- Email: [email protected]
Protein engineering of non-natural enzymes
S114
Abstracts / Journal of Biotechnology 131S (2007) S98–S121
of new enzymes with regard to process parameters (e.g. temperature, pH, potential solvents) to optimize the process total turnover number and the space time yield respectively. However, particularly the determination of the enzyme long term stability is too cost-intensive and laborious to be taken into account in the industrial screening at the state of the art. A new technique, “Enzyme Test Bench”, for the fast enzyme characterization, especially for the long term stability determination, was developed in cooperation between the BASF AG and the Biochemical Engineering of RWTH-Aachen University (BioVT). The concept of the Enzyme Test Bench consists in short enzyme tests under partly extreme conditions to predict the enzyme long term stability under moderate conditions. According to Boy et al. (2001) the technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles by optimal experimental design, the long term deactivation effects can be accelerated and detected within hours with high confidence. During the experiment the enzyme activity is measured online to parameterize the model. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The BASF brings its expertise in modeling biochemical processes, parameter estimation and optimal experimental design into the project. Therefore, temperature profiles are designed to maximize the information content of the experimental results. The BioVT applies its know-how in the development of optical online measurement instrumentation for biochemical applications in micro reactor arrays (Samorski et al., 2005) to engineer the Enzyme Test Bench. For instance, up to 96 samples can be automatically assayed, shaken, homogenously tempered in the range of 10–80 ◦ C ± 0.5 ◦ C whereas the reaction rates are measured online optically without interruption of the shaking. Approved by long time experiments the new fast micro scale method gives exemplary insights into the stability behavior of the important enzyme classes of hydrolases. At the current state of development the method is expanded to the enzyme class of oxidases. Furthermore the influence of different educts and products as well as different operating conditions (temperature, pH-value, additives) on the stability can be tested in few experiments in the range of hours. Consequently, the new technique is expected to support the process of improvement of high throughput screening to high information content screening.
30. Protein engineering of non-natural enzymes Mari Ylianttila a , Mikko Salin b , Marco Casteleijn a , Mirja Krause a , Sampo Mattila c , Marja Lajunen c , Rik K. Wierenga b , Peter Neubauer a,∗ a
c Department
Boy, M., B¨ohm, D., Voss, H., 2001. A new method for rapid determination of biocatalyst process stability. Biocatalysis and Biotransformation 19, 413–425. Samorski, M., M¨uller-Newen, G., B¨uchs, J., 2005. Quasi-continuous combined scattered light and fluorescence measurements: A novel measurement technique for shaken microtiter plates. Biotechnol Bioeng 92 (1), 61–68.
doi:10.1016/j.jbiotec.2007.07.197
of Chemistry, Oulu, Finland
Our challenging aim is to design new artificial enzymes called Kealases by structure-based directed evolution of Triosephosphate Isomerase (TIM) which has a very high substrate specificity. Kealases in difference to the wild type enzyme have different substrate specificity, but still catalyze the efficient conversion of ␣-hydroxyketones into chiral ␣-hydroxyaldehydes. With a rational structure-based approach we have shown that TIM-mutants with a wider substrate binding pocket bind totally new molecules that are not bound by wild type TIM. Furthermore, crystal structures indicate that the new compounds are located so in the active site of the enzyme that biocatalysis could occur. Based on these observations TIM mutants have been further modified by directed evolution to improve their biocatalytic activity for these new substrates. Here we present our new engineered biocatalyst platform for synthesis of chemical building blocks and a new non-standard procedure for the iterative use of mutagenesis and screening /directed evolution of catalytically active molecules (standard technology is the search for binders only). Our study illustrates the powerful approach of creating new biocatalysts by a very cooperative approach, connecting closely molecular engineering, enzymology, structure based biophysical and modelling as well as chemical synthesis competence. doi:10.1016/j.jbiotec.2007.07.198 31. Creating new enzymes—From triosephosphate isomerase to kealases Marco Casteleijn a , Markus Alahuhta b , Matti Vaismaa c , Sampo Mattila c , Marja Lajunen c , Jouni Pursiainen c , Rik K. Wierenga a,∗ , Peter Neubauer a a
References
University of Oulu, P.O. Box 4300, FI-90014, Oulu, Finland
b Department of Biochemistry, University of Oulu, Oulu, Finland
University of Oulu, P.O.Box 4300, FI-90014, Oulu, Finland
b Department of Biochemistry, University of Oulu, Oulu, Finland c Department
of Chemistry, Oulu, Finland
Triosephosphate Isomerase (TIM) catalyses an important chiral conversion which is not easily done with the current chemical methods. Our aim is to build a platform of TIM variants (“Kealases”) with a widened substrate range. Therefore we established by rational design a library of TIM variants and a library of novel chemical substrate analogues. The key molecule for future engineering is a monomeric form of TIM (A-TIM) which has been created and well characterized. A break through observation is that newly designed ligands bind its new binding pocket, and still has a competent active site.
Abstracts / Journal of Biotechnology 131S (2007) S98–S121
of new enzymes with regard to process parameters (e.g. temperature, pH, potential solvents) to optimize the process total turnover number and the space time yield respectively. However, particularly the determination of the enzyme long term stability is too cost-intensive and laborious to be taken into account in the industrial screening at the state of the art. A new technique, “Enzyme Test Bench”, for the fast enzyme characterization, especially for the long term stability determination, was developed in cooperation between the BASF AG and the Biochemical Engineering of RWTH-Aachen University (BioVT). The concept of the Enzyme Test Bench consists in short enzyme tests under partly extreme conditions to predict the enzyme long term stability under moderate conditions. According to Boy et al. (2001) the technique is based on the mathematical modeling of temperature dependent enzyme activation and deactivation. Adapting the temperature profiles by optimal experimental design, the long term deactivation effects can be accelerated and detected within hours with high confidence. During the experiment the enzyme activity is measured online to parameterize the model. Thus, the enzyme activity and long term stability can be calculated as a function of temperature. The BASF brings its expertise in modeling biochemical processes, parameter estimation and optimal experimental design into the project. Therefore, temperature profiles are designed to maximize the information content of the experimental results. The BioVT applies its know-how in the development of optical online measurement instrumentation for biochemical applications in micro reactor arrays (Samorski et al., 2005) to engineer the Enzyme Test Bench. For instance, up to 96 samples can be automatically assayed, shaken, homogenously tempered in the range of 10–80 ◦ C ± 0.5 ◦ C whereas the reaction rates are measured online optically without interruption of the shaking. Approved by long time experiments the new fast micro scale method gives exemplary insights into the stability behavior of the important enzyme classes of hydrolases. At the current state of development the method is expanded to the enzyme class of oxidases. Furthermore the influence of different educts and products as well as different operating conditions (temperature, pH-value, additives) on the stability can be tested in few experiments in the range of hours. Consequently, the new technique is expected to support the process of improvement of high throughput screening to high information content screening.
30. Protein engineering of non-natural enzymes Mari Ylianttila a , Mikko Salin b , Marco Casteleijn a , Mirja Krause a , Sampo Mattila c , Marja Lajunen c , Rik K. Wierenga b , Peter Neubauer a,∗ a
c Department
Boy, M., B¨ohm, D., Voss, H., 2001. A new method for rapid determination of biocatalyst process stability. Biocatalysis and Biotransformation 19, 413–425. Samorski, M., M¨uller-Newen, G., B¨uchs, J., 2005. Quasi-continuous combined scattered light and fluorescence measurements: A novel measurement technique for shaken microtiter plates. Biotechnol Bioeng 92 (1), 61–68.
doi:10.1016/j.jbiotec.2007.07.197
of Chemistry, Oulu, Finland
Our challenging aim is to design new artificial enzymes called Kealases by structure-based directed evolution of Triosephosphate Isomerase (TIM) which has a very high substrate specificity. Kealases in difference to the wild type enzyme have different substrate specificity, but still catalyze the efficient conversion of ␣-hydroxyketones into chiral ␣-hydroxyaldehydes. With a rational structure-based approach we have shown that TIM-mutants with a wider substrate binding pocket bind totally new molecules that are not bound by wild type TIM. Furthermore, crystal structures indicate that the new compounds are located so in the active site of the enzyme that biocatalysis could occur. Based on these observations TIM mutants have been further modified by directed evolution to improve their biocatalytic activity for these new substrates. Here we present our new engineered biocatalyst platform for synthesis of chemical building blocks and a new non-standard procedure for the iterative use of mutagenesis and screening /directed evolution of catalytically active molecules (standard technology is the search for binders only). Our study illustrates the powerful approach of creating new biocatalysts by a very cooperative approach, connecting closely molecular engineering, enzymology, structure based biophysical and modelling as well as chemical synthesis competence. doi:10.1016/j.jbiotec.2007.07.198 31. Creating new enzymes—From triosephosphate isomerase to kealases Marco Casteleijn a , Markus Alahuhta b , Matti Vaismaa c , Sampo Mattila c , Marja Lajunen c , Jouni Pursiainen c , Rik K. Wierenga a,∗ , Peter Neubauer a a
References
University of Oulu, P.O. Box 4300, FI-90014, Oulu, Finland
b Department of Biochemistry, University of Oulu, Oulu, Finland
University of Oulu, P.O.Box 4300, FI-90014, Oulu, Finland
b Department of Biochemistry, University of Oulu, Oulu, Finland c Department
of Chemistry, Oulu, Finland
Triosephosphate Isomerase (TIM) catalyses an important chiral conversion which is not easily done with the current chemical methods. Our aim is to build a platform of TIM variants (“Kealases”) with a widened substrate range. Therefore we established by rational design a library of TIM variants and a library of novel chemical substrate analogues. The key molecule for future engineering is a monomeric form of TIM (A-TIM) which has been created and well characterized. A break through observation is that newly designed ligands bind its new binding pocket, and still has a competent active site.