Function and engineering of the 15β-hydroxylase CYP106A2

2006 ◽  
Vol 34 (6) ◽  
pp. 1215-1218 ◽  
Author(s):  
C. Virus ◽  
M. Lisurek ◽  
B. Simgen ◽  
F. Hannemann ◽  
R. Bernhardt
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Steroid Hormones ◽  
Bacillus Megaterium ◽  
Crystallization Process ◽  
High Yield ◽  
Recent Developments ◽  
Adrenodoxin Reductase ◽  
Hydroxylation Activity

CYP106A2 from Bacillus megaterium ATCC 13368 is a bacterial cytochrome P450 that is capable of transforming steroid hormones. It can be easily expressed in Escherichia coli with a high yield. Its activity in vitro can be achieved by using the adrenal redox proteins adrenodoxin and adrenodoxin reductase. So far, it was not possible to crystallize CYP106A2 because of degradation during the crystallization process. Nevertheless, CYP106A2 is an interesting enzyme for biotechnological use. It hydroxylates pharmaceutically important steroids such as progesterone and 11-deoxycortisol. However, it will be necessary for efficient application of CYP106A2 in biotechnology to improve the hydroxylation activity and manipulate the regiospecificity. The present paper gives an overview of recent developments in protein engineering of CYP106A2.

Cloning, Expression and Characterization of a Monooxygenase P450BM3 from Bacillus megaterium ALA2

2012 ◽  
Vol 518-523 ◽  
pp. 5533-5538
Author(s):  
Ting Wang ◽  
Liang Liang Wang ◽  
Xun Li
Keyword(s):  
Escherichia Coli ◽  
Cytochrome P450 ◽  
Linoleic Acid ◽  
Bacillus Megaterium ◽  
Cytochrome P450 Monooxygenases ◽  
P450 Monooxygenases ◽  
Nadph Oxidation ◽  
First Time ◽  
Optimal Ph

Cytochrome P450 monooxygenases are enzymes which are capable of oxidising saturated and unsaturated substrates. P450BM3 from Bacillus megaterium is one of this family. For the first time, the cyp gene for coding P450BM3 from B. megaterium ALA2 has been cloned and expressed in Escherichia coli. The recombinant enzyme is 120 kDa, containing 1049 aa. The highest activity of purified enzyme is 14.8 U/mg towards palmitic acid by monitoring the NADPH oxidation. The optimal pH and temperature were 9.0 and 40°C. The enzyme has higher activity towards linoleic acid, and 2-Methyl-7-octadecene can also be catalyzed which is a precursor of displar.

Application of Escherichia coli phage K1E DNA-dependent RNA polymerase for in vitro RNA synthesis and in vivo protein production in Bacillus megaterium

2010 ◽  
Vol 88 (2) ◽  
pp. 529-539 ◽  
Author(s):  
Simon Stammen ◽  
Franziska Schuller ◽  
Sylvia Dietrich ◽  
Martin Gamer ◽  
Rebekka Biedendieck ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Bacillus Megaterium ◽  
Protein Production ◽  
Rna Synthesis ◽  
Escherichia Coli Phage

Human CYP27A1 catalyzes hydroxylation of β-sitosterol and ergosterol

2016 ◽  
Vol 397 (6) ◽  
pp. 513-518 ◽  
Author(s):  
Maximilian Ehrhardt ◽  
Adrian Gerber ◽  
Josef Zapp ◽  
Frank Hannemann ◽  
Rita Bernhardt
Keyword(s):  
Bacillus Megaterium ◽  
Current Work ◽  
The Novel ◽  
Nmr Analysis ◽  
Human Diet ◽  
Redox Partners ◽  
Adrenodoxin Reductase ◽  
Novel Products

Abstract β-Sitosterol and ergosterol are the equivalents of cholesterol in plants and fungi, respectively, and common sterols in the human diet. In the current work, both were identified as novel CYP27A1 substrates by in vitro experiments applying purified human CYP27A1 and its redox partners adrenodoxin (Adx) and adrenodoxin reductase (AdR). A Bacillus megaterium based biocatalyst recombinantly expressing the same proteins was utilized for the conversion of the substrates to obtain sufficient amounts of the novel products for a structural NMR analysis. β-Sitosterol was found to be converted into 26-hydroxy-β-sitosterol and 29-hydroxy-β-sitosterol, whereas ergosterol was converted into 24-hydroxyergosterol, 26-hydroxyergosterol and 28-hydroxyergosterol.

scholarly journals Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

2006 ◽  
Vol 34 (19) ◽  
pp. e135-e135 ◽  
Author(s):  
Madina B. Iskakova ◽  
Witold Szaflarski ◽  
Marc Dreyfus ◽  
Jaanus Remme ◽  
Knud H. Nierhaus
Keyword(s):  
Escherichia Coli ◽  
In Vitro Transcription ◽  
High Yield ◽  
Translation System ◽  
Escherichia Coli Cells

Indipendent and Tandem Expression of a Novel Antimicrobial Peptides Plectasin in Escherichia coli

2011 ◽  
Vol 345 ◽  
pp. 134-138 ◽  
Author(s):  
Li Hui Lv ◽  
Xue Gang Luo ◽  
Meng Ni ◽  
Xiao Lan Jing ◽  
Nan Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Expression System ◽  
High Yield ◽  
Gene Synthesis ◽  
Post Translational Modification ◽  
Iptg Induction ◽  
E Coli ◽  
Conventional Antibiotics

Plectasin, a novel antimicrobial peptide, is isolated from a saprophytic fungus Pseudoplectania nigrella. Plectasin showed potent antibacterial activity in vitro against Gram-positive, especially the Streptococcus pneumoniae and Streptococcus pneumoniae, including strains resistant to conventional antibiotics. In our previous study, plectasin had been expressed at a high yield as a thioredoxin (Trx) – fused protein in Escherichia coli. However, it couldn’t exhibit the antimicrobial activity unless the Trx-tag had been cleaved, which made the producing process be complicated. Concerning that plectasin has no complex post-translational modification and toxicity on E. coli, on the basis of the former works, we further establish the independent and tandem expression system of plectasin in E. coli. In the present study, the coding sequence of plectasin was obtained from pET32a-PLEC with four primers to amplify the independent and tandem plectasin fragments by overlapping PCR-based gene synthesis, and then cloned into pET22b (+) vector. The recombinant protein was expressed successfully in E. coli with IPTG induction. These works might throw light on the production or study of plectasin, and contribute to the development of novel anti-infectious drugs in the future.

scholarly journals Bacterial cytochrome P450-catalyzed regio- and stereoselective steroid hydroxylation enabled by directed evolution and rational design

2020 ◽  
Vol 7 (1) ◽  
Author(s):  
Xiaodong Zhang ◽  
Yaqin Peng ◽  
Jing Zhao ◽  
Qian Li ◽  
Xiaojuan Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biological Activity ◽  
Cytochrome P450 ◽  
Directed Evolution ◽  
Rational Design ◽  
Cytochrome P450s ◽  
Steroid Hydroxylation ◽  
Recent Developments ◽  
Low Activity ◽  
High Expression Level

AbstractSteroids are the most widely marketed products by the pharmaceutical industry after antibiotics. Steroid hydroxylation is one of the most important functionalizations because their derivatives enable a higher biological activity compared to their less polar non-hydroxylated analogs. Bacterial cytochrome P450s constitute promising biocatalysts for steroid hydroxylation due to their high expression level in common workhorses like Escherichia coli. However, they often suffer from wrong or insufficient regio- and/or stereoselectivity, low activity, narrow substrate range as well as insufficient thermostability, which hampers their industrial application. Fortunately, these problems can be generally solved by protein engineering based on directed evolution and rational design. In this work, an overview of recent developments on the engineering of bacterial cytochrome P450s for steroid hydroxylation is presented.

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