Part:BBa_K2286006

HheC-P84A: halohydrin dehalogenase site-directed mutant

This is the site-directed mutant of halohydrin dehalogenase. We change the alanine to proline in the 84 site. And this sequence is codon-optimized for E.coli.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 718
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

This part is an improvement of BBa_K1199044 (W249P) in its catalytic activity for 2,3-DCP. We obtained P84A by molecular simulation and single-site saturation mutation. P84A not only had a higher catalytic activity for 2,3-DCP than W249P, but maintained its high catalytic activity for CPD and 1,3-DCP.

Catalytic activity towards to 2，3-DCP

In order to clarify its activity level towards to 2,3-DCP, we tested its activity by the chloride ion method. The results showed that the catalytic activity of P84A to 2,3-DCP was 2.42 times that of W249P.

Figure 1. The catalytic activity of mutants towards to 2,3-DCP. Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.

Catalytic activity towards to CPD

Haloalcohol dehalogenase (HheC) could catalyze o-halide transferred into epoxides and hydrogen halides through intramolecular nucleophilic substitution mechanism. It had a wide range of catalytic substrates. So in order to verify whether P84A is suitable for other substrates under the same conditions, we chose 3-chloropropane-1,2-diol (CPD) as the substrate for further detection respectively. The results show that the catalytic activity of P84A towards to CPD increased a little.

Figure 2. The catalytic activity of mutants on CPD.Data were measured in 50mM Tris-H2SO4 at pH 8.5 and 37℃.

The above results show that P84A not only has a higher catalytic activity towards to 2,3-DCP, but maintains its catalytic activity for CPD.