A Pichia pastoris (P. pastoris) cell surface display system of Bombyx mori acetylcholinesterase (BmAChE) was constructed and its bioactivity was studied. The modified Bombyx mori acetylcholinesterase gene (bmace) was fused with the anchor protein (AGalpha1) from Saccharomyces cerevisiae and transformed into P. pastoris strain GS115. The recombinant strain harboring the fusion gene bmace-AGalpha1 was induced to display BmAChE on the P. pastoris cell surface. Fluorescence microscopy and flow cytometry assays revealed that the BmAChE was successfully displayed on the cell surface of P. pastoris GS115. The enzyme activity of the displayed BmAChE was detected by the Ellman method at 787.7 U/g (wet cell weight). In addition, bioactivity of the displayed BmAChE was verified by inhibition tests conducted with eserine, and with carbamate and organophosphorus pesticides. The displayed BmAChE had an IC50 of 4.17x10(-8) M and was highly sensitive to eserine and five carbamate pesticides, as well as seven organophosphorus pesticides. Results suggest that the displayed BmAChE had good bioactivity.
Title: Functional expression of phaCAB genes from Cupriavidus taiwanensis strain 184 in Escherichia coli for polyhydroxybutyrate production Chien CC, Hong CC, Soo PC, Wei YH, Chen SY, Cheng ML, Sun YM Ref: Appl Biochem Biotechnol, 162:2355, 2010 : PubMed
Polyhydroxyalkanoates are polyesters synthesized by numerous microorganisms. These polyesters are biodegradable and have similar properties to those of conventional plastics. Cupriavidus taiwanensis strain 184 is phylogenetically related to the well-known polyhydroxybutyrate (PHB) producer Ralstonia eutropha (Cupriavidus necator) and is also shown to be able to accumulate significant amounts of PHB. In this study, we cloned the PHB synthesis genes (phaCAB) from C. taiwanensis 184 into Escherichia coli for biosynthesis of PHB. The recombinant E. coli strains were able to synthesize significant amounts of PHB. The PHB amounted to about 66~70% of total cell material of these recombinant strains.
