scholarly journals Cotranslocation of Methyl Parathion Hydrolase to the Periplasm and of Organophosphorus Hydrolase to the Cell Surface of Escherichia coli by the Tat Pathway and Ice Nucleation Protein Display System

2009 ◽  
Vol 76 (2) ◽  
pp. 434-440 ◽  
Author(s):  
Chao Yang ◽  
Roland Freudl ◽  
Chuanling Qiao ◽  
Ashok Mulchandani
Keyword(s):  
Escherichia Coli ◽  
Methyl Parathion ◽  
Ice Nucleation ◽  
Organophosphorus Hydrolase ◽  
Display System ◽  
Ice Nucleation Protein ◽  
Methyl Parathion Hydrolase ◽  
E Coli ◽  
Tat Pathway ◽  
Parathion Hydrolase

ABSTRACT A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E. coli, respectively, by employing the twin-arginine translocation (Tat) pathway and ice nucleation protein (INP) display system. The resulting recombinant strain showed sixfold-higher whole-cell activity than the control strain expressing cytosolic OP hydrolases. The correct localization of MPH and OPH was demonstrated by cell fractionation, immunoblotting, and enzyme activity assays. No growth inhibition was observed for the recombinant E. coli strain, and suspended cultures retained almost 100% of the activity over a period of 2 weeks. Owing to its high level of activity and superior stability, the recombinant E. coli strain could be employed as a whole-cell biocatalyst for detoxification of OPs. This strategy of utilizing dual translocation pathways should open up new avenues for cotranslocating multiple functional moieties to different extracytosolic compartments of a bacterial cell.

scholarly journals Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in E. coli

PLoS ONE ◽  
2016 ◽  
Vol 11 (8) ◽  
pp. e0160367 ◽  
Author(s):  
Zhen Zhang ◽  
Rongxin Tang ◽  
Lu Bian ◽  
Meng Mei ◽  
Chunhua Li ◽  
...  
Keyword(s):  
Ice Nucleation ◽  
Surface Immobilization ◽  
Display System ◽  
Ice Nucleation Protein ◽  
E Coli ◽  
Arginase 1

scholarly journals Whole-cell catalysis by surface display of fluorinase on Escherichia coli using N-terminal domain of ice nucleation protein

2021 ◽  
Author(s):  
Xinming Feng ◽  
Miaomiao Jin ◽  
Wei Huang ◽  
Wei Liu ◽  
Mo Xian
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Surface Display ◽  
Catalytic Efficiency ◽  
Ice Nucleation ◽  
Display System ◽  
Ice Nucleation Protein ◽  
Growth Pressure ◽  
Whole Cell ◽  
Whole Cell Catalysis

Abstract BackgroundFluorinases play a unique role in producing fluorinated organic molecules through a biological method. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates.ResultsA gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused protein was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and Immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase-containing surface display system with improved whole-cell catalytic efficiency and stability showed low growth pressure on the protein expressing host. The conversion rate of 5′-fluorodeoxyadenosine (5′-FDA) from S-adenosyl-L-methionine (SAM) achieved 55%.ConclusionsHere, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of Escherichia coli and maintained its catalytic activity. The surface display offers a new solution for the industrial application of biological fluorination.

scholarly journals Freezing from the inside. Ice nucleation in <i>Escherichia coli</i> and <i>Escherichia coli</i> ghosts by inner membrane bound ice nucleation protein InaZ

2019 ◽  
Author(s):  
Johannes Kassmannhuber ◽  
Sergio Mauri ◽  
Mascha Rauscher ◽  
Nadja Brait ◽  
Lea Schöner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Pseudomonas Syringae ◽  
Survival Rates ◽  
Ice Nucleation ◽  
Free Access ◽  
Ice Nucleation Protein ◽  
Inner Membrane ◽  
E Coli ◽  
Subzero Temperatures

Abstract. An N-terminal truncated form of the ice nucleation protein (INP) of Pseudomonas syringae lacking the transport sequence for the localization of InaZ in the outer membrane was fused to N- and C- terminal inner membrane (IM) anchors and expressed in Escherichia coli C41. The ice nucleation (IN) activity of the corresponding living recombinant E. coli catalyzing heterogeneous ice formation of super-cooled water at high subzero temperatures was tested by droplet freezing assay. Median freezing temperature (T50) of the parental living E. coli C41 cells without INP was detected at −20.1 °C and with inner membrane anchored INPs at T50 value between −7 °C and −9 °C demonstrating that IM anchored INPs facing the luminal IM site are able to induce IN from the inside of the bacterium almost similar to bacterial INPs located at the outer membrane. Bacterial Ghosts (BGs) derived from the different constructs showed first droplet freezing values between −6 °C and −8 °C whereas C41 BGs alone without carrying IM anchored INPs exhibit a T50 of −18.9 °C. The more efficient IN of INP-BGs compared to their living parental strains can be explained by the free access of IM anchored INP constructs to ultrapure water filling the inner space of the BGs. The cell killing rate of -NINP carrying E. coli at subzero temperatures is higher when compared to survival rates of the parental C41 strain.

Crystal Structure of Methyl Parathion Hydrolase from Pseudomonas sp. WBC-3

2005 ◽  
Vol 353 (3) ◽  
pp. 655-663 ◽  
Author(s):  
Yan-Jie Dong ◽  
Mark Bartlam ◽  
Lei Sun ◽  
Ya-Feng Zhou ◽  
Zhi-Ping Zhang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Methyl Parathion ◽  
Pseudomonas Sp ◽  
Methyl Parathion Hydrolase ◽  
Parathion Hydrolase

Freezing from the inside: Ice nucleation in Escherichia coli and Escherichia coli ghosts by inner membrane bound ice nucleation protein InaZ

2020 ◽  
Vol 15 (3) ◽  
pp. 031003
Author(s):  
Johannes Kassmannhuber ◽  
Sergio Mauri ◽  
Mascha Rauscher ◽  
Nadja Brait ◽  
Lea Schöner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ice Nucleation ◽  
Ice Nucleation Protein ◽  
Inner Membrane ◽  
Membrane Bound
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