Part:BBa_K2302006

CALB,an enzyme that can hydrolyze fat to fatty acids.

this sequence encodes a functional protein that can hydrolyze fat to fatty acids. Pseudozyma (Candida) antarctica lipase B (CALB) is one of the most widely used lipases in the world, due to its excellent properties such as high stereoselectivity and stability.

Sequence and Features

Assembly Compatibility:

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

  Illegal BamHI site found at 949
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 670
  Illegal NgoMIV site found at 780
  Illegal AgeI site found at 472
  Illegal AgeI site found at 730
  
• 1000
  COMPATIBLE WITH RFC[1000]

RESULT

To further determine whether the recombinant CALB is functional, we made soft agar containing glyceryl tributyrate on culture plates containing growing bacteria (Fig.1). The result showed that Follower D could secrete degrade glyceryl tributyrate as indicated by transparent ring shaped as “NEFU”, suggesting its production of functional lipase.

Fig.1 The forming transparent area shaped as “NEFU”

Next, we used the culture supernatant of the bacteria to determine whether the lipase can be successfully secreted into culture medium. As shown in Fig.2, the culture supernatant could also cause degradation of glyceryl tributyrate and form transparent arear shaped as “IGEM”. The medium was stained by Sudan III. This result indicate that FD could indeed secret functional lipase into the culture medium, as we expected.

Fig.2 The forming transparent area shaped as “IGEM”.

Reference

Ayana Ujiie,Hideo Nakanoand Yugo Iwasaki.Extracellular production of Pseudozyma (Candida)antarctica lipase B with genuine primary sequence in recombinant Escherichia coli.J Biosci Bioeng,[J],2016,Mar;121(3):303–309.