Description

1) Micro-organisms degrades organophosphorus, and organophosphorus-degradation enzyme opdA Faced with the stress of human pollution such as pesticides, nature itself has evolved many methods to deal with these problems. For example, many natural micro-organisms contain enzymes to degrade organophosphorus pesticides. Currently the micro-organisms which are capable to degrade organophosphorus pesticides include bacteria, fungus, actinomycete and alga. As the research goes further, people find that these degrading effects come from secreting an enzyme, which can hydrolyze phosphoester bonds, organophosphorus degradation enzyme. Because each organophosphorus pesticide has similar structure and protein sequence, one kind of organophosphorus degradation enzyme is capable todegrade multiple kinds of organophosphorus pesticides. Organophosphorus-degradation enzyme has been mostly recognized as the best method to eliminate pesticide residues currently. At present, many enzymes have been identified to be used to degrade organophosphate pesticides. Among these enzymes, the organophosphorus-degradation enzyme (opdA) which comes from Agrobacterium radiobacter P230 has wider targets and higher enzyme-catalyst efficiency. In recent years, the research on the structure and function of organophosphorus-degradation enzyme has gained promising progress,. Thus, it is possible to improve the properties of organophosphorus-degradation enzyme through genetic engineering and protein engineering method, which meet requirements of different applications. 2) In this project, we will use organophosphorus-degradation enzyme opdA to eliminate residual organophosphorus pesticide on fruits and vegetables. The organophosphorus-degradation enzyme (opdA) gene opdA （NCBI genbank：Accession: AY043245.2） programmed by Agrobacterium radiobacter contains 1,155 nucleic acids, programming 384 amino acid residues. The N-terminal of protein sequence is the signal peptide while the C-terminal is the degradation-enzyme sequence. The nucleic acid sequence and amino acid sequence are as follows: Nucleotide sequence at gcaaacgaga agagatgcac ttaagtctgc ggccgcaata actctgctcg gcggcttggc tgggtgtgca agcatggccc gaccaatcgg tacaggcgat ctgattaata ctgttcgcgg ccccattcca gtttcggaag cgggcttcac actgacccat gagcatatct gcggcagttc ggcgggattc ctacgtgcgt ggccggagtt tttcggtagc cgcaaagctc tagcggaaaa ggctgtgaga ggattacgcc atgccagatc ggctggcgtg caaaccatcg tcgatgtgtc gactttcgat atcggtcgtg acgtccgttt attggccgaa gtttcgcggg ccgccgacgt gcatatcgtg gcggcgactg gcttatggtt cgacccgcca ctttcaatgc gaatgcgcag cgtcgaagaa ctgacccagt tcttcctgcg tgaaatccaa catggcatcg aagacaccgg tattagggcg ggcattatca aggtcgcgac cacagggaag gcgaccccct ttcaagagtt ggtgttaaag gcagccgcgc gggccagctt ggccaccggt gttccggtaa ccactcacac gtcagcaagt cagcgcgatg gcgagcagca ggcagccata tttgaatccg aaggtttgag cccctcacgg gtttgtatcg gtcacagcga tgatactgac gatttgagct acctaaccgg cctcgctgcg cgcggatacc tcgtcggttt agatcgcatg ccgtacagtg cgattggtct agaaggcaat gcgagtgcat tagcgctctt tggtactcgg tcgtggcaaa caagggctct cttgatcaag gcgctcatcg accgaggcta caaggatcga atcctcgtct cccatgactg gctgttcggg ttttcgagct atgtcacgaa catcatggac gtaatggatc gcataaaccc agatggaatg gccttcgtcc ctctgagagt gatcccattc ctacgagaga agggcgtccc gccggaaacg ctagcaggcg taaccgtggc caatcccgcg cggttcttgt caccgaccgt gcgggccgtc gtgacacgat ctgaaacttc ccgccctgcc gcgcctattc cccgtcaaga taccgaacga tga Amino acid sequence MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVST FDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSAS QRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFS SYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER DNA SEQUENCE at gcaaacgaga agagatgcac ttaagtctgc ggccgcaata actctgctcg gcggcttggc tgggtgtgca agcatggccc gaccaatcgg tacaggcgat ctgattaata ctgttcgcgg ccccattcca gtttcggaag cgggcttcac actgacccat gagcatatct gcggcagttc ggcgggattc ctacgtgcgt ggccggagtt tttcggtagc cgcaaagctc tagcggaaaa ggctgtgaga ggattacgcc atgccagatc ggctggcgtg caaaccatcg tcgatgtgtc gactttcgat atcggtcgtg acgtccgttt attggccgaa gtttcgcggg ccgccgacgt gcatatcgtg gcggcgactg gcttatggtt cgacccgcca ctttcaatgc gaatgcgcag cgtcgaagaa ctgacccagt tcttcctgcg tgaaatccaa catggcatcg aagacaccgg tattagggcg ggcattatca aggtcgcgac cacagggaag gcgaccccct ttcaagagtt ggtgttaaag gcagccgcgc gggccagctt ggccaccggt gttccggtaa ccactcacac gtcagcaagt cagcgcgatg gcgagcagca ggcagccata tttgaatccg aaggtttgag cccctcacgg gtttgtatcg gtcacagcga tgatactgac gatttgagct acctaaccgg cctcgctgcg cgcggatacc tcgtcggttt agatcgcatg ccgtacagtg cgattggtct agaaggcaat gcgagtgcat tagcgctctt tggtactcgg tcgtggcaaa caagggctct cttgatcaag hgcgctcatcg accgaggcta caaggatcga atcctcgtct cccatgactg gctgttcggg ttttcgagct atgtcacgaa catcatggac gtaatggatc gcataaaccc agatggaatg gccttcgtcc ctctgagagt gatcccattc ctacgagaga agggcgtccc gccggaaacg ctagcaggcg taaccgtggc caatcccgcg cggttcttgt caccgaccgt gcgggccgtc gtgacacgat ctgaaacttc ccgccctgcc gcgcctattc cccgtcaaga taccgaacga tga PROTEIN SEQUENCE MQTRRDALKSAAAITLLGGLAGCASMARPIGTGDLINTVRGPIPVSEAGFTLTHEHICGSSAGFLRAWPEFFGSRKALAEKAVRGLRHARSAGVQTIVDVST FDIGRDVRLLAEVSRAADVHIVAATGLWFDPPLSMRMRSVEELTQFFLREIQHGIEDTGIRAGIIKVATTGKATPFQELVLKAAARASLATGVPVTTHTSAS QRDGEQQAAIFESEGLSPSRVCIGHSDDTDDLSYLTGLAARGYLVGLDRMPYSAIGLEGNASALALFGTRSWQTRALLIKALIDRGYKDRILVSHDWLFGFS SYVTNIMDVMDRINPDGMAFVPLRVIPFLREKGVPPETLAGVTVANPARFLSPTVRAVVTRSETSRPAAPIPRQDTER 3) Genetically engineered E. Colibacteria In this project, we will use E. Coli to construct genetically engineered bacteria which can secrete organophosphorus-degradation enzyme opdA protein, to eliminate the pesticides. Genetically engineered bacteria are bacteria which can channel target gene into bacteria to express the genes and produce required protein. Currently, the mostly used genetically engineered bacteria all over the world are still E. Coli. E. Coli have explicit genetic background, fast growth rate, limited antibiotics resistance, Thus, E. Coli, are easy to be used in any production magnitude from laboratory to industry production. (For example: scientists introduced human insulin gene into E. Coli genome. E. Coli can express functional human insulin protein, which is used as a medicine for diabetes treatment. Human insulin production from E. coli has been applied to industry and this method is also widely used in biotech industry for other drug purposes In 1981, human insulin gene products were put into market and solved the problem of lack of insulin sources). 4) Lac operator The E. Coli wouldn’t produce things that doesn’t belong to itself, the lac operator takes charge of the controlling of the operation of the configuration in E. Coli to be specific the opdA enzyme. 5) Histindine tag The aim of eradicating the organophorphorus residual in vegetables is to help eliminate the potential harm that organophorphorus may done to human’s physical health. Considering the harm that E. Coli itself has, this year’s aim is to purify the opdA enzyme from the E. Coli. In order to extract the opdA out of the bacterium, we use histidine tag to label the opdA enzyme, because the nickle in the resin can form strong molecular bonds with histidine thus grab the opdA which connect with his-tag as well. So when the E. Coli with opdA enzyme pass through resin with nickle, the opdA will cling to the resin and the rest of the E coli will follow the solution be excluded from the resin. The next step to replace the opdA enzyme with his-tag on resin is to add over-dose of histidine, since the nickle in resin can form molecular bonds with histidine, those bonds that histidine+opdA is relatively weak comparing with the bonds between nickle and histidine. In regard of this, the over-dosed histidine will replace the opdA, thus exclude the opdA from the resin. When the opdA is excluded, we can readily obtain the finished product of the enzyme.