RESUMO
Enzyme immobilization is a widespread empiric technology to achieve more stable, active and reusable enzymes. The empiricism can be reduced by the application of rational design procedures employing bioinformatic tools, engineered-proteins and detailed analyses of existent data. In this chapter, we describe relevant approaches to rationalize the design of enzyme immobilization protocols, with special attention to the modulation of immobilization pH to regulate the operational stability of glutaraldehyde cross-linked enzymes and the coating of iron-containing supports to preserve the integrity of iron-sensitive enzymes. Other strategies, such as the use of factorial planning, optimization of specific enzyme orientation through protein engineering and the use of mathematical algorithms and in silico prediction tools are also described to reduce the classical empiricism. Finally, a public repository creation is proposed as a new promising tool to develop an improvement on future rational design procedures of enzyme immobilization.
Assuntos
Enzimas Imobilizadas/química , Animais , Bactérias/química , Bactérias/enzimologia , Biocatálise , Biotecnologia , Reagentes de Ligações Cruzadas/química , Estabilidade Enzimática , Fungos/química , Fungos/enzimologia , Glutaral/química , HumanosRESUMO
PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.
Assuntos
Proteínas de Bactérias/genética , Redes e Vias Metabólicas/genética , Ácidos Fosfatídicos/metabolismo , Fosfolipídeos/biossíntese , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Catálise , Escherichia coli/genética , Escherichia coli/metabolismo , Ácidos Graxos/metabolismo , Lipogênese/genética , Ácidos Fosfatídicos/genética , Fosfolipídeos/genéticaRESUMO
Lipase B from Candida antarctica (CAL-B) is largely employed as a biocatalyst for hydrolysis, esterification, and transesterification reactions. CAL-B is a good model enzyme to study factors affecting the enzymatic structure, activity and/or stability after an immobilization process. In this study, we analyzed the immobilization of CAL-B enzyme on different magnetic nanoparticles, synthesized by the coprecipitation method inside inverse micelles made of zwitterionic surfactants, with distinct carbon chain length: 4 (ImS4), 10 (ImS10) and 18 (ImS18) carbons. Magnetic nanoparticles ImS4 and ImS10 were shown to cross-link to CAL-B enzyme via a Michael-type addition, whereas particles with ImS18 were bond via pyridine formation after glutaraldehyde cross-coupling. Interestingly, the Michael-type cross-linking generated less stable immobilized CAL-B, revealing the influence of a cross-linking mode on the resulting biocatalyst behavior. Curiously, a direct correlation between nanoparticle agglomerate sizes and CAL-B enzyme reuse stability was observed. Moreover, free CAL-B enzyme was not able to catalyze transesterification due to the high methanol concentration; however, the immobilized CAL-B enzyme reached yields from 79.7 to 90% at the same conditions. In addition, the transesterification of lipids isolated from oleaginous yeasts achieved 89% yield, which confirmed the potential of immobilized CAL-B enzyme in microbial production of biodiesel.
Assuntos
Candida/enzimologia , Reagentes de Ligações Cruzadas/química , Enzimas Imobilizadas/metabolismo , Glutaral/química , Lipase/metabolismo , Óleo de Soja/metabolismo , Biocombustíveis , Esterificação , Cinética , Nanopartículas de Magnetita/química , Modelos Moleculares , TermodinâmicaRESUMO
Little is known about the structure-function relationship of membrane-bound lipid desaturases. Using a domain-swapping strategy, we found that the N terminus (comprising the two first transmembrane segments) region of Bacillus cereus DesA desaturase improves Bacillus subtilis Des activity. In addition, the replacement of the first two transmembrane domains from Bacillus licheniformis inactive open reading frame (ORF) BL02692 with the corresponding domain from DesA was sufficient to resurrect this enzyme. Unexpectedly, we were able to restore the activity of ORF BL02692 with a single substitution (Cys40Tyr) of a cysteine localized in the first transmembrane domain close to the lipid-water interface. Substitution of eight residues (Gly90, Trp104, Lys172, His228, Pro257, Leu275, Tyr282, and Leu284) by site-directed mutagenesis produced inactive variants of DesA. Homology modeling of DesA revealed that His228 is part of the metal binding center, together with the canonical His boxes. Trp104 shapes the hydrophobic tunnel, whereas Gly90 and Lys172 are probably involved in substrate binding/recognition. Pro257, Leu275, Tyr282, and Leu284 might be relevant for the structural arrangement of the active site or interaction with electron donors. This study reveals the role of the N-terminal region of Δ5 phospholipid desaturases and the individual residues necessary for the activity of this class of enzymes.
Assuntos
Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Membrana Celular/metabolismo , Ácidos Graxos Dessaturases/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Fases de Leitura Aberta/genética , Domínios Proteicos , Homologia de Sequência de AminoácidosRESUMO
The stringent response is a universal adaptive mechanism to protect bacteria from nutritional and environmental stresses. The role of the stringent response during lipid starvation has been studied only in Gram-negative bacteria. Here, we report that the stringent response also plays a crucial role in the adaptation of the model Gram-positive Bacillus subtilis to fatty acid starvation. B. subtilis lacking all three (p)ppGpp-synthetases (RelBs , RelP and RelQ) or bearing a RelBs variant that no longer synthesizes (p)ppGpp suffer extreme loss of viability on lipid starvation. Loss of viability is paralleled by perturbation of membrane integrity and function, with collapse of membrane potential as the likely cause of death. Although no increment of (p)ppGpp could be detected in lipid starved B. subtilis, we observed a substantial increase in the GTP/ATP ratio of strains incapable of synthesizing (p)ppGpp. Artificially lowering GTP with decoyinine rescued viability of such strains, confirming observations that low intracellular GTP is important for survival of nutritional stresses. Altogether, our results show that activation of the stringent response by lipid starvation is a broadly conserved response of bacteria and that a key role of (p)ppGpp is to couple biosynthetic processes that become detrimental if uncoordinated.
Assuntos
Trifosfato de Adenosina/metabolismo , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Ácidos Graxos/metabolismo , Guanosina Trifosfato/metabolismo , Ligases/genética , Potenciais da Membrana/fisiologia , Inanição/metabolismo , Cerulenina/farmacologia , Inibidores da Síntese de Ácidos Graxos/farmacologia , Ácidos Graxos/biossíntese , Estresse FisiológicoRESUMO
Although homologs of the ATP-dependent Lon protease exist in all domains of life, the relevance of this protease in archaeal physiology remains a mystery. In this study, we have constructed and phenotypically characterized deletion and conditional lon mutants in the model haloarchaeon Haloferax volcanii to elucidate the role of the unusual membrane-bound LonB protease in archaea. Hvlon could be deleted from the chromosome only when a copy of the wild type gene was provided in trans suggesting that Lon is essential for survival in this archaeon. Successful complementation of the lethal phenotype of ΔHvlon was attained by expression of the heterologous protease gene Nmlon from the haloalkaliphilic archaeon Natrialba magadii, meaning that the biological function of Lon is conserved in these organisms. Suboptimal cellular levels of Lon protein affected growth rate, cell shape, cell pigmentation, lipid composition and sensitivity to various antibiotics. The contents of bacterioruberins and some polar lipids were increased in the lon mutants suggesting that Lon is linked to maintenance of membrane lipid balance which likely affects cell viability in this archaeon. The phenotypes associated to a membrane-bound LonB protease mutant were examined for the first time providing insight on the relevance of this protease in archaeal physiology.
Assuntos
Proteínas Arqueais/genética , Haloferax volcanii/enzimologia , Lipídeos de Membrana/metabolismo , Peptídeo Hidrolases/genética , Antibacterianos/farmacologia , Proteínas Arqueais/metabolismo , Bacitracina/farmacologia , Sequência de Bases , Expressão Gênica , Regulação da Expressão Gênica em Archaea , Haloferax volcanii/efeitos dos fármacos , Lovastatina/farmacologia , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Novobiocina/farmacologia , Peptídeo Hidrolases/metabolismo , Pigmentação , Ligação Proteica , Puromicina/farmacologiaAssuntos
Proteínas Arqueais/metabolismo , Halobacteriaceae/metabolismo , Hidrolases/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/isolamento & purificação , Meios de Cultivo Condicionados/metabolismo , Regulação da Expressão Gênica em Archaea , Genes Arqueais , Halobacteriaceae/genética , Halobacteriaceae/crescimento & desenvolvimento , Hidrolases/genética , Hidrolases/isolamento & purificação , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade da Espécie , Transcrição GênicaRESUMO
The ATP-dependent Lon protease is universally distributed in bacteria, eukaryotic organelles and archaea. In comparison with bacterial and eukaryal Lon proteases, the biology of the archaeal Lon has been studied to a limited extent. In this study, the gene encoding the Lon protease of the alkaliphilic haloarchaeon Natrialba magadii (Nmlon) was cloned and sequenced, and the genetic organization of Nmlon was examined at the transcriptional level. Nmlon encodes a 84 kDa polypeptide with a pI of 4.42 which contains the ATPase, protease and membrane targeting domains of the archaeal-type LonB proteases. Nmlon is part of an operon that encodes membrane proteases and it is transcribed as a polycistronic mRNA in N. magadii cells at different growth stages. Accordingly, NmLon was detected in cell membranes of N. magadii throughout growth by Western blot analysis using specific anti-NmLon antibodies. Interestingly, in electrophoretic mobility shift assays, purified NmLon bound double stranded as well as single stranded DNA in the presence of elevated salt concentrations. This finding shows that DNA-binding is conserved in the LonA and LonB subfamilies and suggests that Lon-DNA interaction may be relevant for its function in haloarchaea.