Difference between revisions of "Part:BBa K2856001"

Revision as of 13:05, 11 October 2018

Bifunctional glutamate-cysteine ligase/glutathione synthase (gshF)

The BBa_K2856001 harbors a coding sequence of bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) derived from S.agalactiae. Codon-optimization has been made for Lactococcus Lactis. gshFp catalyzes the conversion of Cys,Glu and Gly to GSH.

Usage and Biology

Bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) is an enzyme involved and responded to synthetic reaction of GSH.In this reaction,one Cys and one Glu are converted to one γ-GC,then one γ-GC and one Gly are converted to one GSH.(Figure 1) The Lactococcus Lactis NZ9000 has inability to synthesis GSH.In our project,we construct a plasmid harboring gshF in order to produnce GSH in Lactococcus Lactis NZ9000.

Figure. 1 Enzymatic reaction catalyzed by gshF

Protein Analysis

SDS-PAGE was performed to detect the protein expression level of gshF.

Figure. 2 gshF protein analysis

Validation of GSH by HPLC analysis

　To further confirm the synthetic GSH in Lactococcus lactis, HPLC was performed to analyze the product. GSH was identified on the basis of retention times related to standard sample. According to the retention time of standard GSH sample, GSH compound of extracts from engineered Lactococcus lactis can be confirmed.Compared with engineered strain,we didn't detect GSH in wild type Lactococcus lactis NZ9000.

Figure. 4　　Validation of GSH by HPLC.
HPLC chromatogram of methanol extracts from theengineered Lactococcus lactis and wild-type Lactococcus lactis. HPLC analysis recorded at 480 nm of extracts.

Sequence and Features