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The problem of antibiotic resistance (AR) in pathogenic bacteria due to the massive use of antibiotics in animals is an underestimated issue. Finding alternatives to reduce or eliminate the appearance and spread of AR is a pressing issue for humanity. Enzymes with antibiotic properties may serve as antimicrobial agents, less prone to generating AR due to their proteic nature and mode of action. Lysozyme is a natural antimicrobial agent, amenable to replacing small-molecule antibiotics in animal farming (poultry and livestock) due to its similar effects on animal health and growth. Moreover, it is safe for human and animal consumption and environmentally friendly due to its biodegradability. The development and testing against pathogens of lysozyme-containing nanomaterials fabricated with biocompatible polymers, particularly polysaccharides is reviewed, focusing on their antimicrobial properties, often superior to lysozyme alone due to a synergistic effect of the nanoscaled materials.

In this work, we present a comprehensive investigation of the entrapment of laccase, a biotechnologically relevant enzyme, into levan-based nanoparticles (LNPs). The entrapment of laccase was achieved concomitantly with the synthesis of LNP, catalyzed by a truncated version of a levansucrase from Leuconostoc mesenteroides. The study aimed to obtain a biocompatible nanomaterial, able to entrap functional laccase, and characterize its physicochemical, kinetic and thermal stability properties. The experimental findings demonstrated that a colloidal stable solution of spherically shaped LNP, with an average diameter of 68 nm, was obtained. An uniform particle size distribution was observed, according to the polydispersity index determined by DLS. When the LNPs synthesis was performed in the presence of laccase, biocatalytically active nanoparticles with a 1.25-fold larger diameter (85 nm) were obtained, and a maximum load of 243 μg laccase per g of nanoparticle was achieved. The catalytic efficiency was 972 and 103 (μM·min)-1, respectively, for free and entrapped laccase. A decrease in kcat values (from 7050 min-1 to 1823 min-1) and an increase in apparent Km (from 7.25 μM to 17.73 μM) was observed for entrapped laccase, compared to the free enzyme. The entrapped laccase exhibited improved thermal stability, retaining 40% activity after 1 h-incubation at 70°C, compared to complete inactivation of free laccase under the same conditions, thereby highlighting the potential of LNPs in preserving enzyme activity under elevated temperatures. The outcomes of this investigation significantly contribute to the field of nanobiotechnology by expanding the applications of laccase and presenting an innovative strategy for enhancing enzyme stability through the utilization of fructan-based nanoparticle entrapments. [ABSTRACT FROM AUTHOR]

dsrP, a gene that encodes a cell-associated dextransucrase produced by Leuconostoc mesenteroides IBT-PQ, was isolated, sequenced and expressed in Escherichia coli. From sequence analysis, seven repeat units in the N-terminal region were found, as well as five cell wall binding repeats in the C-terminal region. A model of the C-terminal domain of dextransucrase was built based on the solenoid structure of the cell wall binding domain already described in LytA. By experiments involving direct interactions of the enzyme with L. mesenteroides cells, as well as among the cells and the single C-terminal domain expressed in E. coli, evidence was obtained concerning the anchoring function of this region in cell-associated dextransucrase, a function which may be independent of its capacity to bind dextran.

The gene coding for inulosucrase in Leuconostoc citreum CW28, islA, was cloned, sequenced, and expressed in Escherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase from Leuconostoc mesenteroides NRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of the islA gene truncated in the C-terminal glucan binding domain were successfully expressed in E. coli and retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.

Two levan distributions are produced typically by Bacillus subtilis levansucrase (SacB): a highmolecular weight (HMW) levan with an average molecular weight of 2300 kDa, and a low-molecular weight (LMW) levan with 7.2 kDa. Previous results have demonstrated how reaction conditions modulate levan molecular weight distribution. Here we demonstrate that the SacB enzyme is able to perform two mechanisms: a processive mechanism for the synthesis of HMW levan and a nonprocessive mechanism for the synthesis of LMW levan. Furthermore, the effect of enzyme and substrate concentration on the elongation mechanism was studied. While a negligible effect of substrate concentration was observed, we found that SacB elongation mechanism is determined by enzyme concentration. A high concentration of enzyme is required to synthesize LMW levan, involving the sequential formation of a wide variety of intermediate size levan oligosaccharides with a degree of polymerization (DP) up to ~70. In contrast, an HMW levan distribution is synthesized through a processive mechanism producing oligosaccharides with DP <20, in reactions occurring at low enzyme concentration. Additionally, reactions where levansucrase concentration was varied while the total enzyme activity was kept constant (using a combination of active SacB and an inactive SacB E342A/D86A) allowed us to demonstrate that enzyme concentration and not enzyme activity affects the final levan molecular weight distribution. The effect of enzyme concentration on the elongation mechanism is discussed in detail, finding that protein-product interactions are responsible for the mechanism shift. [ABSTRACT FROM AUTHOR]

Site-directed mutagenesis was performed on IslA4, a truncated form of inulosucrase (IS) derived from Leuconostoc citreum IS that contains only the IS catalytic domain. This truncated form is more hydrolytic than the wild-type enzyme and produces both high-molecular-weight inulin and fructooligosaccharides (FOS). Among the various mutants obtained from IslA4 by following strategies designed for SacB (the levansucrase from Bacillus subtilis), S425A no longer produces inulin, but instead produces FOS exclusively and hydrolyzes sucrose. Reaction conditions were explored to increase FOS productivity by reducing the hydrolysis, resulting in 65% conversion from a 0.67 M sucrose solution. S425A displays complex kinetic behavior in which the transfructosylation rate is described by first-order kinetics, while sucrose hydrolysis follows Michaelis–Menten behavior. This combined model correctly describes both the overall initial reaction rate as well as the reaction evolution for FOS synthesis. S425A IS may be useful for the synthesis of FOS from sucrose. [ABSTRACT FROM AUTHOR]