Team:KEYSTONE/Improvement
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Overview
The team Brasil-USP designed the part LcpK30 (Part: BBa_K1819001) in 2015, which is a 42kDa enzyme extracted from actinomycete Streptomyces sp. strain K30 that can catalyze rubber cleavage at the cis double bonds to multiple products ranging from C20 tetra-isoprenoid to at least C35 hepta-isoprenoid.
In this year, we improved the pre-existing Lcp(LcpK30) by adding new characterization data for it and designing a new Lcp type- Lcp1VH2 ,which is a rubber oxygenase extracted by a Gram positive rubber degrading bacteria named Gordonia Polyisoprenivorans VH2.
Lcp1VH2 has performed better expression and enzyme activity compared to LcpK30.
Better expression
In order to compare LcpK30 and Lcp1VH2, we tested two proteins using E.coli BL21 with plasmid pET23a:: Lcp1VH2 (C.2 & S.2 presented on figure 1) and pET23a:: LcpK30 (C.3 & S.3 presented on figure 1). From the SDS-Page result in fig.1, Lcp1VH2 expressed a clear and distinct band at 42 kDa, while the band trace of LcpK30 at the same position was obviously much weaker in comparison, indicating that its expression was much inferior to that of Lcp1VH2.
Fig.1 Expression of Lcp1VH2 and LcpK30 when inserted into pET23a plasmid.SDS-PAGE of crude extracts(C) and soluble fractions(S) of E. coli BL21(1) as control, Lcp1VH2 (2) and LcpK30 (3).
Better enzyme activity
The process for Lcp to degrade rubber requires oxygen consumption. It utilizes the process of β oxidation to break down bonds within polyisoprene. During β oxidation, Lcp adds two oxygen molecules in between the chemical bonds of polyisoprene. As shown from the oxygen dissolving results below, the initial dissolved oxygen in the sample is about 8.5 mg/l. After 6 hours, the dissolved oxygen in the sample tube (Supernatant containing Lcp1VH2) creates a downward slope , The value dropped to approximately 0mg/l eventually. In the sample tube containing LcpK30, oxygen was consumed at a slower rate, and after 9.5 hours, oxygen was roughly depleted. However, the dissolved oxygen in the control tube(Supernatant only of BL21) slowly drops to 6mg/l after 24 hours. This indicates that the LCP1VH2 protein has a stronger enzymatic activity.
Fig 2. Dissolved oxygen experiment to verify the activity of Lcp1VH2 and LcpK30
References