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Purification and function analysis of a broad-spectrum organomercurial lyase (MerB3) from mercury resistance transposon, TnMERI1
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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
al., 2009), CH4 and artificial mash gas were used to domesticate the mixed methanotrophic cultures capable of stably and effectively abating CH4 , respectively. Denaturing gradient gel electrophoresis (DGGE) was adopted to analyze the changes of microbial community structure during the enrichment process. Biofilters and the on site removal of methane from coal bed by using the enriched methanotrophic consortia were studied. Clone library and DGGE showed significant changes in microbial communities during the enrichment processes with different gases. By using the stably enriched methanotrophic consortium, the high cell density culture was achieved and the removal efficiency of CH4 by the biofilter and spraying the consortium in the simulated coal bed reached more than 90%. This study demonstrated that the methanotrophic consortium enriched from the coal mine soils could be effective for reduction of methane control in coal mines. Acknowledgment: This work was supported by 863 Project of MOST of China (No. 2006AA02Z203) and the National Key Basic Research Program (973 plan) (No. 2007CB109203). Reference
products (cathecol/hydroquinone molar ratio was nearly 2). The selectivity based on H2 O2 , i.e. mol of dihydroxylated derivatives produced × 100/mol of used hydrogen peroxide. was satisfactory and equal to 48-58%. Pd(II)-EPS was also prepared and its application as potential catalyst in reductive dehalogenation of halo arenes in a bi-phase water/organic solvent mixture and in a Heck-type reaction between bromo-benzene and unsaturated esters in DMA is currently under investigation. This new approach for preparation and application of catalysts seems to be promising with some economic and environment friendly advantages and the number of potential application might be broad. doi:10.1016/j.jbiotec.2010.08.143 [E.48] Purification and function analysis of a broad-spectrum organomercurial lyase (MerB3) from mercury resistance transposon, TnMERI1
Han, B., et al., 2009. FEMS Microbiology Ecology 70, 196–207.
Mei-Fang Chien 1,∗ , Hui-Tzu Lin 2 , Kuo-Hsing Lin 2 , Ginro Endo 1 , Chieh-Chen Huang 2
doi:10.1016/j.jbiotec.2010.08.142
1
Faculty of Engineering, Tohoku Gakuin University, Japan Department of Life Sciences, National Chung Hsing University, Taiwan Keywords: Organomercurial compound; Organomercurial lyase; MerB 2
[E.47] Bio-generated metal-binding polysaccharide as catalyst for synthetic applications and organic pollutant transformations Franco Baldi 1,∗ , Davide Marchetto 1 , Stefano Paganelli 1 , Oreste Piccolo 2 1
Cà Foscari University, Italy SCSOP, Italy Keywords: Metal; Exopolysaccharide; Organic compunds; Transformation 2
Bio-generated metal-binding polysaccharides may be novel potential sustainable catalysts for numerous synthetic applications and environment remediation. Depending on the nature of the metal bound to carbohydrates it is possible to perform hydrogenation, oxidation and C-C bond formation reactions. In this context we are currently investigating the properties of different metals, such as Fe(III) and Pd (II), bound to exopolysaccharides (EPS) produced by a Klebsiella oxytoca BAS-10 isolated from pyrite mines in the Southern Tuscany (Italy). This strain, under anaerobic conditions, during Fe(III)-citrate fermentation produces in the late stationary phase a large amount of colloidal material. EPS production was approximately 6.65 g.l−1 and contains 36% of total iron bound to polysaccharide. EPS can be also prepared without Fe(III) using sodium citrate during the fermentation; by adding an aqueous or organic solution of some suitable metal salts, it is possible to produce Me-EPS by cation exchange. Gel or semi-crystalline products may be easily recovered and characterized. An eptameric unit with 4 ␣-rhamnose, 2 -glucuronic acids and 1  − galactose is repeated to form long polysaccharide molecules of several million Dalton; metals should be located mostly in the proximity of the two glucuronic acids molecules. In some experiment tests Fe(III)-EPS catalyzed the oxidation of phenol with 35% H2 O2 in water or in a mixture of acetonitrile/water nearly 1/1 in the presence or absence of catalytic amount of acetic acid, to afford a mixture of catechol and hydroquinone. In the best reaction conditions, using phenol/H2 O2 in 1.6–2.6 molar ratio, a conversion of 25–18% of phenol was observed with a selectivity ranged 94–96% in dihydroxylated
Organomercurial compounds are potent toxins because of their lipophilicity and affinity for thiol residues of proteins and tend to bioaccumulate in the ecosystem through the food chain. The moststudied microbial mercury resistance mechanism is an enzymatic system which degrades organomercurials to the less toxic elemental mercury. The crucial step is the protonolysis of carbon-mercury bonds by the organomercurial lyase, MerB. However, MerBs identified so far show low similarity and different substrate specificities. In mercury resistant bacterium, Bacillus megaterium MB1, three merB genes are identified in a transposon, TnMERI1, while MerB3 protein confers broad substrate specificity and the fastest removal activity to p-chloromercuribenzoate. In this study, fast performance liquid chromatography was applied to purify the MerB3 of B. megaterium MB1, and subsequent function analysis was performed. The results show that MerB3 catalyzes the protonolysis of the carbon–mercury bonds of both alkyl- and aryl-mercurials and show that MerB3 performs broader substrate specificity than MerB2 does. The existence of thiol groups enhances the detoxification reaction of MerB3, suggesting that the thiol group of MerB3 facilitate the cleavage of carbon-mercury bonds. As the results of enzyme kinetics parameters, the Km of MerB3 to phenylmercury acetate and p-chloromercuribenzoate are 4.5 × 10−3 mM and 7.5 × 10−3 mM, respectively, which both of them are much smaller than the Km of Gram negative MerB. It suggests that the affinity of B. megaterium MB1 MerB3 is better than Gram negative MerB is. These results show the MerB3 is an appropriate candidate when apply it to the bioremediation of organomercurial contamination. doi:10.1016/j.jbiotec.2010.08.144
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
al., 2009), CH4 and artificial mash gas were used to domesticate the mixed methanotrophic cultures capable of stably and effectively abating CH4 , respectively. Denaturing gradient gel electrophoresis (DGGE) was adopted to analyze the changes of microbial community structure during the enrichment process. Biofilters and the on site removal of methane from coal bed by using the enriched methanotrophic consortia were studied. Clone library and DGGE showed significant changes in microbial communities during the enrichment processes with different gases. By using the stably enriched methanotrophic consortium, the high cell density culture was achieved and the removal efficiency of CH4 by the biofilter and spraying the consortium in the simulated coal bed reached more than 90%. This study demonstrated that the methanotrophic consortium enriched from the coal mine soils could be effective for reduction of methane control in coal mines. Acknowledgment: This work was supported by 863 Project of MOST of China (No. 2006AA02Z203) and the National Key Basic Research Program (973 plan) (No. 2007CB109203). Reference
products (cathecol/hydroquinone molar ratio was nearly 2). The selectivity based on H2 O2 , i.e. mol of dihydroxylated derivatives produced × 100/mol of used hydrogen peroxide. was satisfactory and equal to 48-58%. Pd(II)-EPS was also prepared and its application as potential catalyst in reductive dehalogenation of halo arenes in a bi-phase water/organic solvent mixture and in a Heck-type reaction between bromo-benzene and unsaturated esters in DMA is currently under investigation. This new approach for preparation and application of catalysts seems to be promising with some economic and environment friendly advantages and the number of potential application might be broad. doi:10.1016/j.jbiotec.2010.08.143 [E.48] Purification and function analysis of a broad-spectrum organomercurial lyase (MerB3) from mercury resistance transposon, TnMERI1
Han, B., et al., 2009. FEMS Microbiology Ecology 70, 196–207.
Mei-Fang Chien 1,∗ , Hui-Tzu Lin 2 , Kuo-Hsing Lin 2 , Ginro Endo 1 , Chieh-Chen Huang 2
doi:10.1016/j.jbiotec.2010.08.142
1
Faculty of Engineering, Tohoku Gakuin University, Japan Department of Life Sciences, National Chung Hsing University, Taiwan Keywords: Organomercurial compound; Organomercurial lyase; MerB 2
[E.47] Bio-generated metal-binding polysaccharide as catalyst for synthetic applications and organic pollutant transformations Franco Baldi 1,∗ , Davide Marchetto 1 , Stefano Paganelli 1 , Oreste Piccolo 2 1
Cà Foscari University, Italy SCSOP, Italy Keywords: Metal; Exopolysaccharide; Organic compunds; Transformation 2
Bio-generated metal-binding polysaccharides may be novel potential sustainable catalysts for numerous synthetic applications and environment remediation. Depending on the nature of the metal bound to carbohydrates it is possible to perform hydrogenation, oxidation and C-C bond formation reactions. In this context we are currently investigating the properties of different metals, such as Fe(III) and Pd (II), bound to exopolysaccharides (EPS) produced by a Klebsiella oxytoca BAS-10 isolated from pyrite mines in the Southern Tuscany (Italy). This strain, under anaerobic conditions, during Fe(III)-citrate fermentation produces in the late stationary phase a large amount of colloidal material. EPS production was approximately 6.65 g.l−1 and contains 36% of total iron bound to polysaccharide. EPS can be also prepared without Fe(III) using sodium citrate during the fermentation; by adding an aqueous or organic solution of some suitable metal salts, it is possible to produce Me-EPS by cation exchange. Gel or semi-crystalline products may be easily recovered and characterized. An eptameric unit with 4 ␣-rhamnose, 2 -glucuronic acids and 1  − galactose is repeated to form long polysaccharide molecules of several million Dalton; metals should be located mostly in the proximity of the two glucuronic acids molecules. In some experiment tests Fe(III)-EPS catalyzed the oxidation of phenol with 35% H2 O2 in water or in a mixture of acetonitrile/water nearly 1/1 in the presence or absence of catalytic amount of acetic acid, to afford a mixture of catechol and hydroquinone. In the best reaction conditions, using phenol/H2 O2 in 1.6–2.6 molar ratio, a conversion of 25–18% of phenol was observed with a selectivity ranged 94–96% in dihydroxylated
Organomercurial compounds are potent toxins because of their lipophilicity and affinity for thiol residues of proteins and tend to bioaccumulate in the ecosystem through the food chain. The moststudied microbial mercury resistance mechanism is an enzymatic system which degrades organomercurials to the less toxic elemental mercury. The crucial step is the protonolysis of carbon-mercury bonds by the organomercurial lyase, MerB. However, MerBs identified so far show low similarity and different substrate specificities. In mercury resistant bacterium, Bacillus megaterium MB1, three merB genes are identified in a transposon, TnMERI1, while MerB3 protein confers broad substrate specificity and the fastest removal activity to p-chloromercuribenzoate. In this study, fast performance liquid chromatography was applied to purify the MerB3 of B. megaterium MB1, and subsequent function analysis was performed. The results show that MerB3 catalyzes the protonolysis of the carbon–mercury bonds of both alkyl- and aryl-mercurials and show that MerB3 performs broader substrate specificity than MerB2 does. The existence of thiol groups enhances the detoxification reaction of MerB3, suggesting that the thiol group of MerB3 facilitate the cleavage of carbon-mercury bonds. As the results of enzyme kinetics parameters, the Km of MerB3 to phenylmercury acetate and p-chloromercuribenzoate are 4.5 × 10−3 mM and 7.5 × 10−3 mM, respectively, which both of them are much smaller than the Km of Gram negative MerB. It suggests that the affinity of B. megaterium MB1 MerB3 is better than Gram negative MerB is. These results show the MerB3 is an appropriate candidate when apply it to the bioremediation of organomercurial contamination. doi:10.1016/j.jbiotec.2010.08.144