title: Effect of signal peptides on the secretion of β-cyclodextrin glucanotransferase in Lactococcus lactis NZ9000.
abstract: Cyclodextrin glucanotransferase (CGTase) is an extracellular enzyme which catalyzes the formation of cyclodextrin from starch. The production of CGTase using lactic acid bacterium is an attractive alternative and safer strategy to produce CGTase. In this study, we report the construction of genetically modified Lactococcus lactis strains harboring plasmids that secrete the Bacillus sp. G1 β-CGTase, with the aid of the signal peptides (SPs) SPK1, USP45 and native SP (NSP). Three constructed vectors, pNZ:NSP:CGT, pNZ:USP:CGT and pNZ:SPK1:CGT, were developed in this study. Each vector harbored a different SP fused to the CGTase. The formation of halo zones on starch plates indicated the production and secretion of β-CGTase by the recombinants. The expression of this enzyme is shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram analysis. A band size of ∼75 kDa corresponding to β-CGTase is identified in the intracellular and the extracellular environments of the host after medium modification. The replacement of glucose by starch in the medium was shown to induce β-CGTase production in L. lactis. Although β-CGTase production is comparatively low in NZ:SPK1:CGT, the SP SPK1 was shown to have higher secretion efficiency compared to the other SPs used in this study.
authors.affiliation: Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Serdang, Malaysia.
authors.forename: Menaga
authors.initials: M
authors.lastname: Subramaniam
authors.forename: Ali
authors.initials: A
authors.lastname: Baradaran
authors.forename: Md Illias
authors.initials: MI
authors.lastname: Rosli
authors.forename: Mohamad
authors.initials: M
authors.lastname: Rosfarizan
authors.forename: Yusoff
authors.initials: Y
authors.lastname: Khatijah
authors.forename: Abdul Rahim
authors.initials: AR
authors.lastname: Raha
mesh_terms: D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D004586:Electrophoresis; D005822:Genetic Vectors; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics*; D060847:Metabolic Engineering; D008970:Molecular Weight; D030781:Organisms, Genetically Modified; D010957:Plasmids; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013213:Starch / Q000378:metabolism
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013213:Starch; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase