Search Results (6)

Alkaline pectate lyases hold significant promise for various industrial applications, including the degumming processes in papermaking and textiles. In this study, a novel pectinase, PelA, derived from a strain of Paenibacillus borealis, was characterized both at the molecular level and through enzymatic analysis. This enzyme represents a distinct cluster diverging from the well-characterized Bacillus pectinases and exhibits molecular activity under alkaline conditions, with an optimal pH of 9.5. It can be classified as an endo-(1,4)-pectate lyase, capable of cleaving the α-1,4 glycosidic bonds of polygalacturonic acid via a trans-elimination mechanism. Notably, the addition of the metal ion Ca²⁺ did not enhance enzyme activity. To achieve high-level secretory expression and improve its economic viability for bioapplications, the gene copy number of pelA in the host genome was increased by constructing tandem pelA gene expression cassettes. Following optimization of cultivation conditions and monitoring of cell growth, the recombinant strain harboring the multi-copy pelA gene attained an expression level of 7520 U/mL in a bioreactor. This study successfully achieved high-level secretory expression of an alkaline pectinase, thereby enhancing its potential for industrial bioapplications and providing a reference for future research on the heterologous expression of target genes. Full article

Calcium (Ca²⁺) plays a fundamental role in metabolic processes, and it is involved in several structural functions at the cell level, such as vacuole osmotic regulation, cell wall strengthening, and plasma membrane stability, as well as acting as a secondary messenger for several different signals. The role of Ca²⁺ in signal transduction and cell wall organization is crucial for stress responses, cell activity, and plant tissue development. In addition, Ca²⁺ is essential in modulating enzymatic activities, hormonal control, water, and ion transport across the plasma membrane. Although calcium’s role in fruit trees is well studied, many of its specific functions in kiwifruit remain unclear, including the optimal amount of Ca²⁺ in fruit and its distribution in fruit cells for the best pre- and post-harvest fruit quality. Calcium transport to the fruit is mainly regulated by the xylem sap flow; however, the contribution of fruit transpiration and the requirements of fruit cells are not clear. Understanding the kinetics of Ca²⁺ accumulation in fruit under different environmental conditions can help establish correct nutrient management. This review addresses the current knowledge on Ca²⁺ involvement in plant physiology, metabolic processes, structural functions, and fruit growth, quality, and storage, with particular emphasis on Actinidia chinensis. In addition, the different analytical techniques used for the quantification and definition of Ca²⁺ in different plant organs, including stain technology, X-rays, and advanced imaging methods, are here explored. Full article

Calcium (Ca) plays a vital role as a macronutrient in the growth and development of plants. In order of decreasing solubility, Ca can be found in vegetal tissues as soluble Ca (Fraction I), bound Ca (mainly pectates, Fraction II), inorganic insoluble Ca (mainly phosphates and carbonates, Fraction III) and organic insoluble Ca or oxalate (Fraction IV). To explore the impact of Ca fertilizer application on plant growth and its allocation among different fractions, young citrus trees were fed over a complete vegetative cycle with a ⁴⁴Ca labeled fertilizer (T1-Ca), while control plants (T2) received no Ca fertilizer. The results showed that plants receiving Ca exhibited significantly greater biomass. ⁴⁴Ca derived from the fertilizer was localized mainly in sink organs (new flush leaves–twigs and fibrous roots). The primary fraction responsible for total Ca partitioning was Fraction II, followed by Fraction III or IV. Citrus plants, commonly found in calcareous soils, demonstrated improved growth with calcium treatments, indicating a positive link between calcium supplementation and enhanced development. The calcium supplied through the fertilizer (⁴⁴Ca) was predominantly concentrated in sink organs (mainly in Ca-pectate fraction), including new flush leaves and twigs above ground, as well as fibrous roots below ground. Full article

Alkaline pectate lyases have biotechnological applications in plant fiber processing, such as ramie degumming. Previously, we characterized an alkaline pectate lyase from Bacillus clausii S10, named BacPelA, which showed potential for enzymatic ramie degumming because of its high cleavage activity toward methylated pectins in alkaline conditions. However, BacPelA displayed poor thermo-alkaline stability. Here, we report the 1.78 Å resolution crystal structure of BacPelA in apo form. The enzyme has the characteristic right-handed β-helix fold of members of the polysaccharide lyase 1 family and shows overall structural similarity to them, but it displays some differences in the details of the secondary structure and Ca²⁺-binding site. On the basis of the structure, 10 sites located in flexible regions and showing high B-factor and positive ΔT_m values were selected for mutation, aiming to improve the thermo-alkaline stability of the enzyme. Following site-directed saturation mutagenesis and screening, mutants A238C, R150G, and R216H showed an increase in the T₅₀¹⁵ value at pH 10.0 of 3.0 °C, 6.5 °C, and 7.0 °C, respectively, compared with the wild-type enzyme, interestingly accompanied by a 24.5%, 46.6%, and 61.9% increase in activity. The combined mutant R150G/R216H/A238C showed an 8.5 °C increase in the T₅₀¹⁵ value at pH 10.0, and an 86.1% increase in the specific activity at 60 °C, with approximately doubled catalytic efficiency, compared with the wild-type enzyme. Moreover, this mutant retained 86.2% activity after incubation in ramie degumming conditions (4 h, 60 °C, pH 10.0), compared with only 3.4% for wild-type BacPelA. The combined mutant increased the weight loss of ramie fibers in degumming by 30.2% compared with wild-type BacPelA. This work provides a thermo-alkaline stable, highly active pectate lyase with great potential for application in the textile industry, and also illustrates an effective strategy for rational design and improvement of pectate lyases. Full article

Pectate and alginate are among the most important biopolymers able to give rise to ionotropic gelation upon the addition of di- or multivalent counterions. The two ionic polysaccharides exhibit several common aspects of the gelation mechanism with calcium ions, the physiologically and commercially most relevant counterion type. The first one pertains to the role that specific Ca²⁺/polyion interactions play in the establishment of the ion-mediated chain/chain cross-links. Such interactions include both a specific affinity of the territorially condensed Ca²⁺ counterions for the polyuronate(s) and the formation of long-lasting chemical bonding (inner ion–sphere complex) of specific interchain sites accompanied by high conformational ordering. As to the first mechanism, it is dominated by the strong desolvation of the interacting ionic species, with concomitant positive variations in both enthalpy and entropy, the contribution of the latter prevailing over the former due to the favorable liberation of a very large number of water molecules of hydration. Both dilatometric and microcalorimetric data point to the higher affinity of Ca²⁺ for pectate than for alginate. The selective accumulation of calcium ions close to the polyanion(s) favors the onset of the second—chemical bonding—mode, which is associated with charge neutralization at the bonding site. This mode coincides with the largely accepted “egg-box” model for the calcium-mediated interchain junction of pectate and alginate. A new approach was devised for the calculation of the fraction of chemically bound divalent ions; it was based on the available circular dichroism data (further supported by scattering and viscosity results) and successfully tested by comparison with an independently determined fraction in the case of pectate. In detail, the strong bonding mode manifests in two sequential bonding modes. The first one (at low concentrations of added Ca²⁺ ions) entails a cross-link in which only one calcium ions is bracketed in a “twisted” egg-box between two chains; upon further counterion addition, a series of nearest-neighboring “perfect” egg-box structures develops. Both dilatometric and microcalorimetric changes associated with the latter chemical bonding modes are quantitatively larger for pectate than for alginate; clearly the latter polyuronate suffers from the relevant presence of the weakly calcium-binding mannuronic acid repeating units. Light-scattering experiments provided a clear-cut demonstration of the intermolecular bonding of calcium ions from the very beginning of the linker addition. Full article

Cornelian cherries has been grown Balkan peninsula no apparent disease and pest problems for centuries. The most important pomological and technological properties of cornelian cherry genetic resources (eleven local and introduced varieties and selections) in Gornje Polimlje, Montenegro were studied in four-year periods. Fruit weight, stone weight, and mesocarp/stone ratio shows great variation and varied between 1.89 to 6.16 g, 0.32 to 0.64 g and between 76.66 and 90.59%, respectively. Genotypes significantly differed each other in terms of yield determined as per trunk cross section area (TCSA). For better visualization of the agronomical traits important to the yield, i.e., yield per TCSA and fruit weight data is presented in combination as measured in the years of study. The best promising genotypes are Vladimirskiy, Chisinau yellow, and Krupnoplodni NS, which had consistently higher yield and large fruits for sustainable fruit production. Dry matter, total sugars, reducing sugars, sucrose, total acidity, Ca–pectate, vitamin C, total anthocyanins, total polyphenols, and ash content of cornelian cherry cultivars and selections were found between 19.81–24.46%, 10.94–14.47%, 10.44–13.09%, 0.24–1.23%, 1.92–2.51%, 2.76–4.43%, 41.09–101.27 mg/100 g, 0–157.06 mg/100 g, 183.91–307.9 mg/100 g, and 0.89–1.16%, respectively. The amount of potassium, which predominates in percentage of minerals in the ash, ranged from 2888 to 3574 mg/kg. The extensiveness of the study leads, therefore, to several clear conclusions and recommendations. Consistently, the best balance of biochemical elements in combination with good yield and fruit size measurements is found in selection Krupnoplodi NS. If consider fruit size and yield efficiency are priority, Vladimirskiy, Chisinau yellow, Kosten 3, and Krupnoplodni NS have clear advantage over the other genotypes. The study highlights the importance of local cornelian cherry selections for sustainable cornelian cherry production in Montenegro. Full article