- Email: [email protected]
DHAP-dependent aldolases in stereoselective synthesis: Kinetic studies and immobilisation
Abstracts / Journal of Biotechnology 131S (2007) S74–S97
S75
3. Removal of phenol from aqueous solutions using various biomass
4. DHAP-dependent aldolases in stereoselective synthesis: Kinetic studies and immobilisation
Senem Ustun a,∗ , Hanife Buyukgungor b
In´es Ardao, Trinitat Suau, Jordi Ruiz, Sergio Daniel R´ıos, Gre´ gorio Alvaro, Gl`oria Caminal, Maria Dolors Benaiges, Gl`oria Gonz´alez, Josep L´opez-Sant´ın ∗
a
General Directorate of Turkish State Hydrolic Works, Samsun, Turkey b Ondokuz Mayis University, Environmental Engineering Department, Samsun, Turkey E-mail address: [email protected]. Wastewater containing phenolic compounds is a serious problem for the environment. Phenolic compounds are known or suspected human carcinogens. Phenols contamination of drinking water even at 1 g/l concentration can cause significant taste and odor problems. So, using new treatment and recovery technologies is essential to reduce phenols pollution. Recently, biosorbents have emerged as an eco-friendly effective and low cost material option. These biosorbents include some fungus, agricultural wastes, algae and bacteria. Biosorption attacks attention at recent year as an alternative to conventional methods for phenolic compounds removal from water and wastewater. Fungal cell walls and their components have major role in the biosorption. Fungal biomass can also take up considerable quantities of pollutants from aqueous solutions by adsorption or a related process, even in the absence of physiological activity. This study, investigated the use of non-viable pretreated cells of Aspergillus niger, Rhizopus arrhizus and activated sludge to remove phenol from aqueous solutions in batch reactors. Three types of died pretreated A. niger, R. arrhizus and activated sludge biomasses powders were used as a biosorbent to remove phenol present in an aqueous solution at a concentration of 50 mg/l. It was observed that initial pH, initial biosorbent concentration and adsorption time affected adsorption rates. It was observed that, sulfuric acid-pretreated died A. niger biomass powder was the most effective in three types of used microorganisms to remove phenol. The maximum removal of phenol was observed at in an initial pH of 5.3 for the sulfuric acid-pretreated biomass. Approximately, 85–90% phenol was removed for the sulphuric acid pretreated A. niger biomass and an initial concentration of 50 mg/l of phenol within 50 min.
Universitat Aut`onoma de Barcelona, Departament d”Enginyeria Qu´ımica, Escola T`ecnica Superior d”Enginyeria. Edifici Q, Universitat Aut`onoma de Barcelona, Campus de Bellaterra, 08193 Bellaterra (Cerdanyola del Vall`es), Catalunya, Spain E-mail address: [email protected]. Aldolases are biocatalysts of great interest in chiral synthetic processes because they are able to catalyze C–C bond formation controlling the regio and stereoselectivity of aldol addition reactions. The four enzymes of the family of dihydroxyacetone phosphate (DHAP) -dependent aldolases, fructose-1,6-bisphosphate aldolase (FruA, EC 4.1.2.13), fuculose-1-phosphate aldolase (FucA, EC 4.1.2.17), rhamnulose-1-phosphate aldolase (RhuA, EC 4.1.2.19) and tagatose-1,6-bisphosphate aldolase (TagA, EC 4.1.2.40), have been proven to be useful in synthesis because they catalyze the aldol addition of DHAP with a wide range of acceptor aldehydes conducting to products with two new stereogenic centers. Depending of the employed enzyme, ␣,dihydroxyketones of complementary stereochemistry can be obtained (Samkland and Sprenger, 2006; Fessner and Helaine, 2001). Most of the published work in its application for the synthesis of valuable products cover the selection of suitable acceptor aldehydes and stereoselectivity, with a focus on organic synthesis (Fessner and Helaine, 2001; Sukuraman and Hanefeld, 2005). Nevertheless, there is a lack of papers concerning kinetic behavior and immobilization, necessary information for bioreactor design. DHAP-dependent aldolases, DERA and two treonine aldolases have been cloned and overexpressed in E. coli as fusion proteins with a hexahistidine tag. DHAP-dependent aldolases have been used for the synthesis of aminopolyols, precursors of iminocyclitols, products with a great therapeutical potential. The aldol addition between DHAP and (S)-Cbz-alaninal has been selected:
doi:10.1016/j.jbiotec.2007.07.131
Using FucA as a model, the synthesis in cosolvent medium (DMF/water 20%, v/v), produced the expected 3R,4R,5S diastereomer with around 100% enatiomeric excess. Chemical and enzymatic secondary DHAP degradation reactions have been identified. The enzymatic non-synthetic DHAP degradation has been demonstrated for the type II aldolases as proceeding through the same enediolate intermediate than the synthesis one. Optimal operation conditions for maximum product conversion were identified (4 ◦ C in DHAP fed-batch
S76
Abstracts / Journal of Biotechnology 131S (2007) S74–S97
operation) and good yields (90%) and selectivities (>90%) obtained (Suau et al., 2006). With the above information, a two-reaction kinetic model has been proposed, involving twosubstrate and one-substrate Michaelis model, respectively. The inhibitory effects of substrates and products have been identified, and the model has been calibrated and validated, describing properly the reaction behavior. The aldolases have been immobilitzed either on glyoxalagarose supports (Suau et al., 2005) or on metal chelate supports by a one step purification and immobilization procedure (Ardao et al., 2006). In this last case, the influence of the divalent metal on activity has been determined, and Co2+ containing biocatalysts selected as the more active. Both immobilized derivatives have been shown to be able to catalyze the synthesis reaction in batch and fed-batch mode. Acknowledgements Spanish Plan Nacional I + D + I. Project number CTQ200501706/PPQ. DURSI 2005SGR 00698 Generalitat de Catalunya. Xarxa de Refer`encia en Biotecnologia (XRBa) and Xarxa XIT Transfer`encia de Tecnologia. CIDEM (Generalitat de Catalunya). I. Ardao acknowledges a predoctoral grant from DURSI (Generalitat de Catalunya). References ´ Ardao, I., Benaiges, M.D., Caminal, G., Alvaro, G., 2006. Enzyme Microb. Technol. 39, 22–27. Fessner, W.D., Helaine, V., 2001. Curr. Opin. Biotechnol. 12, 574–586. Samkland, A.K., Sprenger, G.A., 2006. Appl. Microbiol. Biotechnol. 71, 253–264. ´ Suau, T., Alvaro, G., Benaiges, M.D., L´opez-Sant´ın, J., 2006. Biotechnol. Bioeng. 93, 48–55. ´ Suau, T., Calveras, J., Clap´es, P., Benaiges, M.D., Alvaro, G., 2005. Biocatal. Biotransfor. 23, 241–250. Sukuraman, J., Hanefeld, U., 2005. Chem. Soc. Rev. 34, 530–542.
doi:10.1016/j.jbiotec.2007.07.132 5. Sorbitol co-feeding an efficient strategy to reduce metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris
To increase cell density and process productivity, as well as to reduce the induction time, a typical approach is the use of a multicarbon substrate in addition to methanol, generally glycerol or sorbitol (Cos et al., 2006). Recently, specific production rate of Rhizopus oryzae lipase (ROL) in P. pastoris, a protein that triggers the unfolded protein response in this host microorganism, has been improved by the co-feeding of sorbitol/methanol mixed substrates in batch and preliminary fed-batch cultures (Ram´on et al., 2007). The mixed substrate strategy applied to a Muts ROL single copy fed-batch culture at a low residual methanol concentration set-point (0.5 g l−1 ) has allowed to increase the production of heterologous protein in Muts phenotype, avoiding the drastic reduction of the specific production rate observed after the start of the induction phase when methanol is used as a sole carbon source. This fact, together with the higher specific growth rate achieved resulted in a significant increase of volumetric and specific productivities (Ram´on et al., 2007). Furthermore, sorbitol co-feeding in Muts ROL multicopy strain fed-batch cultivations allowed sustained cell growth and lipolytic productivity, whereas a dramatic viability loss had been observed in cells growing on pure methanol (Cos et al., 2005), strongly suggesting that cells growing on mixed substrates can – at least partially – overcome the stress caused by ROL overexpression in Muts cells. Also, this observation reveals that the cellular (stress) response to heterologous protein overexpression and secretion is interconnected with the energetic state of the cells. In this work we present a systematic study of the influence of methanol set-point and sorbitol feeding rate in fed-batch operation with Muts phenotype. Four experiments were made at a constant methanol set-point of 0.5 g l−1 and 2 g l−1 with two different sorbitol feeding rate to assure a constant specific rate of µmax and µmax /2 by means of a pre-programmed exponential feeding rate strategy. Lipolytic activity, yields, productivity and specific productivity, but also specific growth, consumption and production rates were analyzed showing that the best conditions were reached when the methanol set-point was 2 g l−1 with a low influence of the constant specific growth tested. Acknowledgements
Carolina Arnau ∗ , Ram´on Ram´on, Pau Ferrer, Francisco Valero
Spanish program on Chemical Science and Technology (CTQ2004-00300) and grant 2005-SGR-00698 (Generalitat de Catalunya).
Departament d’Enginyeria Qu´ımica. ETSE. Universitat Aut`onoma de Barcelona, Campus de Bellaterra, Bellaterra, Barcelona, Spain
References
E-mail address: [email protected]. The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for both basic research and industrial use. There are two main types of P. pastoris host strains with regard to their ability to assimilate methanol: Mut+ and Muts (Cereghino Lin and Cregg, 2000; Macauley-Patrick et al., 2005). Although Muts is not as sensitive to residual methanol in the cultivation media ad Mut+ strains, the lower specific growth rate of Muts limits the productivity of the process.
Cereghino Lin, J., Cregg, J.M., 2000. FEMS Microbiol. Rev. 24, 45–66. Cos, O., Serrano, A., Montesinos, J.L., Ferrer, P., Cregg, J.M., Valero, F., 2005. J. Biotechnol. 116, 321–335. Cos, O., Ram´on, R., Montesinos, J.L., Valero, F., 2006. Microb. Cell Factories 5, 17. Macauley-Patrick, S., Fazenda, M.L., McNEil, B., Harvey, L.M., 2005. Yeast 22, 249–270. Ram´on, R., Ferrer, P., Valero, F., 2007. Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a R. oryzae lipase in P. pastoris. J. Biotechnol. 130, 39–46.
doi:10.1016/j.jbiotec.2007.07.133
S75
3. Removal of phenol from aqueous solutions using various biomass
4. DHAP-dependent aldolases in stereoselective synthesis: Kinetic studies and immobilisation
Senem Ustun a,∗ , Hanife Buyukgungor b
In´es Ardao, Trinitat Suau, Jordi Ruiz, Sergio Daniel R´ıos, Gre´ gorio Alvaro, Gl`oria Caminal, Maria Dolors Benaiges, Gl`oria Gonz´alez, Josep L´opez-Sant´ın ∗
a
General Directorate of Turkish State Hydrolic Works, Samsun, Turkey b Ondokuz Mayis University, Environmental Engineering Department, Samsun, Turkey E-mail address: [email protected]. Wastewater containing phenolic compounds is a serious problem for the environment. Phenolic compounds are known or suspected human carcinogens. Phenols contamination of drinking water even at 1 g/l concentration can cause significant taste and odor problems. So, using new treatment and recovery technologies is essential to reduce phenols pollution. Recently, biosorbents have emerged as an eco-friendly effective and low cost material option. These biosorbents include some fungus, agricultural wastes, algae and bacteria. Biosorption attacks attention at recent year as an alternative to conventional methods for phenolic compounds removal from water and wastewater. Fungal cell walls and their components have major role in the biosorption. Fungal biomass can also take up considerable quantities of pollutants from aqueous solutions by adsorption or a related process, even in the absence of physiological activity. This study, investigated the use of non-viable pretreated cells of Aspergillus niger, Rhizopus arrhizus and activated sludge to remove phenol from aqueous solutions in batch reactors. Three types of died pretreated A. niger, R. arrhizus and activated sludge biomasses powders were used as a biosorbent to remove phenol present in an aqueous solution at a concentration of 50 mg/l. It was observed that initial pH, initial biosorbent concentration and adsorption time affected adsorption rates. It was observed that, sulfuric acid-pretreated died A. niger biomass powder was the most effective in three types of used microorganisms to remove phenol. The maximum removal of phenol was observed at in an initial pH of 5.3 for the sulfuric acid-pretreated biomass. Approximately, 85–90% phenol was removed for the sulphuric acid pretreated A. niger biomass and an initial concentration of 50 mg/l of phenol within 50 min.
Universitat Aut`onoma de Barcelona, Departament d”Enginyeria Qu´ımica, Escola T`ecnica Superior d”Enginyeria. Edifici Q, Universitat Aut`onoma de Barcelona, Campus de Bellaterra, 08193 Bellaterra (Cerdanyola del Vall`es), Catalunya, Spain E-mail address: [email protected]. Aldolases are biocatalysts of great interest in chiral synthetic processes because they are able to catalyze C–C bond formation controlling the regio and stereoselectivity of aldol addition reactions. The four enzymes of the family of dihydroxyacetone phosphate (DHAP) -dependent aldolases, fructose-1,6-bisphosphate aldolase (FruA, EC 4.1.2.13), fuculose-1-phosphate aldolase (FucA, EC 4.1.2.17), rhamnulose-1-phosphate aldolase (RhuA, EC 4.1.2.19) and tagatose-1,6-bisphosphate aldolase (TagA, EC 4.1.2.40), have been proven to be useful in synthesis because they catalyze the aldol addition of DHAP with a wide range of acceptor aldehydes conducting to products with two new stereogenic centers. Depending of the employed enzyme, ␣,dihydroxyketones of complementary stereochemistry can be obtained (Samkland and Sprenger, 2006; Fessner and Helaine, 2001). Most of the published work in its application for the synthesis of valuable products cover the selection of suitable acceptor aldehydes and stereoselectivity, with a focus on organic synthesis (Fessner and Helaine, 2001; Sukuraman and Hanefeld, 2005). Nevertheless, there is a lack of papers concerning kinetic behavior and immobilization, necessary information for bioreactor design. DHAP-dependent aldolases, DERA and two treonine aldolases have been cloned and overexpressed in E. coli as fusion proteins with a hexahistidine tag. DHAP-dependent aldolases have been used for the synthesis of aminopolyols, precursors of iminocyclitols, products with a great therapeutical potential. The aldol addition between DHAP and (S)-Cbz-alaninal has been selected:
doi:10.1016/j.jbiotec.2007.07.131
Using FucA as a model, the synthesis in cosolvent medium (DMF/water 20%, v/v), produced the expected 3R,4R,5S diastereomer with around 100% enatiomeric excess. Chemical and enzymatic secondary DHAP degradation reactions have been identified. The enzymatic non-synthetic DHAP degradation has been demonstrated for the type II aldolases as proceeding through the same enediolate intermediate than the synthesis one. Optimal operation conditions for maximum product conversion were identified (4 ◦ C in DHAP fed-batch
S76
Abstracts / Journal of Biotechnology 131S (2007) S74–S97
operation) and good yields (90%) and selectivities (>90%) obtained (Suau et al., 2006). With the above information, a two-reaction kinetic model has been proposed, involving twosubstrate and one-substrate Michaelis model, respectively. The inhibitory effects of substrates and products have been identified, and the model has been calibrated and validated, describing properly the reaction behavior. The aldolases have been immobilitzed either on glyoxalagarose supports (Suau et al., 2005) or on metal chelate supports by a one step purification and immobilization procedure (Ardao et al., 2006). In this last case, the influence of the divalent metal on activity has been determined, and Co2+ containing biocatalysts selected as the more active. Both immobilized derivatives have been shown to be able to catalyze the synthesis reaction in batch and fed-batch mode. Acknowledgements Spanish Plan Nacional I + D + I. Project number CTQ200501706/PPQ. DURSI 2005SGR 00698 Generalitat de Catalunya. Xarxa de Refer`encia en Biotecnologia (XRBa) and Xarxa XIT Transfer`encia de Tecnologia. CIDEM (Generalitat de Catalunya). I. Ardao acknowledges a predoctoral grant from DURSI (Generalitat de Catalunya). References ´ Ardao, I., Benaiges, M.D., Caminal, G., Alvaro, G., 2006. Enzyme Microb. Technol. 39, 22–27. Fessner, W.D., Helaine, V., 2001. Curr. Opin. Biotechnol. 12, 574–586. Samkland, A.K., Sprenger, G.A., 2006. Appl. Microbiol. Biotechnol. 71, 253–264. ´ Suau, T., Alvaro, G., Benaiges, M.D., L´opez-Sant´ın, J., 2006. Biotechnol. Bioeng. 93, 48–55. ´ Suau, T., Calveras, J., Clap´es, P., Benaiges, M.D., Alvaro, G., 2005. Biocatal. Biotransfor. 23, 241–250. Sukuraman, J., Hanefeld, U., 2005. Chem. Soc. Rev. 34, 530–542.
doi:10.1016/j.jbiotec.2007.07.132 5. Sorbitol co-feeding an efficient strategy to reduce metabolic burden caused by the overexpression of a Rhizopus oryzae lipase in Pichia pastoris
To increase cell density and process productivity, as well as to reduce the induction time, a typical approach is the use of a multicarbon substrate in addition to methanol, generally glycerol or sorbitol (Cos et al., 2006). Recently, specific production rate of Rhizopus oryzae lipase (ROL) in P. pastoris, a protein that triggers the unfolded protein response in this host microorganism, has been improved by the co-feeding of sorbitol/methanol mixed substrates in batch and preliminary fed-batch cultures (Ram´on et al., 2007). The mixed substrate strategy applied to a Muts ROL single copy fed-batch culture at a low residual methanol concentration set-point (0.5 g l−1 ) has allowed to increase the production of heterologous protein in Muts phenotype, avoiding the drastic reduction of the specific production rate observed after the start of the induction phase when methanol is used as a sole carbon source. This fact, together with the higher specific growth rate achieved resulted in a significant increase of volumetric and specific productivities (Ram´on et al., 2007). Furthermore, sorbitol co-feeding in Muts ROL multicopy strain fed-batch cultivations allowed sustained cell growth and lipolytic productivity, whereas a dramatic viability loss had been observed in cells growing on pure methanol (Cos et al., 2005), strongly suggesting that cells growing on mixed substrates can – at least partially – overcome the stress caused by ROL overexpression in Muts cells. Also, this observation reveals that the cellular (stress) response to heterologous protein overexpression and secretion is interconnected with the energetic state of the cells. In this work we present a systematic study of the influence of methanol set-point and sorbitol feeding rate in fed-batch operation with Muts phenotype. Four experiments were made at a constant methanol set-point of 0.5 g l−1 and 2 g l−1 with two different sorbitol feeding rate to assure a constant specific rate of µmax and µmax /2 by means of a pre-programmed exponential feeding rate strategy. Lipolytic activity, yields, productivity and specific productivity, but also specific growth, consumption and production rates were analyzed showing that the best conditions were reached when the methanol set-point was 2 g l−1 with a low influence of the constant specific growth tested. Acknowledgements
Carolina Arnau ∗ , Ram´on Ram´on, Pau Ferrer, Francisco Valero
Spanish program on Chemical Science and Technology (CTQ2004-00300) and grant 2005-SGR-00698 (Generalitat de Catalunya).
Departament d’Enginyeria Qu´ımica. ETSE. Universitat Aut`onoma de Barcelona, Campus de Bellaterra, Bellaterra, Barcelona, Spain
References
E-mail address: [email protected]. The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for both basic research and industrial use. There are two main types of P. pastoris host strains with regard to their ability to assimilate methanol: Mut+ and Muts (Cereghino Lin and Cregg, 2000; Macauley-Patrick et al., 2005). Although Muts is not as sensitive to residual methanol in the cultivation media ad Mut+ strains, the lower specific growth rate of Muts limits the productivity of the process.
Cereghino Lin, J., Cregg, J.M., 2000. FEMS Microbiol. Rev. 24, 45–66. Cos, O., Serrano, A., Montesinos, J.L., Ferrer, P., Cregg, J.M., Valero, F., 2005. J. Biotechnol. 116, 321–335. Cos, O., Ram´on, R., Montesinos, J.L., Valero, F., 2006. Microb. Cell Factories 5, 17. Macauley-Patrick, S., Fazenda, M.L., McNEil, B., Harvey, L.M., 2005. Yeast 22, 249–270. Ram´on, R., Ferrer, P., Valero, F., 2007. Sorbitol co-feeding reduces metabolic burden caused by the overexpression of a R. oryzae lipase in P. pastoris. J. Biotechnol. 130, 39–46.
doi:10.1016/j.jbiotec.2007.07.133