Difference between revisions of "Part:BBa K398027"

Revision as of 22:22, 27 October 2010

LadA Protein Generator (High expression)

Figure 1 - Protein [http://www.ncbi.nlm.nih.gov/protein/165761309?report=genbank&log$=prottop&blast_rank=1&RID=B6XCTCJ2014 ladA]

Figure 1:Complete Alkane degradation pathway, ladA is one of the 1st steps herein

BBa_K398027 was shown to have an increased activity of 3.33x10^-3U/mg total protein compared to the negative control (E.coli without BBa_K398027) had which had an activity of 5.49x10^-4 U/mg total protein.

NOTE This part contains the same protein as BBa_K398017 but with a stronger promoter-RBS region. The following text is the same that can be found on the BBa_K398017 parts page.

Introduction

For the first step in the long-chain alkane degradation pathway ladA was implemented; a flavoprotein alkane monooxygenase native to Geobacillus thermodinitrificans NG-80-2. It has been found to specifically oxidize the terminal regions of alkanes ranging from C₁₅ up to at least C₃₆. The product is the corresponding primary alkanol. LadA forms a catalytic complex with flavin mononucleotide (FMN) which utilizes dioxygen to insert an oxygen atom into the substrate.

The general catalytic function involves three chemical processes:

• Reduction of the cofactor flavin mononucleotide (FMN to FMNH₂) by NAD(P)H
• Reaction of FMNH2 with O₂
• Binding, orienting, and activating the substrate for its oxygenation

LadA's ability to preferentially capture long-chain alkanes for oxidation sets it apart from other flavoprotein monooxygenases.

Characterization

Enzyme activity assay based on NADH absorbance

A less favored, yet very well accepted method for enzyme activity determination is by following the consumption of NADH at an absorption wavelength of 340 nm. By using a buffer at the appropriate pH, containing FMN and proper amounts of NADH the kinetics could easily be monitored by a 96-well plate reader. The protocol is described in detail [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Enzyme_activity_assay_for_LadA_by_NADH_absorption here].

Enzyme activity assay based on GC-analysis

The most favorable way to analyse the activity of the LadA enzyme is by creating an environment in which it can properly function in-vitro. As explained earlier the enzyme required NADH and flavin mononucleotide as cofactors. Furthermore the optimal activity was found at a temperature of 60 degrees and a pH of 7.5. In accordance with these properties a [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Enzyme_activity_assay_for_LadA_by_GC protocol] was set up for the conversion of hexadecane using the lysates of the E.coli cells carrying the ladA constructs. After the reaction the hydrocarbons are extracted with an apolar solvent and analysed by gas chromatography (click [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=General_gas_chromatography_program_for_alkanes_and_alkanols here] for the GC program and the obtained GC calibration graph).

Results

From figure 3 it can be concluded that BBa_K398027 had a activity of 3.33x10^-3 U/mg dry weight while the negative control (E.coli without BBa_K398017) had an activity of 5.49x10^-4 U/mg dry weight.

Enzyme activity [U/mg dry weight] of the alkane monooxygenase LadA as compared to the negative control E.coli K12 strain

References

1. Liu Li, Xueqian Liu, Wen Yang, Feng Xu, Wei Wang, Lu Feng, Mark Bartlam, Lei Wang and Zihe Rao. Crystal Structure of Long-Chain Alkane Monooxygenase (LadA) in Complex with Coenzyme FMN: Unveiling the Long-Chain Alkane Hydroxylase. Journal of molecular biology, 376: 453-465 (2008)

Sequence and Features