Binding Specificity of a Novel Cyclo/Maltodextrin-Binding Protein and Its Role in the Cyclodextrin ABC Importer System from Thermoanaerobacterales.

Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo/maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and ß-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria.

Mining for novel cyclomaltodextrin glucanotransferases unravels the carbohydrate metabolism pathway via cyclodextrins in Thermoanaerobacterales.

Carbohydrate metabolism via cyclodextrins (CM-CD) is an uncommon starch-converting pathway that thoroughly depends on extracellular cyclomaltodextrin glucanotransferases (CGTases) to transform the surrounding starch substrate to α-(1,4)-linked oligosaccharides and cyclodextrins (CDs). The CM-CD pathway has emerged as a convenient microbial adaptation to thrive under extreme temperatures, as CDs are functional amphipathic toroids with higher heat-resistant values than linear dextrins. Nevertheless, although the CM-CD pathway has been described in a few mesophilic bacteria and archaea, it remains obscure in extremely thermophilic prokaryotes (Topt ≥ 70 °C). Here, a new monophyletic group of CGTases with an exceptional three-domain ABC architecture was detected by (meta)genome mining of extremely thermophilic Thermoanaerobacterales living in a wide variety of hot starch-poor environments on Earth. Functional studies of a representative member, CldA, showed a maximum activity in a thermoacidophilic range (pH 4.0 and 80 °C) with remarkable product diversification that yielded a mixture of α:ß:γ-CDs (34:62:4) from soluble starch, as well as G3-G7 linear dextrins and fermentable sugars as the primary products. Together, comparative genomics and predictive functional analysis, combined with data of the functionally characterized key proteins of the gene clusters encoding CGTases, revealed the CM-CD pathway in Thermoanaerobacterales and showed that it is involved in the synthesis, transportation, degradation, and metabolic assimilation of CDs.

Fungal co-culture increases ligninolytic enzyme activities: statistical optimization using response surface methodology.

The optimization of ligninolytic enzyme (LE) activities in a novel fungal co-culture between Pycnoporus sanguineus and Beauveria brongniartii were studied using a Plackett-Burman experimental design (PBED) and a central composite design (CCD). In addition, H2O2 role was analyzed. Laccase (EC. 1.10.3.2) and MnP (EC 1.11.1.14) activities of P. sanguineus increased 6.0- and 2.3-fold, respectively, in the co-culture with B. brongniartii. The H2O2 content was higher in the co-culture (0.33-7.12-fold) than in the P. sanguineus monoculture. The PBED revealed that yeast extract (YE), FeSO4, and inoculum amount were significant factors for laccase and MnP activities and H2O2 production in the co-culture, which increased by 8.2-, 5.2- and 1.03-fold, respectively. The YE and FeSO4 were studied using a CCD to optimize the studied response variables. Laccase activity was enhanced 1.5-fold by CCD, the optimal amount of YE was 0.366 g L-1. Quadratic term of FeSO4 modulated MnP activity and was associated with a 4.28-fold increase compared to the PBED. Both YE and its quadratic term significantly affected H2O2 production; however, the CCD did not enable an increase in H2O2 production. Pearson correlation indicated an increase in laccase (r2=0.4411, p = 0.0436) and MnP (r2=0.5186, p = 0.0198) activities following increases in H2O2 in the co-culture system.

Gene expression of an arabinogalactan lysine-rich protein CaAGP18 during vegetative and reproductive development of bell pepper (Capsicum annuum L.).

Lysine-rich (Lys-rich) proteins encoded by AGP17, AGP18, and AGP19 genes are cell wall-associated glycopeptides related to sexual reproduction in flowering plants. This subclass belongs to classical arabinogalactan proteins (AGPs) widely studied in model plants like Arabidopsis. In this study, we identified the CaAGP18 cDNA from bell pepper (Capsicum annuum L.), as well as its expression pattern during vegetative and reproductive development. The deduced amino acid sequence revealed a Lys-rich AGP18 protein of 238 amino acids residues in length with an estimated molecular mass of 22.85 kDa and an isoelectric point of 9.7. The protein is predicted as canonical AGP due to the presence of a small Lys-rich region and a C-terminal sequence essential for posttranslational modification with a glycosylphosphatidylinositol (GPI). Phylogenetic analysis showed that CaAGP18 is clustered together with NtAGP18, SpAGP18, StAGP18 and NaAGP18 from Solanaceae species. CaAGP18 expression through plant phenological stages had the highest transcription level in leaves at the seedling stage, whereas in reproductive organs there was a significant up-regulation in pistils during anthesis, also in petals 2 days post-anthesis (DPA), and in fruit at the expansion stage. Our results open future research for possible roles of CaAGP18 in cell expansion as a wall-associated plasticizer and reproductive processes like pistil interactions and petal cell death.

Purification and biochemical characterization of 11S globulin from chan (Hyptis suaveolens L. Poit) seeds.

Chan (Hyptis suaveolens) is a Mesoamerican crop highly appreciated since the pre-Hispanic cultures. Its proteins are a good source of essential amino acids; however, there are no reports on the properties of its individual proteins. In this study, the 11S globulin (Hs11S) was purified and biochemically characterized. The molecular weight of native Hs11S was about 150-300 kDa with isoelectric points of 5.0-5.3, composed by four monomers of 53.5, 52, 51.1 and 49.5 kDa, each formed by one acidic subunit and one basic subunit linked by a disulfide bond. Dynamic light scattering, size exclusion chromatography and native PAGE show that Hs11S is assembled in different oligomeric forms. LC-MS/MS analysis confirmed its identity. Hs11S presents antigenic determinants in common with lupin 11S globulin. Carbohydrate moieties or phosphate groups linked to Hs11S were not detected. This information is very useful in order to exploit and utilize rationally chan 11S globulin in food systems.

Modification of solubility and heat-induced gelation of amaranth 11S globulin by protein engineering.

The primary structure of amaranth 11S globulin (Ah11S) was engineered with the aim to improve its functional properties. Four continuous methionines were inserted in variable region V, obtaining the Ah11Sr+4M construction. Changes on protein structure and surface characteristics were analyzed in silico. Solubility and heat-induced gelation of recombinant amaranth 11S proglobulin (Ah11Sr and Ah11Sr+4M) were compared with the native protein (Ah11Sn) purified from amaranth seed flour. The Ah11Sr+4 M showed the highest surface hydrophobicity, but as consequence the solubility was reduced. At low ionic strength (µ = 0.2) and acidic pH (<4.1), the recombinant proteins Ah11Sr and Ah11Sr+4 M had the highest and lowest solubility values, respectively. All globulins samples formed gels at 90 °C and low ionic strength, but Ah11Sn produced the weakest and Ah11Sr the strongest gels. Differential scanning calorimetry analysis under gel forming conditions revealed only exothermic transitions for all amaranth 11S globulins analyzed. In conclusion, the 3D structure analysis has revealed interesting molecular features that could explain the thermal resistance and gel forming ability of amaranth 11S globulins. The incorporation of four continuous methionines in amaranth increased the hydrophobicity, and self-supporting gels formed had intermediate hardness between Ah11Sn and Ah11Sr. These functional properties could be used in the food industry for the development of new products based on amaranth proteins.

Optimization of the isoelectric precipitation method to obtain protein isolates from amaranth (Amaranthus cruentus) seeds.

This research was conducted to evaluate the effect of extraction pH (7.8-9.2) and precipitation pH (4.3-5.7) on four selected quality attributes of protein isolates from amaranth seeds (Amaranthus cruentus) such as protein content (PC), whiteness index (WI), enthalpy of transition (EN), and denaturation temperature (DT). Ten different treatments involving extraction and precipitation pH combinations were analyzed by a central composite design; the experimental data were fitted by a second-order model using a least-squares method for each one of the four dependent variables. Response surface methodology was used for the optimization process; in addition, a common optimum value for the four dependent variables was obtained utilizing the desirability method. A confirmatory test showed that the generated regression equations could adequately predict performance of this isoelectric precipitation method. The results indicate that extraction pH and precipitation pH showed an important effect on PC, WI, and EN. However, the different combinations did not significantly affect the DT. Values of 9.2 and 8.0 for extraction pH and 5.7 for precipitation pH produced the best overall result for all responses. Finally, the results have shown that it is possible to obtain protein isolates from A. cruentus seeds at optimized values of extraction pH and precipitation pH, which presented a high protein content and good physicochemical properties.