Heterologous expression and characterization of soluble recombinant 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Actinosynnema pretiosum ssp. auranticum ATCC31565 through co-expression with Chaperones in Escherichia coli

2012 ◽  
Vol 82 (2) ◽  
pp. 263-269 ◽  
Author(s):  
Na Ma ◽  
Liujing Wei ◽  
Yuxiang Fan ◽  
Qiang Hua
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Actinosynnema Pretiosum ◽  
Phosphate Synthase

Heterologous expression and characterization of l-amino acid deaminases from Proteus mirabilis in Escherichia coli

2008 ◽  
Vol 136 ◽  
pp. S300 ◽  
Author(s):  
Jin-Oh Baek ◽  
Jeong-Woo Seo ◽  
Ohsuk Kwon ◽  
Su-Il Seong ◽  
Ik-Hwan Kim ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Heterologous Expression ◽  
Proteus Mirabilis

scholarly journals A Lysine Substitute for K+

2002 ◽  
Vol 277 (51) ◽  
pp. 49651-49654 ◽  
Author(s):  
Georgiy A. Belogurov ◽  
Reijo Lahti
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Phylogenetic Analyses ◽  
Functional Characterization ◽  
Membrane Vesicles ◽  
Thermophilic Bacterium ◽  
Inorganic Pyrophosphatase ◽  
Inner Membrane ◽  
Carboxydothermus Hydrogenoformans

The H+proton-translocating inorganic pyrophosphatase (H+-PPase) family is composed of two phylogenetically distinct types of enzymes: K+-dependent and K+-independent. However, to date, the sequence criteria governing this dichotomy have remained unknown. In this study, we describe the heterologous expression and functional characterization of H+-PPase from the thermophilic bacteriumCarboxydothermus hydrogenoformans. Both PPi-hydrolyzing and PPi-energized H+translocation activities of the recombinant enzyme inEscherichia coliinner membrane vesicles are strictly K+-dependent. Here we deduce the K+requirement of all available H+-PPase sequences based on the K+dependence ofC. hydrogenoformansH+-PPase in conjunction with phylogenetic analyses. Our data reveal that K+-independent H+-PPases possess conserved Lys and Thr that are absent in K+-dependent H+-PPases. We further demonstrate that a A460K substitution inC. hydrogenoformansH+-PPase is sufficient to confer K+independence to both PPihydrolysis and PPi-energized H+translocation. In contrast, a A463T mutation does not affect the K+dependence of H+-PPase.

scholarly journals Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

2004 ◽  
Vol 70 (1) ◽  
pp. 370-376 ◽  
Author(s):  
Leandro Padilla ◽  
Reinhard Krämer ◽  
Gregory Stephanopoulos ◽  
Eduardo Agosin
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Corynebacterium Glutamicum ◽  
Metabolic Flux ◽  
Krebs Cycle ◽  
Exponential Growth Phase ◽  
Fourfold Increase ◽  
Potential Applications ◽  
Defined Culture ◽  
Phosphate Synthase

ABSTRACT Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.

scholarly journals Heterologous Expression and Characterization of Thermostable Lipases from Geobacillus thermoleovorans PPD2 through Escherichia coli

2017 ◽  
Vol 14 (3) ◽  
pp. 1081-1088 ◽  
Author(s):  
Anita H. Permana ◽  
Fida Madayanti Warganegara ◽  
Deana Wahyuningrum ◽  
Made Puspasari Widhiastuty ◽  
Akhmaloka Akhmaloka
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Lipolytic Activity ◽  
Escherichia Coli Bl21 ◽  
Geobacillus Thermoleovorans ◽  
Medium Chain Length ◽  
Dominant Band ◽  
Acetone Fractionation ◽  
Thermostable Lipases

ABSTRACT: Heterologous expression and purification of thermostable lipase from Geobacillus thermoleovorans PPD2 had been carried out through Escherichia coli BL21 as host. Two bands obtained showed lipolytic activity with the size at around 51 (LipA) and 43 (LipB) kDa, respectively. The activities were identified by zymogram analysis, while the control protein from Escherichia coli BL21(DE3) do not show any lipolytic activity. Purification of crude extract using chromatography affinity Ni-NTA resulted one dominant band of LipA, meanwhile LipB did not appeared on the gel. Another purification for LipB was carried out by acetone fractionation. Both of LipA and LipB showed high activity toward medium chain length substrates, with optimum activity at 50oC and pH 8.5. The activities of LipA and LipB showed tolerance toward short chain alcohols, such as methanol, ethanol, n-propanol, and isopropanol.

scholarly journals Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2521 ◽  
Author(s):  
Birgit Grill ◽  
Maximilian Glänzer ◽  
Helmut Schwab ◽  
Kerstin Steiner ◽  
Daniel Pienaar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Functional Expression ◽  
Target Proteins ◽  
High Ph ◽  
Escherichia Coli Bl21 ◽  
Activity Profiling ◽  
Nitrile Hydratases ◽  
Target Sequences

Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were—in addition—able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.

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