Part:BBa_K398018

ADH generator

Figure 1 - Protein [http://www.ncbi.nlm.nih.gov/protein/34810608?report=genbank&log$=prottop&blast_rank=2&RID=B6X48AND01S ADH]

Figure 1:Complete Alkane degradation pathway, ADH is the 2nd step herein

BBa_K398018 showed an increased enzymatic dehydrogenase activity using dodecanol-1 as substrate. The strain showed an enzymatic activity of 2.93e-11 kat/mg (1.76 mU/mg) whereas the negative control had an enzymatic activity of 9.64e-12 kat/mg (0.58 mU/mg).

Introduction

ADH, an alcohol dehydrogenase isolated from Bacillus thermoleovorans B23, is capable of converting longer chain n-alkanols into the corresponding n-alkanal, the second step in the biodegradation of alkanes.

Characterization

This part was characterized using NAD/NADH enzyme assay. By disrupting the cells in the exponential growth phase (through sonication), adding the substrate dodecanol-1 and analyzing the NADH production using spectrophotometrical analysis (absorbance at 340nm), the activity could be determined. The protein utilizes NAD⁺, thus by determining the NADH production compared to a negative control the activity of the protein can be determined.

The [http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation characterization of BBa_K398029] has been described on the TU Delft iGEM Team 2010 wiki.

Results

According to our results, the E. coli cell extract has a dodecanol-1 dehydrogenase activity of 9.64e-12 kat/mg (0.58 mU/mg); whereas our recombinant strain 018A has an activity of 2.93e-11 kat/mg (1.76 mU/mg). According to our analysis, the enzymatic activities of both strains are statistically different at confidence level of 0.95, which means that the part BBa_K398018 increases 2 times the alcohol dehydrogenase activity in the cell extract. We can conclude from our data that the parts BBa_K398005 and BBa_K398018 have catalytic activity; particularly when we used BBa_K398018 the enzyme activity of E. coli cell extracts was equivalent to 3% of the in vitro activity of the positive control (Pseudomonas putida).

Figure 2: Comparison between E. coli ADH activity and our recombinant strain.

References

1. Tomohisa Kato, Asuka Miyanaga, Mitsuru Haruki, Tadayuki Imanaka, Masaaki Morikawa & Shigenori Kanaya. Gene Cloning of an Alcohol Dehydrogenase from Thermophilic Alkane-Degrading Bacillus thermoleovorans B23. Journal of Bioscience and Bioengineering 91(1):100-102 (2001)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]