Part:BBa_K3990001
LCP-K30
We expressed the enzyme LCP in BL21(DE3) E.coli to decompose the polyisoprene.
Lcp oxidatively attacks the double bonds of polyisoprene molecules with the help of ferrous ion Fe2+ (see Fig.1) to give cleavage products that have aldehyde and keto end groups as illustrated in Fig.2. The products of oxidized polyisoprene by LCP range from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid as shown in Fig.2, which indicates that LCP works like endonuclease that can cleave the substrate at different positions.
Fig.1 The mechanism of oxydizing polyisoprene by Lcp</br>
Fig.2 The cleavage products of polyisoprene by LCP and A UV(210-nm) absorbance spectrum of LCP products(gray line)
Fig.3 The plasmid map of pET28b::LCP-K30
The plasmid integrated complete coding sequence of LCP from Streptomyces sp. K30 with a His-tag sequence at the C-terminus of LCP sequence, pET28b::LCP-K30, would be synthesized(Fig.4). We chose E.coli BL21 to express LCP, since literature supported heterologous expression in E.coli. Besides, modeling can help us predict the expression curve of LCP to predict the time for harvesting cells. Limited by experimental conditions, we couldn't follow the protocol of testing enzymes activity on the document, which qualitatively and quantitively characterize the activity of LCP. We also came up with a new measurement of testing the activity of LCP by utilizing the reaction of Schiff reagent indicating the formation of aldehyde groups. At the absorption peak of Schiff reagent, the absorbance of reaction mixture, including buffer, natural latex and LCP, should regard with the enzyme activity.
The original coding sequence of Latex Clearing Protein was from Genbank dataase (reference code: AY387589.1). The plasmid was transformed into E.coli BL21(DE3). The overnight culture in Luria-Bertani(LB) medium was inoculated 1:100 with fresh medium containing kanamycin and cultured at 37°C with shaking 220rpm for about 4 hours until the OD600 reaches 0.5-0.6. Then the cells were induced with 0.1mM isopropyl-b-D-thiogalactoside(IPTG) and supplemented with 8uM HemeB. Cells were cultured at 16°C/220rpm for about 16hours before harvestation.
After being centrifuged at 4000g, 4°C, for 20 minutes, sedimented cells were resuspended in lysis buffer containing lysozyme and protease inhibitor. After an hour, cells were sonicated on ice. Disrupted cells were removed by centrifugation(10000g for 20-30 minutes at 4°C). The supernatent was utilized to confirm the success of expression when carrying out SDS-PAGE. A standard curve of formaldehyde was established to measure the number of aldehyde groups formed as a result of oxidation of polyisoprene. The assay mixture is made up of LCP, 1% natural latex solved in n-hexane, and 100 mM potassium phosphate buffer(pH 7). The mixture was incubated at room temperature for 2 hours. The LCP activity was measured by the increase in absorbance at 540nm where the absorption peak of Schiff reagent is.
Fig.4 The SDS-PAGE gel of LCP
Fig.5 The enzymatic activity of LCP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1074
Illegal NgoMIV site found at 1083 - 1000COMPATIBLE WITH RFC[1000]
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