present in the ecological environment.They have high toxicity,environmental persistence,bioaccumulation,semi-volatileness,and long-distance migration.There are various inconveniences in the current commonly used detection methods for PCBs,so there is an urgent need to develop new detection methods that are fast,sensitive,simple,and efficient.The biosensor can directly measure samples with high accuracy,so it has become a new type of environmental pollutant detection method.The whole-cell biosensor is a device that uses whole microbial cells as sensitive elements and can quickly sense pollutants in the environment.Because of its fast response,small size,low cost,and in-situ monitoring,it has shown great potential in the fields of environmental monitoring,drug research and development,and the food industry.In this thesis,microorganisms are used as the sensing center to construct a whole-cell microbial sensor for detecting PCBs.The main results are as follows:1.The XylS/Pm system derived from the TOL plasmid of P.putida was fused with green fluorescent protein(e GFP)to construct a chlorobenzoic acid sensor system.Then explored the most suitable bacteria for the system toward E.coli XL10-gold,DH10 B,Top10,DH5α.It was found that when E.coli DH5α was used as the host bacteria for expression,the detection limit of benzoic acid and 3-chlorobenzoic acid was 10 μM,and 2-chlorobenzoic acid and4-chlorobenzoic acid could not be detected;2.The upstream pathway of PCBs aerobic degradation was constructed in E.coli DH5α,combined with the chlorobenzoic acid sensing pathway,so that PCBs were degraded into chlorobenzoic acid,thereby inducing the expression of the Pm promoter to produce a green fluorescent signal.The sensitivity of this pathway to different concentrations of2,2-dichlorobiphenyl,2,6-dichlorobiphenyl,4,4’-dichlorobiphenyl and2,4,4’-trichlorobiphenyl was tested.It was found that it can only respond to2,2-dichlorobiphenyl above 0.5 m M,but cannot do with 2,6-dichlorobiphenyl,4,4’-dichlorobiphenyl and 2,4,4’-trichlorobiphenyl;3.In order to improve the degradation ability of PCBs,based on the T335A/F336 M mutants reported before,17 single-point mutations were designed using the online software Fireprot,and then the mutants were obtained experimentally and the degradation ability of different mutants on PCBs was determined.Compared with T335A/F336 M,the T335A/F336M/G389 M mutant has increased the degradation ability of 2,6-dichlorobiphenyl and 4,4’-dichlorobiphenyl by 71% and 44%,respectively;4.In order to improve the sensitivity of the chlorobenzoic acid sensing pathway,XylS was molecularly modified based on structural analysis,molecular docking results and existing research.A library of single-point saturation mutants at the A111 site and five mutants of St Ep13 were constructed.It was found that A111 V reduced the detection limit of benzoic acid and 3-chlorobenzoic acid from 10 μM to 1 μM,but it still failed to respond to 2-chlorobenzoic acid and 4-Chlorobenzoic acid.The detection limit of the five mutants of St Ep13 for the four chlorobenzoic acids was reduced to 1 μM.Subsequently,we combined the XylS-St Ep13 five mutations with the three mutants of biphenyl dioxygenase Bph A1-T335A/F336M/G389 M,and found that the recombinant strain Cb3x5 is paired with 2,2-dichlorobiphenyl and2,4,4’-trichloro The detection limit of biphenyl is 0.01 m M,and the detection limit of4,4’-dichlorobiphenyl and 2,6-dichlorobiphenyl is 0.05 m M;5.In addition,we found that when the chlorobenzoic acid sensor system was expressed in E.coli DH5α,the fluorescence signal of p-chlorobenzoic acid was increased by about 4times after the strain was subcultured,and then the subculture was continued with the fluorescence-enhancing strain DX-H.The sensitivity of benzoic acid is weakened,and compared with the original strain,the growth rate of DX-H is slower.When the plasmid is extracted with the same amount of bacteria,the plasmid concentration of DX-H is lower,and it will be extracted from DX-H.The resulting plasmid was sequenced and found no mutations.Why this strain cannot be passaged stably still needs to be further explored;6.The tolerance of E.coli,B.subtilis,B.velezensis,and P.putida to PCBs was explored,and the PCBs whole-cell biosensor was constructed in Bacillus subtilis SCK6.First,the upstream pathway of PCBs aerobic degradation was reconstructed,and PCBs can be successfully degraded by HPLC detection.The chlorobenzoic acid-sensing pathway was subsequently reconstructed,but the fluorescence signal could not be detected by3-chlorobenzoic acid induction culture.The analysis believed that SCK6 lack of the sigma factor required for Pm transcription,and subsequent experiments need to modification of the sequence identified by the Pm promoter for sigma factor.The next step is to replace the reporter protein e GFP in the PCB whole-cell biosensor with protease so that the signal cascade can be amplified to enhance the detection sensitivity and provide options for the detection of PCBs.On the other hand,in the industrial production of enzyme preparations,high-efficiency expression of economically significant proteins in Pichia pastoris GS115 is an important means.Chitinase and chitin oligosaccharides can be widely used in many fields,so the development of high-producing chitinase strains is significant.The specific strategies and results are as follows:1.Construct the chitinase gene derived from Bacillus licheniformis WX-02 strain into the p HBM905 M expression vector for the tandem expression of the gene expression cassette,and then construct the multi-copy tandem module through the biological brick strategy and integrate it into the Pichia pastoris GS115 genome Induced expression at the his4 gene locus on the above;2.Shake flasks of recombinant chitinase strains containing 1,2,3,4,6 multi-copy expression cassette plasmids,and found that P.pastoris A4 C co-expressed with HAC1 gave the highest expression level.The activity of ChiA in the fermentation supernatant reached168.78 U/m L with 12.7 mg/m L protein,which is the highest data reported until now;3.The ability to hydrolyze substrates of different concentrations was explored,and it was found that the chitinase can hydrolyze 30% of colloidal chitin with a conversion rate of 74%.The hydrolyzed product is mainly Glc NAc,followed by N,N’-diacetylchitobiose.In addition,combined ChiA with Bs Nag Z,Glc NAc can be obtained as the only product with a conversion rate of 88%.4.In this study,the hydrolysate of ChiA can obviously promote the germination and rooting of rice and wheat and can increase the seedling length and root length of seedlings by at least 1.6 times within 10 days. |
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