Part:BBa_K1195001

Amylase A

The fundamental component of bacterial biofilms is the extracellular polymeric substance, which is composed primarily of exopolysaccharides and proteins. The EPS forms the framework matrix for bacterial biofilms, and composes 50% to 90% of the total organic matter of the biofilm.

α-Amylase is a calcium metalloenzyme that hydrolyzes alpha bonds of long-chain carbohydrates. As a metalloenzyme, α-Amylase is nonfunctional in the absence of calcium. In the presence of calcium, α-Amylase is capable of inhibiting biofilm growth by hydrolyzing the polysaccharides that help compose the extracellular polymeric substance that is the fundamental component of bacterial biofilms.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 874
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 874
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 874
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 874
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization Data

The enzymatic activity of α-Amylase was characterized to determine its capacity to inhibit biofilm formation by V. cholerae. Samples were prepared by adding 50 µL of V. cholerae culture to 1 mL of a high salt LB. The samples were then treated by the addition of purified α-Amylase in various concentrations and allowed to incubate at 30°C for 48 hours. After 48 hours the samples were examined and a distinct difference was seen in the amount of biofilm formed in the treated and untreated samples.

The samples were then transferred to eppendorf tubes and centrifuged at 16,000 × g for two minutes, the supernatant was discarded to remove the growth media, and the samples were resuspended in 200 µL ddH2O. The samples were then stained with 50 µL of a 0.03% CV solution and allowed to incubate for five minutes. The samples were again centrifuged at 16,000 × g for two minutes and the supernatant containing excess CV was discarded. The pelleted biofilm was then washed with 800 µL of 95% EtOH without resuspension, centrifuged for 30 seconds, and the EtOH was discarded. The EtOH wash was repeated twice more for a total of three washes. The samples were then resuspended in 200 µL EtOH, transferred to a 96-well plate, and incubated for five minutes. The plate was then shaken for ten seconds and absorbance readings were taken for each sample at 540 nm.

The above graph shows the average absorbance readings for samples treated with 0, 2.5, 12.5, and 25 µL of α-Amylase. There is a distinct reduction in the amount of biofilm growth between untreated and treated samples, with samples treated with 25 µL α-Amylase showing a 65.8% decrease in biofilm formation after 48 hours. While this clearly shows the ability of α-Amylase to inhibit biofilm formation by V. cholerae, further characterization is needed to determine the capacity of α-Amylase to degrade preexisting biofilms.