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.

Improvement from iGEM22_Worldshaper-NJBIOX

1 Overview

This year team Worldshaper-NJBIOX has upgraded (BBa_K2302006)to a better version, the improved parts was registered as BBa_K4408007. BBa_K2302006 is the coding region of Candida antarctica: lipase B (CALB). CALB is a widely used lipase with high stereoselectivity and stability. In BBa_K2302006, CALB was used to hydrolyze fat into fatty acid. However, CALB can also catalyze the esterification of many esters, including butyl butyrate.[1] We used CALB to catalyze the synthesis of butyl butyrate. Furthermore, we have added ChBD, which is an affinity tag for chitin purification of proteins. ChBD-fusion CALB can facilitate the purification of CALB, thus enhancing the reaction rate of CALB catalyzed esterification. We used T7 promoter (BBa_K3633015) and Cpa fdx terminator (BBa_K3585002) to express ChBD-fusion CALB. We used pet25b as the linear vector to construct the recombination plasmid pet25b-T7-pelB-CALB-ChBD. We used E.coli JM109 for recombination plasmid construction and E.coli Rosetta(DE3) for exogenous protein expression. We improved the CALB into a fusion protein ChBD-CALB, which can be immobilized by chitin pellets. With the chitin immobilization of ChBD-CALB, we have demonstrated that the enzyme activity of CALB was increased by 24% by enzyme activity assay.

2 Results

Protein gel electrophoresis confirmed that E.coli Rosetta(DE3) transformed with pet25b-T7-pelB-CALB-ChBD plasmid expressed ChBD-fusion CALB (Figure 1).

We used enzyme activity assay to evaluate the lipase activity of the lipase, ChBD-fusion CALB, expressed by the engineered E.coli. In enzyme activity assay, butyric acid and butanol (5 g/L each) were used as substrates. Through two-phase catalysis and hexadecane as extractant, butyl butyrate generated in unit time was measured by gas phase to determine the lipase activity. Enzyme activity assay showed that the exogenously expressed lipase had good activity, with the highest enzyme activity of 110 U/mL at 120 min (Figure 2). 220 mg/L butyl butyrate was obtained by catalyzing C. tyrobutyricum transformed with pMTL-Pthl-adhE2. After immobilization by chitin pellets, the enzymatic activity of CALB increased by 24% to 136 U/mL. And the final catalytic butyl butyrate production by catalyzing C. tyrobutyricum transformed with pMTL-Pthl-adhE2 was increased by 28% to 280 mg/L.

Figure 1 Expression of ChBD-fusion CALB in E. coli

Figure 2 Enzyme activity assay of exogenously expressed lipase CALB

Reference

[1] Noh H J, Lee S Y, Jang Y S. Microbial production of butyl butyrate, a flavor and fragrance compound[J]. Applied microbiology and biotechnology, 2019, 103(5): 2079-2086.