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

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

Biosciences Biotechnology Research Asia ◽

10.13005/bbra/2545 ◽

2017 ◽

Vol 14 (3) ◽

pp. 1081-1088 ◽

Cited By ~ 2

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.

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Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

Scientific Reports ◽

10.1038/s41598-021-94181-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Masuzu Kikuchi ◽

Keiichi Kojima ◽

Shin Nakao ◽

Susumu Yoshizawa ◽

Shiho Kawanishi ◽

...

Keyword(s):

Escherichia Coli ◽

Proton Pump ◽

Expression System ◽

Spectroscopic Characterization ◽

Functional Expression ◽

Proton Pumping ◽

E Coli ◽

Oxyrrhis Marina ◽

Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

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

Journal of Biotechnology ◽

10.1016/j.jbiotec.2008.07.1881 ◽

2008 ◽

Vol 136 ◽

pp. S300 ◽

Cited By ~ 1

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

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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

Protein Expression and Purification ◽

10.1016/j.pep.2012.01.013 ◽

2012 ◽

Vol 82 (2) ◽

pp. 263-269 ◽

Cited By ~ 8

Author(s):

Na Ma ◽

Liujing Wei ◽

Yuxiang Fan ◽

Qiang Hua

Keyword(s):

Escherichia Coli ◽

Heterologous Expression ◽

Actinosynnema Pretiosum ◽

Phosphate Synthase

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A Lysine Substitute for K+

Journal of Biological Chemistry ◽

10.1074/jbc.m210341200 ◽

2002 ◽

Vol 277 (51) ◽

pp. 49651-49654 ◽

Cited By ~ 67

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.

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