Improvement of low-temperature caseinolytic activity of a thermophilic subtilase by directed evolution and site-directed mutagenesis

2009 ◽  
Vol 104 (5) ◽  
pp. 862-870 ◽  
Author(s):  
Chuan-Qi Zhong ◽  
Shengli Song ◽  
Nan Fang ◽  
Xiaoliang Liang ◽  
Hui Zhu ◽  
...  
Keyword(s):  
Low Temperature ◽  
Directed Evolution ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Caseinolytic Activity

Photoreaction of Cyanobacterial BLUF Protein PixD Studied by Low Temperature Spectroscopy and Site-Directed Mutagenesis

2008 ◽  
pp. 1291-1294
Author(s):  
Yoshimasa Fukushima ◽  
Yoshiya Murai ◽  
Koji Okajima ◽  
Masahiko Ikeuchi ◽  
Shigeru Itoh
Keyword(s):  
Low Temperature ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis

2021 ◽  
Author(s):  
◽  
Kelsi Hall
Keyword(s):  
Enzyme Activity ◽  
Directed Evolution ◽  
Negative Selection ◽  
Nitroaromatic Compounds ◽  
Degenerative Disease ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Cell Ablation ◽  
Wide Range

<p>Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish.  There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels.  The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell ablation system. Using a dual positive-negative selection, we recovered several NfsA_Ec variants that exhibited preferential nitrofurazone activation over metronidazole. Our lead variants for both applications are currently being trialed in zebrafish for their utility in generating degenerative disease models.</p>

scholarly journals Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

2019 ◽  
Vol 20 (24) ◽  
pp. 6224 ◽  
Author(s):  
Bangqiao Yin ◽  
Qinyan Hui ◽  
Muhammad Kashif ◽  
Ran Yu ◽  
Si Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Directed Evolution ◽  
Catalytic Efficiency ◽  
Raw Material ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
The Novel ◽  
Wild Type

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM−1 s−1) toward ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to β-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel β-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

scholarly journals Investigating the evolvability of 'Escherichia coli nfsA' via simultaneous site-directed mutagenesis

2021 ◽  
Author(s):  
◽  
Kelsi Hall
Keyword(s):  
Enzyme Activity ◽  
Directed Evolution ◽  
Negative Selection ◽  
Nitroaromatic Compounds ◽  
Degenerative Disease ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Cell Ablation ◽  
Wide Range

<p>Bacterial nitroreductases are flavoenzymes able to catalyse the reduction of nitroaromatic compounds. The research presented in this thesis focused on NfsA_Ec, a nitroreductase from E. coli. NfsA_Ec is a promiscuous enzyme that can reduce a wide range of nitroaromatic antibiotics and prodrugs. This research sought to use NfsA_Ec as a model to improve our understanding of directed evolution, and also to identify NfsA_Ec variants exhibiting improved activation with a range of nil-bystander prodrugs for use in a targeted cell ablation system in zebrafish.  There is a substantial gap between the levels of enzyme activity that nature can achieve and those that scientists can evolve in the lab. This suggests that conventional directed evolution techniques involving incremental improvements in enzyme activity may frequently fail to ascend even local fitness maxima. We sought to contrast such approaches with simultaneous site-directed mutagenesis, employing a library of 252 million unique nfsA variants. To determine whether two superior NfsA_Ec variants recovered from this library could have been identified using a conventional stepwise approach we generated all possible intermediates of these two enzyme variants and recreated the most logical evolutionary trajectory for each enzyme variant. This revealed that a stepwise mutagenesis approach could indeed have yielded both of these variants, but also that very few evolutionary trajectories were accessible due to complex epistatic interactions between substitutions in these enzymes. Moreover, many conventional stepwise mutagenesis approaches such as iterative saturation mutagenesis would have failed to identify key substitutions in these variants. We also investigated the “black-box” effect of directed evolution, using NfsA_Ec and a panel of nitroaromatic compounds to model the off-target effects an evolved enzyme can have within an existing metabolic network. We found that selection for improved niclosamide and chloramphenicol detoxification also improved activity with some structurally distinct prodrugs, but not others. Using a dual positive-negative selection, we recovered NfsA_Ec variants that were more specialised for their primary activities, however this came at a cost in terms of overall activity levels.  The simultaneous site-directed nfsA_Ec mutagenesis library also had practical applications, enabling recovery of NfsA_Ec variants for targeted cell ablation in zebrafish models. These models involve the selective ablation of nitroreductase expressing cells without harming adjacent cells, to mimic a degenerative disease. Several NfsA_Ec variants were identified which were highly active with the nil-bystander prodrugs metronidazole, tinidazole, RB6145 and misonidazole when expressed in E. coli. However, these NfsA_Ec variants had inconsistent activities in our eukaryotic cell model (HEK-293). To expand the utility of the core ablation system, we sought to identify pairs of nitroreductases with non-overlapping prodrug specificities, suitable for use in a multiplex cell ablation system. Using a dual positive-negative selection, we recovered several NfsA_Ec variants that exhibited preferential nitrofurazone activation over metronidazole. Our lead variants for both applications are currently being trialed in zebrafish for their utility in generating degenerative disease models.</p>

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