RESUMO
Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.
Assuntos
Bacillus licheniformis , Domínio Catalítico , Mutagênese Sítio-Dirigida , Bacillus licheniformis/enzimologia , Bacillus licheniformis/genética , Cristalografia por Raios X , Simulação de Dinâmica Molecular , Manosidases/química , Manosidases/genética , Manosidases/metabolismo , Especificidade por Substrato , Hidrólise , Tetroses/química , Tetroses/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Conformação Proteica , Mananas/química , Mananas/metabolismo , beta-Manosidase/química , beta-Manosidase/genética , beta-Manosidase/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , OligossacarídeosRESUMO
MAIN CONCLUSION: Blue light exposure delays tomato seed germination by decreasing endosperm-degrading hydrolase activities, a process regulated by CRY1a-dependent signaling and the hormonal balance between ABA and GA. The germination of tomato seeds (Solanum lycopersicum L.) is tightly controlled by an internal hormonal balance, which is also influenced by environmental factors such as light. In this study, we investigated the blue light (BL)-mediated impacts on physiological, biochemical, and molecular processes during the germination of the blue light photoreceptor CRYPTOCHROME 1a loss-of-function mutant (cry1a) and of the hormonal tomato mutants notabilis (not, deficient in ABA) and procera (pro, displaying a GA-constitutive response). Seeds were germinated in a controlled chamber in the dark and under different intensities of continuous BL (ranging from 1 to 25 µmol m-2 s-1). In general, exposure to BL delayed tomato seed germination in a fluency rate-dependent way due to negative impacts on the activities of endosperm-degrading hydrolases, such as endo-ß-mannanase, ß-mannosidase, and α-galactosidase. However, not and pro mutants presented higher germination speed index (GSI) compared to WT despite the BL influence, associated with higher hydrolase activities, especially evident in pro, indicating that the ABA/GA hormonal balance is important to diminish BL inhibition over tomato germination. The cry1a germination percentage was higher than in WT in the dark but its GSI was lower under BL exposure, suggesting that functional CRY1a is required for BL-dependent germination. BL inhibits the expression of GA-biosynthetic genes, and induces GA-deactivating and ABA-biosynthetic genes. The magnitude of the BL influence over the hormone-related transcriptional profile is also dependent upon CRY1a, highlighting the complex interplay between light and hormonal pathways. These results contribute to a better understanding of BL-induced events behind the photoregulation of tomato seed germination.
Assuntos
Endosperma , Solanum lycopersicum , Endosperma/genética , Endosperma/metabolismo , Solanum lycopersicum/genética , Germinação , Sementes/fisiologia , Criptocromos/genética , Criptocromos/metabolismo , beta-Manosidase/genética , beta-Manosidase/metabolismo , Percepção , Ácido Abscísico/metabolismo , Giberelinas/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
After being isolated from a sugarcane pile, the bacterium Chitinophaga sp. CB10 demonstrated to be a rich source of carbohydrases, with 350 predicted CAZyme domains. CB10 was able to grow on carbohydrates of different structural complexities: glucose, carboxymethylcellulose, corn starch, galactomannan, Aloe vera gum and sugarcane bagasse. The sugarcane bagasse is a rich source of complex polymers, and the diversity of metabolites released by its enzymatic hydrolysis has an important role for green chemistry, including minority pathways such as the degradation of mannan conjugates. In this sense, CB10 demonstrated considerable levels of gene expression for mannanases, and was stable for a period of 96-144 hours in the presence of sugarcane bagasse as sole carbon source. The bacterium showed respectively 4.8x and 5.6x expression levels for two genes predicted for GH2 ß-mannosidase: one located within a gene cluster identified as "polysaccharide utilization loci" (PUL), and another a classic ß-mannosidase. These enzymes shared less than 45% of identity with enzymes characterized from the genus Chitinophaga belonging to the phylum Bacteroidetes. The degree of novelty-as demonstrated by the low identity with previously characterized enzymes; the remarkable capability to grow in different substrates; mannanase activity, evidenced by the release of residual oligosaccharides in the cultivation with galactomannan (HPLC-RID, 12.3 mMol); associated to the ability of mannanases expression in a low concentration of inductor conditions (sugarcane bagasse, 0.2%) indicate the high potential for the application of CB10 as a source of enzymes in the production of oligosaccharides from biomass. This capacity might prove to be very valuable for the biorefinery process of pre-biotic precursors and other functional oligosaccharides focused on the food and pharmaceutical industries.
Assuntos
Proteínas de Bactérias/metabolismo , Bacteroidetes/enzimologia , Bacteroidetes/crescimento & desenvolvimento , Celulose/metabolismo , beta-Manosidase/metabolismo , Proteínas de Bactérias/genética , Bacteroidetes/genética , Expressão Gênica , Hidrólise , Reação em Cadeia da Polimerase em Tempo Real , beta-Manosidase/genéticaRESUMO
Coffee seeds begin to develop shortly after fertilization and can take 6 to 8 months to complete their formation, a period during which all the characteristics of the mature seed are determined, directly influencing physiological quality. However, little is known about the molecular mechanisms that act during coffee seed maturation. The objective of the current study was to analyze expression of the ß-tubulin (TUB) and endo-ß-mannanase (MAN) genes during different phases at the end of development and in different tissues of Coffea arabica seeds. The transcription levels of the TUB and MAN genes were quantified in a relative manner using qRT-PCR in whole seeds, and dissected into embryos and endosperms at different developmental stages. Greater expression of MAN was observed in whole seeds and in endosperms during the green stage, and in the embryo during the over-ripe stage. High TUB gene expression was observed in whole seeds during the green stage and, in the embryos, there were peaks in expression during the over-ripe stage. In endosperms, the peak of expression occurred in both the green stage and in the cherry stage. These results suggest participation of endo-ß-mannanase during the initial seed developmental stages, and in the stages of physiological maturity in the embryo tissues. TUB gene expression varied depending on the developmental stage and section of seed analyzed, indicating the participation of ß-tubulin during organogenesis and coffee seed maturation.
Assuntos
Coffea/genética , Endosperma/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tubulina (Proteína)/genética , beta-Manosidase/genética , Coffea/crescimento & desenvolvimento , Coffea/metabolismo , Primers do DNA/química , Endosperma/crescimento & desenvolvimento , Endosperma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Germinação/genética , Proteínas de Plantas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transcrição Gênica , Tubulina (Proteína)/metabolismo , beta-Manosidase/metabolismoRESUMO
Phytohormones have different characteristics and functions, and they may be subject to changes in their gene expression and synthesis during seed development. In this study, we evaluated the physiological qualities of habanero peppers (Capsicum chinense Jacquin) during seed development and the expression of genes involved in germination. Seeds were obtained from fruits harvested at different stages of development [i.e., 14, 21, 28, 35, 42, 49, 56, 63, and 70 days after anthesis (DAA)]. Immediately after harvesting, the seeds were subjected to various tests to determine moisture content, germination, first count germination, and seedling emergence. Real-time polymerase chain reaction was used to evaluate the expression of various genes, including MAN2, NCED, B73, ICL6, and GA3ox. Electrophoresis was used to assess the expression of various enzymes, including α-amylase, isocitrate-lyase, and endo-ß-mannanase. Habanero peppers harvested at 70 DAA and subjected to 7 days of rest exhibited higher germination rates and vigor compared to those harvested at all other developmental stages. Peppers harvested at 63 DAA without drying exhibited higher α amylase and AmyB73 gene expression levels. Peppers harvested at 70 DAA with 7 days of rest exhibited higher endo-ß-mannanase expression levels. MAN2 gene expression increased during the development of non-dried seeds until 70 DAA. Peppers harvested at 42 DAA exhibited the highest isocitrate-lyase and ICL6 gene activity levels in comparison to those at all other developmental stages.
Assuntos
Capsicum/genética , Frutas/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Sementes/genética , Capsicum/crescimento & desenvolvimento , Capsicum/metabolismo , Frutas/crescimento & desenvolvimento , Frutas/metabolismo , Germinação , Isocitrato Liase/genética , Isocitrato Liase/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismo , beta-Manosidase/genética , beta-Manosidase/metabolismoRESUMO
The anaerobic fungus Orpinomyces sp. strain PC-2 produces a broad spectrum of glycoside hydrolases, most of which are components of a high molecular mass cellulosomal complex. Here we report about a cDNA (manA) having 1924 bp isolated from the fungus and found to encode a polypeptide of 579 amino acid residues. Analysis of the deduced sequence revealed that it had a mannanase catalytic module, a family 1 carbohydrate-binding module, and a noncatalytic docking module. The catalytic module was homologous to aerobic fungal mannanases belonging to family 5 glycoside hydrolases, but unrelated to the previously isolated mannanases (family 26) of the anaerobic fungus Piromyces. No mannanase activity could be detected in Escherichia coli harboring a manA-containing plasmid. The manA was expressed in Saccharomyces cerevisiae and ManA was secreted into the culture medium in multiple forms. The purified extracellular heterologous mannanase hydrolyzed several types of mannan but lacked activity against cellulose, chitin, or beta-glucan. The enzyme had high specific activity toward locust bean mannan and an extremely broad pH profile. It was stable for several hours at 50 degrees C, but was rapidly inactivated at 60 degrees C. The carbohydrate-binding module of the Man A produced separately in E. coli bound preferably to insoluble lignocellulosic substrates, suggesting that it might play an important role in the complex enzyme system of the fungus for lignocellulose degradation.