Part:BBa_K4408007

Lipase CALB connected with chitin binding domain

This part is the coding region of the fusion protein of Candida antarctica: lipase B (CALB) and chitin-binding domain (ChBD). It consists CALB (BBa_K2302006), Linker 2 (BBa_K4408004) and ChBD (BBa_T2028) sequences. Candida antarctica lipase B (CALB) is a widely used lipase with high stereoselectivity and stability. ChBD 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.

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
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1025
  Illegal SapI site found at 964
  Illegal SapI site found at 994
  

1 Overview

This year team Worldshaper-NJBIOX has upgraded (BBa_K2302006)to a better version.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.