The Experts below are selected from a list of 321 Experts worldwide ranked by ideXlab platform
Jeifu Shaw - One of the best experts on this subject based on the ideXlab platform.
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role of the c terminal domain of thermus thermophilus trehalose synthase in the thermophilicity Thermostability and efficient production of trehalose
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Jia Hung Wang, Meng Yin Tsai, Jen Jye Chen, Jeifu ShawAbstract:: Trehalose synthase (TS) from Thermus thermophilus (TtTS) is a thermostable enzyme that catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation. It has a relatively higher thermophilicity and Thermostability and a better conversion ratio for trehalose production than other known TSs from different sources at present. By amino acid sequences and the schematic motif alignment of trehalose synthase-related enzymes, it was found that TtTS (965 amino acid residues) contains a particular C-terminal fragment that is not found in most other TSs. To verify the function of this fragment, C-terminal deletion and enzyme fusion were respectively performed to explain the important role this fragment plays in the formation of trehalose. First, the C terminus (TtTSDeltaN, 415 amino acid residues) of TtTS is deleted to construct a TtTSDeltaC containing 550 amino acids. Furthermore, a novel cold-active TS was cloned and purified from Deinococcus radiodurans (DrTS, 552 amino acid residues) and then a fusion protein was created with TtTSDeltaN at the C terminus of DrTS (DrTS-TtTSDeltaN). It was found that the recombinant TtTStriangle upC enzyme had a lower Thermostability and a higher byproduct than TtTS in catalyzing the conversion of maltose into trehalose. On the other hand, the recombinant DrTS-TtTSDeltaN enzyme had a higher Thermostability and a lower byproduct than DrTS in their reactions. The above-mentioned results allowed the inference that the C terminus of TtTS plays a key role in maintaining its Thermostability and hence in modulating the side reaction to reduce glucose production at a high temperature. A new, simple, and fast method to improve thermophilicity by fusing this fragment with particular conformation to a thermolabile enzyme is offered.
Jia Hung Wang - One of the best experts on this subject based on the ideXlab platform.
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role of the c terminal domain of thermus thermophilus trehalose synthase in the thermophilicity Thermostability and efficient production of trehalose
Journal of Agricultural and Food Chemistry, 2007Co-Authors: Jia Hung Wang, Meng Yin Tsai, Jen Jye Chen, Jeifu ShawAbstract:: Trehalose synthase (TS) from Thermus thermophilus (TtTS) is a thermostable enzyme that catalyzes the conversion of maltose into trehalose by intramolecular transglucosylation. It has a relatively higher thermophilicity and Thermostability and a better conversion ratio for trehalose production than other known TSs from different sources at present. By amino acid sequences and the schematic motif alignment of trehalose synthase-related enzymes, it was found that TtTS (965 amino acid residues) contains a particular C-terminal fragment that is not found in most other TSs. To verify the function of this fragment, C-terminal deletion and enzyme fusion were respectively performed to explain the important role this fragment plays in the formation of trehalose. First, the C terminus (TtTSDeltaN, 415 amino acid residues) of TtTS is deleted to construct a TtTSDeltaC containing 550 amino acids. Furthermore, a novel cold-active TS was cloned and purified from Deinococcus radiodurans (DrTS, 552 amino acid residues) and then a fusion protein was created with TtTSDeltaN at the C terminus of DrTS (DrTS-TtTSDeltaN). It was found that the recombinant TtTStriangle upC enzyme had a lower Thermostability and a higher byproduct than TtTS in catalyzing the conversion of maltose into trehalose. On the other hand, the recombinant DrTS-TtTSDeltaN enzyme had a higher Thermostability and a lower byproduct than DrTS in their reactions. The above-mentioned results allowed the inference that the C terminus of TtTS plays a key role in maintaining its Thermostability and hence in modulating the side reaction to reduce glucose production at a high temperature. A new, simple, and fast method to improve thermophilicity by fusing this fragment with particular conformation to a thermolabile enzyme is offered.
Manfred T Reetz - One of the best experts on this subject based on the ideXlab platform.
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multiparameter optimization in directed evolution engineering Thermostability enantioselectivity and activity of an epoxide hydrolase
ACS Catalysis, 2016Co-Authors: Manfred T Reetz, Guangyue Li, Hui ZhangAbstract:The challenge of optimizing several parameters in the directed evolution of enzymes remains a central issue. In this study we address the Thermostability, enantioselectivity, and activity of limonene epoxide hydrolase (LEH) as the catalyst in the hydrolytic desymmetrization of cyclohexene oxide with formation of (R,R)- and (S,S)-cyclohexane-1,2-diol. Wild type LEH shows a Thermostability of T5030 = 41 °C and an enanioselectivity of 2% ee (S,S). Two approaches are described herein. In one strategy, the mutations generated previously by Janssen, Baker, and co-workers for notably increased Thermostability are combined with mutations evolved earlier for enhanced enantioselectivity. Although highly enantioselective R,R and S,S variants (92–93% ee) with increases in T5030 by 10–11 °C were obtained, relative to wild type LEH the tradeoff in activity was significant. The second strategy based on the simultaneous optimization of both parameters using iterative saturation mutagenesis (ISM) with minimized tradeoff i...
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iterative saturation mutagenesis ism for rapid directed evolution of functional enzymes
Nature Protocols, 2007Co-Authors: Manfred T Reetz, Jose Daniel CarballeiraAbstract:Iterative saturation mutagenesis (ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary molecular biological work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of saturation mutagenesis at rationally chosen sites in an enzyme, a given site being composed of one, two or three amino acid positions. The basis for choosing these sites depends on the nature of the catalytic property to be improved, e.g., enantioselectivity, substrate acceptance or Thermostability. In the case of Thermostability, sites showing highest B-factors (available from X-ray data) are chosen. The pronounced increase in Thermostability of the lipase from Bacillus subtilis (Lip A) as a result of applying ISM is illustrated here.
A.j. Rader - One of the best experts on this subject based on the ideXlab platform.
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Endoglucanases: insights into Thermostability for biofuel applications.
Biotechnology for Biofuels, 2013Co-Authors: Ragothaman M. Yennamalli, A.j. Rader, Adam J. Kenny, Jeffrey D. WoltAbstract:Obtaining bioethanol from cellulosic biomass involves numerous steps, among which the enzymatic conversion of the polymer to individual sugar units has been a main focus of the biotechnology industry. Among the cellulases that break down the polymeric cellulose are endoglucanases that act synergistically for subsequent hydrolytic reactions. The endoglucanases that have garnered relatively more attention are those that can withstand high temperatures, i.e., are thermostable. Although our understanding of Thermostability in endoglucanases is incomplete, some molecular features that are responsible for increased Thermostability have been recently identified. This review focuses on the investigations of endoglucanases and their implications for biofuel applications.
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Sequence, Structure and Dynamics Analysis of Thermostability in Endoglucanases
Biophysical Journal, 2011Co-Authors: Ragothaman M. Yennamalli, Jeffrey D. Wolt, A.j. RaderAbstract:Endoglucanases are crucial enzymes used in the production of biofuels from cellulosic biomass, a process which requires Thermostability at high processing temperatures. Despite the economic importance of these industrial proteins, we currently lack a basic understanding of how some endoglucanases can efficiently function at elevated processing temperatures, while others with the same fold have substantial reduction in activity.Here we explore the origins of Thermostability in endoglucanases from sequence, structure, and dynamics perspectives using thermostable and mesostable protein sets. We performed a comparative sequence and structure analysis for thermophilic and mesophilic endoglucanases in (α/β)8, β-jelly roll, and (α/α)6 folds, followed by a dynamics analysis of the (α/β)8 fold using elastic network models. We observed that thermophilic endoglucanases and their mesophilic counterparts differ significantly in their amino acid compositions. Interestingly, these compositional differences are specific to protein folds and enzyme families and lead to modification in hydrophobic, aromatic, and ionic interactions in a fold-dependent fashion.We then focused specifically on a pair of thermostable and mesostable endoglucanases for a detailed dynamics analysis. It is often the case that thermophiles have shorter loops than their mesophilic counterparts, which was suggested to impart Thermostability. In our case, however, the thermophile surprisingly possessed three insertions in the mesophilic loop regions and therefore has longer loops. The comparative structural dynamics analysis using elastic network models of (α/β)8 fold indicate that these three loops may contribute to the Thermostability by modulating the direction of correlated motions between the catalytic residues (acid/base donor and nucleophile). We also observed that the thermostable protein showed larger dynamic domains than its mesostable counterpart, which suggests that cooperative dynamics is a critical contributing factor to Thermostability.
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Thermostability in rubredoxin and its relationship to mechanical rigidity
Physical Biology, 2009Co-Authors: A.j. RaderAbstract:The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features possibly responsible for such Thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. Experimental evidence exists to both support and contradict this supposition. We computationally investigate the relationship between Thermostability and rigidity using rubredoxin as a case study. The mechanical rigidity is calculated using atomic models of homologous rubredoxin structures from the hyperthermophile Pyrococcus furiosus and mesophile Clostridium pasteurianum using the FIRST software. A global increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in Thermostability. Locally, rigidity differences (between mesophilic and thermophilic structures) agree with differences in protection factors.
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Thermostabilization Due to Rigidity: A Case Study of Rubredoxin
Biophysical Journal, 2009Co-Authors: A.j. RaderAbstract:The source of increased stability in proteins from organisms that thrive in extreme thermal environments is not well understood. Previous experimental and theoretical studies have suggested many different features responsible for such Thermostability. Many of these thermostabilizing mechanisms can be accounted for in terms of structural rigidity. Thus a plausible hypothesis accounting for this remarkable stability in thermophilic enzymes states that these enzymes have enhanced conformational rigidity at temperatures below their native, functioning temperature. This study investigates the relationship between Thermostability and rigidity using rubredoxin as a case study. The FIRST software is used to calculate local (residue level) and global rigidity for available rubredoxin structures and simulated mutants. Quantitative global rigidity measures indicate that an increase in structural rigidity (equivalently a decrease in flexibility) corresponds to an increase in Thermostability. At the level of individual residues, hydrogen deuterium exchange experiments level indicate differential changes in flexibility between mesophilic and thermophilic rubredoxin structures that agree with computational flexibility analysis from the FIRST software.
Digvijay Verma T. Satyanarayana - One of the best experts on this subject based on the ideXlab platform.
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Improvement in Thermostability of metagenomic GH11 endoxylanase (Mxyl) by site-directed mutagenesis and its applicability in paper pulp bleaching process
Journal of Industrial Microbiology & Biotechnology, 2013Co-Authors: Digvijay Verma T. SatyanarayanaAbstract:An attempt has been made for enhancing the Thermostability of xylanase (Mxyl) retrieved from a compost-soil-based metagenomic library. The analysis of the structure of xylanase by molecular dynamics simulation revealed more structural fluctuations in β-sheets. When the surface of β-sheets was enriched with arginine residues by substituting serine/threonine by site-directed mutagenesis, the enzyme with four arginine substitutions (MxylM4) exhibited enhanced Thermostability at 80 °C. The T _1/2 of MxylM4 at 80 °C, in the presence of birchwood xylan, increased from 130 to 150 min at 80 °C without any alteration in optimum pH and temperature and molecular mass. Improvement in Thermostability of MxylM4 was corroborated by increase in T _m by 6 °C over that of Mxyl. The K _m of MxylM4, however, increased from 8.01 ± 0.56 of Mxyl to 12.5 ± 0.32 mg ml^−1, suggesting a decrease in the affinity as well as specific enzyme activity. The Mxyl as well as MxylM4 liberated chromophores and lignin-derived compounds from kraft pulp, indicating their applicability in pulp bleaching.
