Photobiocatalytic Oxyfunctionalization with High Reaction Rate using a Baeyer-Villiger Monooxygenase from Burkholderia xenovorans in Metabolically Engineered Cyanobacteria

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to lactones under very mild reaction conditions. This enzymatic route is hindered by the requirement of a stoichiometric supply of auxiliary substrates for cofactor recycling and difficulties with supplying the necessary oxygen. The recombinant production of BVMO in cyanobacteria allows the substitution of auxiliary organic cosubstrates with water as an electron donor and the utilization of oxygen generated by photosynthetic water splitting. Herein, we report the identification of a BVMO from Burkholderia xenovorans (BVMOXeno) that exhibits higher reaction rates in comparison to currently identified BVMOs. We report a 10-fold increase in specific activity in comparison to cyclohexanone monooxygenase (CHMOAcineto) in Synechocystis sp. PCC 6803 (25 vs 2.3 U g(DCW)(-1) at an optical density of OD750 = 10) and an initial rate of 3.7 +/- 0.2 mM h(-1). While the cells containing CHMOAcineto showed a considerable reduction of cyclohexanone to cyclohexanol, this unwanted side reaction was almost completely suppressed for BVMOXeno, which was attributed to the much faster lactone formation and a 10-fold lower KM value of BVMOXeno toward cyclohexanone. Furthermore, the whole-cell catalyst showed outstanding stereoselectivity. These results show that, despite the self-shading of the cells, high specific activities can be obtained at elevated cell densities and even further increased through manipulation of the photosynthetic electron transport chain (PETC). The obtained rates of up to 3.7 mM h-1 underline the usefulness of oxygenic cyanobacteria as a chassis for enzymatic oxidation reactions. The photosynthetic oxygen evolution can contribute to alleviating the highly problematic oxygen mass-transfer limitation of oxygendependent enzymatic processes

Identification of Residues Involved in Substrate Specificity and Cytotoxicity of Two Closely Related Cutinases from <em>Mycobacterium tuberculosis</em>

International audienceThe enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. Bysite-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production

Modifying the substrate specificity of Mycobacterium tuberculosis enzymes

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Control the substrate specificity of Mycobacterium tuberculosis Rv1984c and Rv3452 cutinase-like proteins by site-directed mutagenesis

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Primers and plasmids used in this study.

<p>Nucleotides of the primers that differ from the wild type sequence are presented in bold.</p

Lipase Specific Activities (SAs) of the Cfp21 mutants on fluorescent triglycerides.

<p>SAs were calculated from the velocity slope obtained for 10 minutes using 10 µg of enzymes. Results are the means obtained in at least 3 independent experiments. Only the mutants with lipase activity are presented. Cfp21 and Cut4 were used as positive and negative standards, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066913#pone.0066913-Schue1" target="_blank">[7]</a>.</p

Development of a high-throughput assay for measuring lipase activity using natural triacylglycerols coated on microtiter plates.

International audienceWe have designed a convenient, specific, sensitive and continuous lipase assay based on the use of natural triacylglycerols (TAGs) from the Aleurites fordii seed oil which contains α-eleostearic acid (9,11,13,cis,trans,trans-octadecatrienoic acid) and which was coated in the wells of microtiter plates. The coated TAG film cannot be desorbed by the various buffers used during the lipase assay. Upon lipase action, α-eleostearic acid is liberated and desorbed from the interface and then solubilized into the micellar phase. Consequently, the UV absorbance of the α-eleostearic acid is considerably enhanced due to the transformation from an adsorbed to a water soluble state. The lipase activity can be measured continuously by recording the variations with time of the UV absorption spectra. The rate of lipolysis was monitored by measuring the increase of OD at 272 nm, which was found to be linear with time and directly proportional to the amount of added lipase. This microtiter plate lipase assay, based on coated TAGs, presents various advantages as compared to the classical systems: (i) coated TAGs on the microtiter plates could be stored for a long-time at 4 °C, (ii) higher sensitivity in lipase detection, (iii) good reproducibility, and (iv) increase of signal to noise ratio due to high UV absorption after transfer of α-eleostearic acid from an adsorbed to a soluble state. Low concentrations, down to 1 pg mL(-1) of pure Thermomyces lanuginosus or human pancreatic lipase, could be detected under standard assay conditions. The detection sensitivity of this coated method is around 1000 times higher as compared to those obtained with the classical emulsified systems. This continuous high throughput lipase assay could be used to screen new lipases and/or lipase inhibitors present in various biological samples

Esterase specific activities (SA) of the Cfp21 mutant proteins.

<p>The SAs of all the recombinant enzymes obtained using Vinyl-esters were determined by titrating the free fatty acids released using the pH-Stat technique and expressed in international units per mg of protein (U/mg). Vinyl esters with alkyl chains ranging from 4 to 16 carbon atoms were assayed. Only substrates with alkyl chains ranging from 4 to 10 carbon atoms are presented, since no significant activity was detected with longer alkyl chains.</p><p>All substrates were assayed at concentrations above their solubility limits as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066913#pone.0066913-MullerSantos1" target="_blank">[46]</a>. ND: not detected.</p

SDS-PAGE and CD spectra analysis of purified proteins.

<p>A) Protein purity assessed on SDS-PAGE. MW: molecular weight standard from Euromedex; 1: Cfp21 WT; 2: Cfp21-A1; 3: Cfp21-A2; 4: Cfp21-A3; 5: Cfp21-A1A2; 6: Cfp21-A2A3; 7: Cfp21-A1A3; 8: Cfp21-TM. 1 µg of each protein was loaded onto a 15% polyacrylamide gel and stained with Coomassie Blue. B) Far UV spectra of the Cfp21 mutant proteins.</p