RESUMO
The tomato (Solanum lycopersicum) MADS box FRUITFULL homologs FUL1 and FUL2 act as key ripening regulators and interact with the master regulator MADS box protein RIPENING INHIBITOR (RIN). Here, we report the large-scale identification of direct targets of FUL1 and FUL2 by transcriptome analysis of FUL1/FUL2 suppressed fruits and chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) targeting tomato gene promoters. The ChIP-chip and transcriptome analysis identified FUL1/FUL2 target genes that contain at least one genomic region bound by FUL1 or FUL2 (regions that occur mainly in their promoters) and exhibit FUL1/FUL2-dependent expression during ripening. These analyses identified 860 direct FUL1 targets and 878 direct FUL2 targets; this set of genes includes both direct targets of RIN and nontargets of RIN. Functional classification of the FUL1/FUL2 targets revealed that these FUL homologs function in many biological processes via the regulation of ripening-related gene expression, both in cooperation with and independent of RIN. Our in vitro assay showed that the FUL homologs, RIN, and tomato AGAMOUS-LIKE1 form DNA binding complexes, suggesting that tetramer complexes of these MADS box proteins are mainly responsible for the regulation of ripening.
Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Sítios de Ligação , Imunoprecipitação da Cromatina , Flavonoides/metabolismo , Perfilação da Expressão Gênica , Solanum lycopersicum/metabolismo , Proteínas de Domínio MADS/metabolismo , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de RNARESUMO
We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.
Assuntos
Desoxiglucose/metabolismo , Mutagênese , Trichoderma/genética , DNA Polimerase III/genética , Microscopia Eletrônica de Varredura , Trichoderma/metabolismo , Trichoderma/ultraestruturaRESUMO
The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preß1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preß1-HDL ELISA. Here, we purified preß1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preß1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preß1-HDL and original plasma preß1-HDL, or between the preß1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preß1-HDL MAbs, reactivity with intact plasma preß1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preß1-HDL is lipid-free monomolecular apoA-1.
Assuntos
Apolipoproteína A-I/sangue , Apolipoproteína A-I/química , Sequência de Aminoácidos , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Apolipoproteína A-I/imunologia , Epitopos/imunologia , Lipoproteínas de Alta Densidade Pré-beta/química , Lipoproteínas de Alta Densidade Pré-beta/isolamento & purificação , Humanos , Dados de Sequência MolecularRESUMO
Certain MADS-box transcription factors play central roles in regulating fruit ripening. RIPENING INHIBITOR (RIN), a tomato MADS-domain protein, acts as a global regulator of ripening, affecting the climacteric rise of ethylene, pigmentation changes, and fruit softening. Previously, we showed that two MADS-domain proteins, the FRUITFULL homologs FUL1 and FUL2, form complexes with RIN. Here, we characterized the FUL1/FUL2 loss-of-function phenotype in co-suppressed plants. The transgenic plants produced ripening-defective fruits accumulating little or no lycopene. Unlike a previous study on FUL1/FUL2 suppressed tomatoes, our transgenic fruits showed very low levels of ethylene production, and this was associated with suppression of the genes for 1-aminocyclopropane-1-carboxylic acid synthase, a rate-limiting enzyme in ethylene synthesis. FUL1/FUL2 suppression also caused the fruit to soften in a manner independent of ripening, possibly due to reduced cuticle thickness in the peel of the suppressed tomatoes.
Assuntos
Etilenos/biossíntese , Frutas/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Homologia de Sequência de Aminoácidos , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/metabolismo , Frutas/anatomia & histologia , Solanum lycopersicum/anatomia & histologia , Solanum lycopersicum/genética , Fenótipo , Proteínas de Plantas/genética , Plantas Geneticamente ModificadasRESUMO
The tomato MADS-box transcription factor RIN acts as a master regulator of fruit ripening. Here, we identified MADS-box proteins that interact with RIN; we also provide evidence that these proteins act in the regulation of fruit ripening. We conducted a yeast two-hybrid screen of a cDNA library from ripening fruit, for genes encoding proteins that bind to RIN. The screen identified two MADS-box genes, FUL1 and FUL2 (previously called TDR4 and SlMBP7), both of which have high sequence similarity to Arabidopsis FRUITFULL. Expression analyses revealed that the FUL1 mRNA and FUL1 protein accumulate in a ripening-specific manner in tomato fruits and FUL2 mRNA and protein accumulate at the pre-ripening stage and throughout ripening. Biochemical analyses confirmed that FUL1 and FUL2 form heterodimers with RIN; this interaction required the FUL1 and FUL2 C-terminal domains. Also, the heterodimers bind to a typical target DNA motif for MADS-box proteins. Chromatin immunoprecipitation assays revealed that FUL1 and FUL2 bind to genomic sites that were previously identified as RIN-target sites, such as the promoter regions of ACS2, ACS4 and RIN. These findings suggest that RIN forms complexes with FUL1 and FUL2 and these complexes regulate expression of ripening-related genes. In addition to the functional redundancy between FUL1 and FUL2, we also found they have potentially divergent roles in transcriptional regulation, including a difference in genomic target sites.
Assuntos
Frutas/metabolismo , Proteínas de Domínio MADS/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Frutas/genética , Regulação da Expressão Gênica de Plantas , Solanum lycopersicum/genética , Proteínas de Domínio MADS/genética , Proteínas de Plantas/genéticaRESUMO
Abscission in plants is a crucial process used to shed organs such as leaves, flowers, and fruits when they are senescent, damaged, or mature. Abscission occurs at predetermined positions called abscission zones (AZs). Although the regulation of fruit abscission is essential for agriculture, the developmental mechanisms remain unclear. Here, we describe a novel transcription factor regulating the development of tomato (Solanum lycopersicum) pedicel AZs. We found that the development of tomato pedicel AZs requires the gene MACROCALYX (MC), which was previously identified as a sepal size regulator and encodes a MADS-box transcription factor. MC has significant sequence similarity to Arabidopsis (Arabidopsis thaliana) FRUITFULL, which is involved in the regulation of fruit dehiscent zone development. The MC protein interacted physically with another MADS-box protein, JOINTLESS, which is known as a regulator of fruit abscission; the resulting heterodimer acquired a specific DNA-binding activity. Transcriptome analyses of pedicels at the preabscission stage revealed that the expression of the genes involved in phytohormone-related functions, cell wall modifications, fatty acid metabolism, and transcription factors is regulated by MC and JOINTLESS. The regulated genes include homologs of Arabidopsis WUSCHEL, REGULATOR OF AXILLARY MERISTEMS, CUP-SHAPED COTYLEDON, and LATERAL SUPPRESSOR. These Arabidopsis genes encode well-characterized transcription factors regulating meristem maintenance, axillary meristem development, and boundary formation in plant tissues. The tomato homologs were specifically expressed in AZs but not in other pedicel tissues, suggesting that these transcription factors may play key roles in pedicel AZ development.
Assuntos
Frutas/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Domínio MADS/metabolismo , Proteínas de Plantas/genética , Solanum lycopersicum/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Homeodomínio/genética , Proteínas de Domínio MADS/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
This review discusses sugar isomerization with organogermanium compounds. Organogermanium compounds markedly increase the aldose-ketose (glucose-fructose or lactose-lactulose) isomerization ratio, double the initial reaction rate, and significantly reduce the base-catalyzed degradation of sugars. 1H-nuclear magnetic resonance analysis reveals that the affinity of organogermanium compounds with a 3-(trihydroxygermyl)propanoic acid (THGP) structure toward ketoses is 20-40 times stronger than that toward aldoses; thus, such organogermanium compounds form complexes more readily with ketoses than with aldoses. Stable ketose complexes, which contain multiple cis-diol structures and high fractions of furanose structures, suppress the reverse ketose-aldose reaction, thereby shifting the equilibrium toward the ketose side. These complexes also protect sugar molecules from alkaline degradation owing to the repulsion between anionic charges. The increased rate of the initial reaction in the alkaline isomerization process results from stabilizing the transition state by forming a complex between THGP and a cis-enediol intermediate. The cyclic pentacoordinate or hexacoordinate THGP structures give rise to a conjugated system of germanium orbitals, which is extended through dπ-pπ interactions, thereby improving the stability of the complex. Based on these results, we have developed a bench-scale lactulose syrup manufacturing plant incorporating a system to separate, recover, and reuse organogermanium poly-trans-[(2-carboxyethyl)germasesquioxane]. This manufacturing plant can be used as a model of an alkaline isomerization accelerator for continuous industrial production.
RESUMO
The physiological and biochemical changes in fruit ripening produce key attributes of fruit quality including color, taste, aroma and texture. These changes are driven by the highly regulated and synchronized activation of a huge number of ripening-associated genes. In tomato (Solanum lycopersicum), a typical climacteric fruit, the MADS-box transcription factor RIN is one of the earliest-acting ripening regulators, required for both ethylene-dependent and ethylene-independent pathways. Although we previously identified several direct RIN targets, many additional targets remain unidentified, likely including key ripening-associated genes. Here, we report the identification of novel RIN targets by transcriptome and chromatin immunoprecipitation (ChIP) analyses. Transcriptome comparisons by microarray of wild-type and rin mutant tomatoes identified 342 positively regulated genes and 473 negatively regulated genes by RIN during ripening. Most of the positively regulated genes contained possible RIN-binding (CArG-box) sequences in their promoters. Subsequently, we selected six genes from the positively regulated genes and a ripening regulator gene, CNR, and assayed their promoters by quantitative ChIP-PCR to examine RIN binding. All of the seven genes, which are involved in cell wall modification, aroma and flavor development, pathogen defense and transcriptional regulation during ripening, are targets of RIN, suggesting that RIN may control multiple diverse ripening processes. In particular, RIN directly regulates the expression of the ripening-associated transcription factors, CNR, TDR4 and a GRAS family gene, providing an important clue to elucidate the complicated transcriptional cascade for fruit ripening.
Assuntos
Imunoprecipitação da Cromatina/métodos , Perfilação da Expressão Gênica/métodos , Proteínas de Plantas/metabolismo , Solanum lycopersicum/crescimento & desenvolvimento , Solanum lycopersicum/genética , Transcriptoma/genética , Sequência de Bases , Sítios de Ligação , Etilenos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/genética , Estudos de Associação Genética , Solanum lycopersicum/efeitos dos fármacos , Modelos Biológicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/metabolismo , Transcriptoma/efeitos dos fármacosRESUMO
A fungal strain, Penicillium sp. AZ, produced the azaphilone Monascus pigment homolog when cultured in a medium composed of soluble starch, ammonium nitrate, yeast extract, and citrate buffer, pH 5.0. One of the typical features of violet pigment PP-V [(10Z)-12-carboxyl-monascorubramine] is that pyranoid oxygen is replaced with nitrogen. In this study, we found that ammonia and nitrate nitrogen are available for PP-V biosynthesis, and that ammonia nitrogen was much more effective than nitrate nitrogen. Further, we isolated nitrate assimilation gene cluster, niaD, niiA, and crnA, and analyzed the expression of these genes. The expression levels of all these genes increased with sodium nitrate addition to the culture medium. The results obtained here strongly suggest that Penicillium sp. AZ produced PP-V using nitrate in the form of ammonium reduced from nitrate through a bioprocess assimilatory reaction.
Assuntos
4-Butirolactona/análogos & derivados , Regulação Fúngica da Expressão Gênica , Família Multigênica/genética , Nitratos/metabolismo , Nitrogênio/metabolismo , Penicillium/genética , Penicillium/metabolismo , 4-Butirolactona/biossíntese , Isoquinolinas , OxirreduçãoRESUMO
We previously demonstrated that the organogermanium compound 3-(trihydroxygermyl)propanoic acid (THGP) enhances the enzymatic and alkaline isomerization of an aldose to a ketose through cis-diol complex formation by multiple mechanisms. Its higher affinity for the ketose than the aldose protects the ketose complex from alkaline decomposition. Furthermore, it has been reported that the aldose-ketose alkaline isomerization pathway includes 1,2-enediol. Therefore, we speculated that the complex-forming ability of THGP could also be applied to enediol, a transient intermediate of alkaline isomerization. To test this prediction, we analyzed the initial rates of glucose or lactose isomerization in a region where there was no substantial difference in pH with and without THGP addition. The results showed that THGP enhanced the rate of fructose or lactulose formation per unit time by approximately 2-fold compared to the control. This finding indicated that THGP could form a complex with the transition state of aldose-ketose alkaline isomerization.
RESUMO
The potential of erythritol as a platform chemical in biomass refinery is discussed in terms of erythritol production and utilization. Regarding erythritol production, fermentation of sugar or starch has been already commercialized. The shift of the carbon source from glucose to inexpensive inedible waste glycerol is being investigated, which will decrease the price of erythritol. The carbon-based yield of erythritol from glycerol is comparable to or even higher than that from glucose. The metabolic pathway of erythritol biosynthesis has become clarified: erythrose-4-phosphate, which is one of the intermediates in the pentose phosphate pathway, is dephosphorylated and reduced to erythritol. The information about the metabolic pathway may give insights to improve the productivity by bleeding. Regarding erythritol utilization, chemical conversions of erythritol, especially deoxygenation, have been investigated in these days. Erythritol is easily dehydrated to 1,4-anhydroerythritol, which can be also used as the substrate for production of useful C4 chemicals. C-O hydrogenolysis and deoxydehydration using heterogeneous catalysts are effective reactions for erythritol/1,4-anhydroerythritol conversion.
RESUMO
Awamori is a distilled spirit produced in Okinawa Prefecture, in southern Japan. Awamori contains the volatile organic compound 1-octen-3-ol, an important flavor component. Here, using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GCMS), we demonstrate that the black koji mold Aspergillus luchuensis produces 1-octen-3-ol in rice koji. To examine the role of the fatty acid oxygenase genes ppoA and ppoC in 1-octen-3-ol biosynthesis by A. luchuensis, we constructed ppoA and ppoC disruptants, ΔppoA and ΔppoC, respectively, via protoplast-PEG transformation. No clear differences in growth and conidiation were observed between the transformants and the parent strain. Volatile compounds in rice koji prepared using these gene disruptants were analyzed by SPME-GCMS. The amount of 1-octen-3-ol contained in koji produced by the ΔppoA strain was the same as that produced by the parental strain. In contrast, although the ΔppoC strain grew on the rice koji, 1-octen-3-ol was not detected. These results indicate that ppoC is involved in 1-octen-3-ol biosynthesis in A. luchuensis.
Assuntos
Aspergillus/metabolismo , Octanóis/metabolismo , Oxigenases/metabolismo , Aspergillus/genética , Ácidos Graxos/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Oryza/microbiologia , Oxigenases/genética , Microextração em Fase SólidaRESUMO
The RIN gene encodes a putative MADS box transcription factor that controls tomato fruit ripening, and its ripening inhibitor (rin) mutation yields non-ripening fruit. In this study, the molecular properties of RIN and the rin mutant protein were clarified. The results revealed that the RIN protein accumulates in ripening fruit specifically and is localized in the nucleus of the cell. In vitro studies revealed that RIN forms a stable homodimer that binds to MADS domain-specific DNA sites. Analysis of binding site selection experiments revealed that the consensus binding sites of RIN highly resemble those of the SEPALLATA (SEP) proteins, which are Arabidopsis MADS box proteins that control the identity of floral organs. RIN exhibited a transcription-activating function similar to that exhibited by the SEP proteins. These results indicate that RIN exhibits similar molecular functions to SEP proteins although they play distinctly different biological roles. In vivo assays revealed that RIN binds to the cis-element of LeACS2. Our results also revealed that the rin mutant protein accumulates in the mutant fruit and exhibits a DNA-binding activity similar to that exhibited by the wild-type protein, but has lost its transcription-activating function, which in turn would inhibit ripening in mutant fruit.
Assuntos
DNA de Plantas/metabolismo , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Transcrição Gênica/fisiologia , Transporte Ativo do Núcleo Celular/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Frutas/citologia , Solanum lycopersicum/citologia , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional/fisiologiaRESUMO
Lactulose, a keto-type disaccharide widely used in pharmaceuticals and functional foods, is produced by the isomerization of lactose. The organogermanium compound poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) is an effective reaction promoter for the conversion of lactose to lactulose because of its high affinity to ketoses. Herein, an effective method for the continuous production of lactulose syrup was developed using Ge-132 through the alkaline isomerization of lactose in a bench-scale plant. This plant carried out a continuous isomerization process using Ge-132, continuous two-step separation process for separating the sugar and Ge-132, a continuous purification and concentration processes for the lactulose syrup, and separation and purification processes for the recovery of Ge-132. In this bench-scale plant, lactulose-containing syrup (350 g/L lactulose, 92 g/L lactose, and 31 g/L galactose) was prepared. The syrup was produced at a rate of 37.7 mL/h, and the content of residual Ge-132 in the syrup was 2 mg/L. The separation process was a two-step separation system requiring an ordinary electrodialyzer and an electro deionizer, which allowed the separation of more than 99.6 % Ge-132 from the reaction mixture. Moreover, the majority of Ge-132 and sodium hydroxide were recovered through electrodialysis using a bipolar membrane. The proposed system is the first to represent the novel development of an effective continuous production system for lactulose-containing syrup on the basis of the use of organogermanium compounds and incorporation of the electrodialysis technology.
RESUMO
In the current study, we attempted to enhance the xylanase activity of Trichoderma reesei ATCC66589 by using disparity mutagenesis, wherein a plasmid harboring proofreading-impaired DNA polymerase δ was inserted. Following selection on xylan-rich media and successive plasmid curing, a mutant showing conidiospores strikingly different from those of the parent strain, with many small humped-surface spheres, was generated. Xylanase and ß-xylosidase activities of the mutant XM1, cultivated in xylan medium, were 15.8- and 11.0-fold higher than those of the parent strain, respectively. Furthermore, xylanase activity was generated approximately 24 h in advance compared to that in the parent. In contrast, when cultivated in Avicel medium, its xylanase and ß-xylosidase activities were 0.14- and 0.33-fold, respectively, compared to those in the parent. Among the xylan component sugars and related polyols, D-xylose and xylobiose exerted a distinct inductive effect on the xylanase activity in Avicel media, while xylitol and L-arabinose did not. Mutagenesis involved in xylose catabolism is suggestive of changes at the gene transcription level. Although the induction mechanism remains unclear in details, disparity mutagenesis may be useful for obtaining T. reesei mutants with high xylanase activity.
RESUMO
Lactulose, a disaccharide widely used in pharmaceuticals and functional foods, is produced by lactose isomerization. Lactose and lactulose have an aldose-ketose relationship. Less than 25 % conversion of lactose into lactulose is achieved using the Lobry de Bruyn-Alberda van Ekenstein transformation with heating, whereas the conversion is increased to 80 % by the addition of an approximately equimolar concentration of the organogermanium compound 3-(trihydroxygermyl)propanoic acid (THGP) to the reaction mixture. To further understand this phenomenon, in this study, we analyzed the affinity between THGP and sugar isomers using 1H nuclear magnetic resonance spectroscopy. For the dimethyl derivative of THGP with lactose and lactulose, the complex formation ratios at 0.1 M (1:1 mixing ratio) were 14 and 59 %, respectively, with complex formation constants of 1.8 and 43 M-1, respectively. The complex formation capacity was approximately 24-fold higher for lactulose than for lactose. Moreover, THGP is considered to protect lactulose from alkaline degradation, resulting in high production yield of lactulose. Therefore, we concluded that high affinity for the isomerization product may promote isomerization and that promotion of sugar isomerization using an organogermanium compound is an effective method for converting lactose to lactulose.
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Two transketolase isogenes, MmTKL1 and MmTKL2, isolated from Moniliella megachiliensis were investigated for their roles in stress response and erythritol biosynthesis. The encoded proteins were highly homologous in amino acid sequence and domain structure. Two stress response elements (STREs) were found upstream of MmTKL1, while no STRE was found upstream of MmTKL2. In contrast, two Ap-1 elements were present upstream of MmTKL2, but none were detected upstream of MmTKL1. MmTKL2 partially complemented the aromatic amino acid auxotrophy of a Saccharomyces cerevisiae tkl1 deletion mutant, suggesting that at least one of the MmTKLs functioned as a transketolase in vivo. In response to short-term osmotic stress (20% glucose or 1.2 M NaCl) in Moniliella cells, MmTKL1 expression increased rapidly through the first 40 min before subsequently decreasing gradually, while MmTKL2 expression showed no significant change. In contrast, short-term oxidative stress (0.15 mM menadione) induced considerable increases in MmTKL2, while MmTKL1 expression remained low under the same conditions. Long-term osmotic stress (20% glucose) yielded increased expression of both genes starting at 12 h and continuing through 72 h. During either osmotic or oxidative stress, intracellular erythritol accumulation could clearly be correlated with the pattern of expression of either MmTKL1 or MmTKL2. These results strongly suggested that MmTKL1 is responsible primarily for the response to osmotic stress, while MmTKL2 is responsible primarily for the response to oxidative stress. Thus, we postulate that the two transketolase isoforms of M. megachiliensis play distinct and complementary roles in coordinating erythritol production in response to distinct environmental stresses.
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Rice straw was evaluated as a carbon source for the fungi, Trichoderma reesei and Humicola insolens, to produce enzymes for rice straw hydrolysis. The enzyme activity of T. reesei and H. insolens cultivated in medium containing non-treated rice straw were almost equivalent to the enzyme of T. reesei cultivated in Avicel medium, a form of refined cellulose. The enzyme activity of T. reesei cultivated in medium containing NH4OH-treated rice straw was 4-fold higher than enzyme from cultures grown in Avicel medium. In contrast, H. insolens enzyme from cultures grown in NH4OH-treated rice straw had significantly lower activity compared with non-treated rice straw or Avicel. The combined use of T. reesei and H. insolens enzymes resulted in a significant synergistic enhancement in enzymatic activity. Our data suggest that rice straw is a promising low-cost carbon source for fungal enzyme production for rice straw hydrolysis.
Assuntos
Oryza , Trichoderma/enzimologia , Carbono , Celulase , Celulose , HidróliseRESUMO
Tripeptidases from Lactococcus lactis subsp. lactis (L9PepTR), L. lactis subsp. cremoris (L6PepTR), and L. lactis subsp. hordniae (hTPepTR) were cloned, overexpressed, purified, and characterized. Although these enzymes contained three to seven naturally occurring amino acid differences, both metal-binding and catalytic sites were highly conserved. The k(cat) values of hTPepTR were approximately 1.5- to 2-fold higher than those of L9PepTR, while, for L6PepTR, they were approximately 0.8- to 1.4-times the L9PepTR values. The K(m) of tripeptidase from subsp. lactis (L9PepTR) was considerably larger when glycine was the amino acid located at both the N- and C-terminus of the peptide substrate. In addition, the K(m) values of L9PepTR increased in the following order for YGG, LGG, FGG, SGG, and alpha-aminoisobutyrylglycylglycine, while the k(cat)/K(m) decreased in the same order. These results suggest that the dipole moment and steric hindrance of the N-terminal amino acid side chain may be the most important factors controlling substrate specificity.
Assuntos
Aminopeptidases/metabolismo , Proteínas de Bactérias/metabolismo , Lactococcus lactis/enzimologia , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Especificidade por Substrato , TemperaturaRESUMO
D-Glucose and D-fructose are isomers of commonly consumed monosaccharides. The ratio of conversion of D-glucose to D-fructose by glucose isomerase (xylose isomerase) is not more than 50 %. However, addition of an equimolar ratio of the organogermanium compound poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) or its derivative increases the conversion ratio to 80 %. In contrast, use of the Lobry de Bruyn-Alberda van Ekenstein transformation with heating results in a lower conversion ratio, less than 30 %, whereas addition of an equimolar concentration of Ge-132 or its derivative to this reaction mixture increases the ratio to 73 %. Therefore, in this study, we aimed to further analyze the affinity between organogermanium compounds (i.e., Ge-132 and its derivatives) and sugar using 1H-nuclear magnetic resonance (NMR) spectrometry. For the dimethyl derivative of Ge-132, the complex formation ratios at 0.25 M (mixing ratio 1:1) were 19 and 74 % for D-glucose and D-fructose, respectively. Additionally, the complex formation constants between monosaccharides and Ge-132 were 1.2 and 46 M-1 for D-glucose and D-fructose, respectively. The complex formation capacity was approximately 40-fold higher for D-fructose than for D-glucose. Therefore, we concluded that the high affinity for the product of isomerization may promote isomerization, and that promotion of sugar isomerization using organogermanium compounds is an effective method for conversion of D-glucose to D-fructose.