Difference between revisions of "Part:BBa K4408011"

Revision as of 11:35, 1 October 2022

T7-CALB-ChBD, Expression of CALB connected with chitin binding domain

This part is responsible for expressing CALB and ChBD fusion protein. The expression is controlled by a T7 promoter with an ribosome binding site (RBS), and a Cpa fdx terminator. pelB leader sequence codes for a sequence of amino acid that can attach to the CALB protein expressed in E.coli to the surface of the cell, where the sequence is removed by pelB peptidase. It can direct CALB protein to the medium from the cell. It consists of BBa_K3633015 (T7 promoter), BBa_K3940012 (RBS), BBa_J32015 (pelB), BBa_K2302006 (CALB), BBa_K4408004(Linker 2), BBa_T2028 (ChBD) and BBa_K3585002(Cpa fdx terminator). 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. This part can be used to express ChBD-fusion CALB in E.coli.

Sequence and Features

Results

The linear vector pet25b was obtained by double digestion of pet25b using NdeI and EcoRⅠ enzymes. CALB fragment (966 bp) was amplified from the Pseudomonas aeruginosa genome by PCR. ChBD fragment (417 bp) was obtained from a synthetic gene by PCR. The two fragments were fused together by fusion PCR, and then ligated to the linearized vector pet25b by T4 ligation. The pet25b-T7-pelB-CALB-ChBD recombinant plasmid was transformed into E. coli Rosetta(DE3). Colony PCR and DNA electrophoresis was performed to confirm the positive colonies. These colonies were transferred and expanded. Plasmids extracted from the colonies were confirmed by gene sequencing.

Protein gel electrophoresis showed that E. coli was successfully constructed to produce Candida antarctica Lipase B (CALB) (Figure 1). In the enzyme activity assay, the exogenously expressed lipase was found to have good activity, showing 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 was increased by 28% to 280 mg/L.

Figure 1 Lipase expression in E. coli

Figure 2 Enzyme activity assay of exogenously expressed lipase CALB