Enzymatically Functionalized Composite Materials Based on Nanocellulose and Poly(Vinyl Alcohol) Cryogel and Possessing Antimicrobial Activity

Abstract

In the present work, innovative composite biomaterials possessing bactericidal properties and based on the hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) entrapped in the poly(vinyl alcohol) cryogel (PVA-CG)/bacterial cellulose (BC) were developed. His₆-OPH possesses lactonase activity, with a number of N-acyl homoserine lactones being the inducers of Gram-negative bacterial resistance. The enzyme can also be combined with various antimicrobial agents (antibiotics and antimicrobial peptides) to improve the efficiency of their action. In this study, such an effect was shown for composite biomaterials when His₆-OPH was entrapped in PVA-CG/BC together with β-lactam antibiotic meropenem or antimicrobial peptides temporin A and indolicidin. The residual catalytic activity of immobilized His₆-OPH was 60% or more in all the composite samples. In addition, the presence of BC filler in the PVA-CG composite resulted in a considerable increase in the mechanical strength and heat endurance of the polymeric carrier compared to the BC-free cryogel matrix. Such enzyme-containing composites could be interesting in the biomedical field to help overcome the problem of antibiotic resistance of pathogenic microorganisms.

Keywords: antimicrobial peptides; bacterial cellulose; bactericidal activity; immobilized hexahistidine-tagged organophosphorus hydrolase; poly(vinyl alcohol) cryogel; β-lactam antibiotic.

Figure 1

The appearance of a bacterial cellulose (BC) mat biosynthesized by immobilized K. xylinum cells in fructose-containing medium (a) before and (b) after cutting into pieces, which were further used to immobilize the enzyme. (c) The general view of poly(vinyl alcohol) cryogel (PVA-CG)/BC composites. SEM images of (d) BC, (e) PVA-CG, and (f) PVA-CG/BC/hexahistidine-tagged organophosphorus hydrolase (His₆-OPH) composites. Individual nanofibers of BC, marked by red triangles, are seen within the initial material and its composite.

Figure 2

Enzymatic activity of composite biomaterials PVA-CG/His₆-OPH (samples 2,6,10, and 14) and PVA-CG/BC/His₆-OPH (samples 4,8,12, and 16) in the absence (samples 2 and 4) or in the presence of meropenem (samples 6 and 8), temporin A (samples 10 and 12), and indolicidin (samples 14 and 16). White and patterned bars represent activity at zero time and after 24 h, respectively. The same samples prepared without His₆-OPH were used as controls and had no measurable hydrolase activity. Initial activity of free His₆-OPH was accepted as 100%.

Figure 3

Concentration of Pseudomonas sp. in a suspension after 24 h exposure with PVA-CG (sample 1), PVA-CG/His₆-OPH (samples 2,6,10, and 14), PVA-CG/BC (sample 3), or PVA-CG/BC/His₆-OPH (samples 4,8,12, and 16) in the absence (samples 1,2,3, and 4) or in the presence of meropenem (samples 6 and 8), temporin A (samples 10 and 12), or indolicidin (samples 14 and 16). The dashed line indicates the cell concentration at zero time. Concentration of cells in the control without any additions after 24 h was assumed as 100%. Cultures treated by composite samples without His₆-OPH but with antimicrobial agents for 24 h had almost the same bacterial concentration as the initial level.