Part:BBa_K4940002
GlcDH-II-his
GlcDH-II is a coding sequence of glucose dehydrogenase2 from Bacillus megaterium. This enzyme can convert the unphosphorylated glucose in the cytoplasm to gluconolactone, which can further hydrolyze to gluconate spontaneously. During the whole process, NADPH can also be generated (1). Combined with GLF and P450BM-3 QM, a system can be constructed to effectively and continuously degrade α-pinene to α-pinene oxide.
Usage and Biology
The gene encodes a NADP-dependent glucose dehydrogenase that oxidizes unphosphorylated glucose in the cytoplasm to gluconolactone while converting NADP+ to NADPH (1). In our project, GlcDH was involved in a NADH regenerative system, allowing for more efficient α-pinene oxidation.
Plasmid design and construction
We inserted P450BM-3 QM and GlcDH-II into pCDFDUET-1 vector to form pCDFDUET-1 p450bm-3qm glcdh, achieving efficient α-pinene oxidation.
Figure 1. The construction of plasmid pCDFDUET-1 p450bm-3qm glcdh.
Cultivation, Purification and SDS-PAGE
The recombinant E.coli was cultured and induced by IPTG to allow GlcDH-II protein expression. Since the protein was fused to a his tag, we purified the protein via affinity chromatography. The protein samples were tested by SDS-PAGE. We have already confirmed the expression of GlcDH-II based on the corresponding bands.
Figure 2. Transformation results of three plasmids in the LB agar plate. Three plasmids were all successfully transformed into bacteria, indicated by the colonies on the agar plate. (A) E. coli BL21 star (DE3) with pQE-80L-Kan glf; (B) E. coli BL21 star (DE3) with pCDFDUET-1 p450bm-3qm glcdh-II; (C) E. coli BL21 star (DE3) pET-28a prα-pol.
Figure 3. The results of the SDS-PAGE. The corresponding protein bands were indicated by the red box.
References
1. Schewe H, Kaup BA, Schrader J. Improvement of P450BM-3 whole-cell biocatalysis by integrating heterologous cofactor regeneration combining glucose facilitator and dehydrogenase in E. coli. Appl Microbiol Biotechnol. 2008 Feb 1;78(1):55–65.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 371
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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