| 1. |
- Bolivar, Juan M, et al.
(författare)
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Coating of soluble and immobilized enzymes with ionic polymers : full stabilization of the quaternary structure of multimeric enzymes
- 2009
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 10:4, s. 742-747
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Tidskriftsartikel (refereegranskat)abstract
- This paper shows a simple and effective way to avoid the dissociation of multimeric enzymes by coating their surface with a large cationic polymer (e.g., polyethylenimine (PEI)) by ionic exchange. As model enzymes, glutamate dehydrogenase (GDH) from Thermus thermophilus and formate dehydrogenase (FDH) from Pseudomonas sp. were used. Both enzymes are very unstable at acidic pH values due to the rapid dissociation of their subunits (half-life of diluted preparations is few minutes at pH 4 and 25 degrees C). GDH and FDH were incubated in the presence of PEI yielding an enzyme-PEI composite with full activity. To stabilize the enzyme-polymer composite, a treatment with glutaraldehyde was required. These enzyme-PEI composites can be crosslinked with glutaraldehyde by immobilizing previously the composite onto a weak cationic exchanger. The soluble GDH-PEI composite was much more stable than unmodified GDH at pH 4 and 30 degrees C (retaining over 90% activity after 24 h incubation) with no effect of the GDH concentration in the inactivation course. The composite could be very strongly, but reversibly, adsorbed on cationic exchangers. Similarly, FDH could be treated with PEI and glutaraldehyde after adsorption on cationic exchangers, This permitted a stabilized FDH preparation. In this way, the coating of the enzymes surfaces with PEI is used as a simple and efficient strategy to prevent enzyme dissociation of multimeric enzymes. These composites can be used as a soluble catalyst or reversibly immobilized onto a cationic exchanger (e.g., CM-agarose).
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| 2. |
- Bolivar, Juan M, et al.
(författare)
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Immobilization-stabilization of a new recombinant glutamate dehydrogenase from Thermus thermophilus
- 2008
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Ingår i: Applied Microbiology and Biotechnology. - : Springer Science and Business Media LLC. - 0175-7598 .- 1432-0614. ; 80:1, s. 49-58
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Tidskriftsartikel (refereegranskat)abstract
- The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.
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| 3. |
- Hidalgo, Aurelio, et al.
(författare)
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Thermus thermophilus as a cell factory for the production of a thermophilic Mn-dependent catalase which fails to be synthesized in an active form in Escherichia coli
- 2004
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 70:7, s. 3839-3844
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Tidskriftsartikel (refereegranskat)abstract
- Thermostable Mn-dependent catalases are promising enzymes in biotechnological applications as H(2)O(2)-detoxifying systems. We cloned the genes encoding Mn-dependent catalases from Thermus thermophilus HB27 and HB8 and a less thermostable mutant carrying two amino acid replacements (M129V and E293G). When the wild-type and mutant genes were overexpressed in Escherichia coli, unmodified or six-His-tagged proteins of the expected size were overproduced as inactive proteins. Several attempts to obtain active forms or to activate the overproduced proteins were unsuccessful, even when soluble and thermostable proteins were used. Therefore, a requirement for a Thermus-specific activation factor was suggested. To overcome this problem, the Mn-dependent catalase genes were overexpressed directly in T. thermophilus under the control of the Pnar promoter. This promoter belongs to a respiratory nitrate reductase from of T. thermophilus HB8, whose transcription is activated by the combined action of nitrate and anoxia. Upon induction in T. thermophilus HB8, a 20- to 30-fold increase in catalase specific activity was observed, whereas a 90- to 110-fold increase was detected when the laboratory strain T. thermophilus HB27::nar was used as the host. The thermostability of the overproduced wild-type catalase was identical to that previously reported for the native enzyme, whereas decreased stability was detected for the mutant derivative. Therefore, our results validate the use of T. thermophilus as an alternative cell factory for the overproduction of thermophilic proteins that fail to be expressed in well-known mesophilic hosts.
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| 4. |
- Kuppler, Jonas, et al.
(författare)
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Global gradients in intraspecific variation in vegetative and floral traits are partially associated with climate and species richness
- 2020
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Ingår i: Global Ecology and Biogeography. - : Wiley. - 1466-822X .- 1466-8238. ; 29:6, s. 992-1007
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Tidskriftsartikel (refereegranskat)abstract
- AimIntraspecific trait variation (ITV) within natural plant communities can be large, influencing local ecological processes and dynamics. Here, we shed light on how ITV in vegetative and floral traits responds to large‐scale abiotic and biotic gradients (i.e., climate and species richness). Specifically, we tested whether associations of ITV with temperature, precipitation and species richness were consistent with any of four hypotheses relating to stress tolerance and competition. Furthermore, we estimated the degree of correlation between ITV in vegetative and floral traits and how they vary along the gradients.LocationGlobal.Time period1975–2016.Major taxa studiedHerbaceous and woody plants.MethodsWe compiled a dataset of 18,401 measurements of the absolute extent of ITV (measured as the coefficient of variation) in nine vegetative and seven floral traits from 2,822 herbaceous and woody species at 2,372 locations.ResultsLarge‐scale associations between ITV and climate were trait specific and more prominent for vegetative traits, especially leaf morphology, than for floral traits. The ITV showed pronounced associations with climate, with lower ITV values in colder areas and higher values in drier areas. The associations of ITV with species richness were inconsistent across traits. Species‐specific associations across gradients were often idiosyncratic, and covariation in ITV was weaker between vegetative and floral traits than within the two trait groups.Main conclusionsOur results show that, depending on the traits considered, ITV either increased or decreased with climate stress and species richness, suggesting that both factors can constrain or enhance ITV, which might foster plant‐population persistence in stressful conditions. Given the species‐specific responses and covariation in ITV, associations can be hard to predict for traits and species not yet studied. We conclude that consideration of ITV can improve our understanding of how plants cope with stressful conditions and environmental change across spatial and biological scales.
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| 5. |
- Orrego, Alejandro H., et al.
(författare)
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One‐step Synthesis of α‐Keto Acids from Racemic Amino Acids by A Versatile Immobilized Multienzyme Cell‐free System
- 2018
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Ingår i: ChemCatChem. - : Wiley-VCH Verlagsgesellschaft. - 1867-3880 .- 1867-3899. ; 10:14, s. 3002-3011
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Tidskriftsartikel (refereegranskat)abstract
- The elevated value of α‐keto acids has pushed scientists to explore more efficient and less expensive alternatives for their synthesis. In this work, an immobilized tri‐enzyme system that produced α‐keto acids in “one‐pot” from l‐ or racemic mixtures of diverse amino acids was presented. The system combined a broad‐spectrum amino acid racemase (BsrV), a d‐amino acid oxidase (DAAO) and catalase (CAT). BsrV racemized l‐amino acids into their d‐enantiomers, DAAO catalyzed the stereospecific oxidative deamination of the d‐amino acids into their corresponding α‐keto acids, ammonium ion, and H2O2. Finally, CAT converted the inactivating H2O2 into H2O and O2, which can be reused by the oxidase reaction. BsrV thermal stability was improved 3,300‐fold by immobilizing the enzyme on glyoxyl‐activated agarose beads. DAAO and CAT were co‐immobilized on agarose beads functionalized with glutaraldehyde groups for enhancing their stabilities and eliminating H2O2 in a much more effective way. To show the versatility of this system, racemic mixtures of amino acids were converted in their corresponding α‐keto acids. The coupling of the three immobilized enzymes permitted conversions of approximately 99 % through a dynamic kinetic resolution process. This system conserved 100 % of its initial effectiveness after 8 reaction cycles. Collectively, our innovative tri‐enzyme system for the synthesis of α‐keto acids opens the door for a cheapening in the production of many pharmaceutical and cosmetics.
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| 6. |
- Pearman, Peter B., et al.
(författare)
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Monitoring of species' genetic diversity in Europe varies greatly and overlooks potential climate change impacts
- 2024
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Ingår i: Nature Ecology & Evolution. - : Springer Nature. - 2397-334X. ; 8:2, s. 267-281
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Tidskriftsartikel (refereegranskat)abstract
- Genetic monitoring of populations currently attracts interest in the context of the Convention on Biological Diversity but needs long-term planning and investments. However, genetic diversity has been largely neglected in biodiversity monitoring, and when addressed, it is treated separately, detached from other conservation issues, such as habitat alteration due to climate change. We report an accounting of efforts to monitor population genetic diversity in Europe (genetic monitoring effort, GME), the evaluation of which can help guide future capacity building and collaboration towards areas most in need of expanded monitoring. Overlaying GME with areas where the ranges of selected species of conservation interest approach current and future climate niche limits helps identify whether GME coincides with anticipated climate change effects on biodiversity. Our analysis suggests that country area, financial resources and conservation policy influence GME, high values of which only partially match species' joint patterns of limits to suitable climatic conditions. Populations at trailing climatic niche margins probably hold genetic diversity that is important for adaptation to changing climate. Our results illuminate the need in Europe for expanded investment in genetic monitoring across climate gradients occupied by focal species, a need arguably greatest in southeastern European countries. This need could be met in part by expanding the European Union's Birds and Habitats Directives to fully address the conservation and monitoring of genetic diversity. Comparing data on genetic monitoring efforts across Europe with the distributions of areas at species' climatic niche margins, the authors show that monitoring efforts should be expanded to populations at trailing niche margins to include genetic variation that may prove important for adaptation to ongoing climate warming.
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