Construction And Characterization Of Cell-Surface Displayed Engineering Strains For Organophosphate Biodegradation

Organophosphates pesticides are used extensively in agriculture.However,some of them bring great threat to the human being health and entironment owing to their high toxicity and residua.Microbes act as the principal role for biodegradation of organophosphates pesticides in environment.Most studies of organophosphorus degrading enzymes have focused on organophosphorus hydrolase(OPH) from Flavobacterium sp.ATCC27551 and Pseudomonas diminuta MG,and the gene opd of OPH have been investigated deeply in the molecular biology.Many recombinant strains acting as whole cell biocatalyst for organophosphates degradation have been constructed using traditional DNA recombinant technology,whose whole cell activity of OPH,however,is always inhibited by the cell membrane which prevents full attachment of enzyme and substrate. Cell surface display is a new biotechnology developed in recent ten years.Protein can be transported and anchored onto the cell surface using different anchoring system.Such cells can convert substrate more efficiently without the diffusion limitations.This new technology provides an idea to resolve the obstacle of OPH across the cell membrane.In this research,the work can be divided into three parts.The first part is focus on isolation and identification of ice-nucleating strain.We have isolated an ice-nucleating strain from plant tissue of frost injury,named HY-1.This strain is Gram-negative,bacillus, obvious mobility and obligate aerobic.Its ice-nucleating activity is obvious;the average ice-nucleating rate in 2 min is 92.5%under -8℃.According to the result of biological and chemical identification and 16S rDNA sequence analysis,HY-1 belongs to Pseudomonas sp.,and it's the most similar to P.fluorescens on genetic distance.The organophosphorus hydrolase from Flavobacterium species was expressed on the cell surface of E.coli JM109 using N-terminate anchor(INPN) of ice nucleation protein (InaK) from Pseudomonas syringae KCTC1832.The recombinant plasmid pMPL405 was constructed containing the 1553 bp fusion gene of inpn and opd.An obvious band of approximately 57 ku,corresponding to INPN-OPH fusion was detected in SDS-PAGE of total cell protein.The maximum whole cell OPH activity was acquired when cells were grown in M9 medium suppemented with ampicillin to a final concentration of 50μg/mL at 20℃and induced for 8 h by adding IPTG and cobalt chloride to a final concentration 0.1mmol/L,respectively.More than 91%of the OPH activity was located on the cell surface as determined by protease accessibility.OPH was displayed and anchored onto the cell surface of Pseudomonas putida AB92019 using ice nucleation protein anchor linked to the N-terminal region of OPH. The construction of plasmid pOpr-nopd was realized by a series of digestion and ligation. Double digestion confirmed that the 1553bp fragment of inpn-opd fusion gene have been inserted into the recombinant plasmid.SDS-PAGE profile confirmed that the fusion protein of approximately 80 ku was expressed in the engineering strain.91%of OPH whole-cell activity was detected on the cell surface by protease accessibility assay.The maximum activity was obtained when cells were grown in LB medium supplement carbencillin to a final concentration of 500μg/mL at 28℃for 48 h,and cobalt chloride was added at 100μmol/L.In the absence of antibotic selection,plasmids were still favorably maintained 100%in seven days culturing.Suspended cultures also exhibited good stability,retaining almost 93%of whole cell OPH activity over a period of 30 days.