Biochemical Properties and Anti-Biofilm Activity of Chitosan-Immobilized Papain

Abstract

Chitosan, the product of chitin deacetylation, is an excellent candidate for enzyme immobilization purposes. Here we demonstrate that papain, an endolytic cysteine protease (EC: 3.4.22.2) from Carica papaya latex immobilized on the matrixes of medium molecular (200 kDa) and high molecular (350 kDa) weight chitosans exhibits anti-biofilm activity and increases the antimicrobials efficiency against biofilm-embedded bacteria. Immobilization in glycine buffer (pH 9.0) allowed adsorption up to 30% of the total protein (mg g chitosan−1) and specific activity (U mg protein−1), leading to the preservation of more than 90% of the initial total activity (U mL−1). While optimal pH and temperature of the immobilized papain did not change, the immobilized enzyme exhibited elevated thermal stability and 6–7-fold longer half-life time in comparison with the soluble papain. While one-half of the total enzyme dissociates from both carriers in 24 h, this property could be used for wound-dressing materials design with dosed release of the enzyme to overcome the relatively high cytotoxicity of soluble papain. Our results indicate that both soluble and immobilized papain efficiently destroy biofilms formed by Staphylococcus aureus and Staphylococcus epidermidis. As a consequence, papain, both soluble and immobilized on medium molecular weight chitosan, is capable of potentiating the efficacy of antimicrobials against biofilm-embedded Staphylococci. Thus, papain immobilized on medium molecular weight chitosan appears a presumably beneficial agent for outer wound treatment for biofilms destruction, increasing antimicrobial treatment effectiveness.

Improving the Efficacy of Antimicrobials against Biofilm-Embedded Bacteria Using Bovine Hyaluronidase Azoximer (Longidaza®)

“…While embedded into the biofilm matrix, bacterial cells become largely inaccessible to both antibiotics and biocides. The above data suggest that biofilm matrix destruction with Longidaza ® could facilitate the penetration of antimicrobials into biofilm-embedded bacteria, as has been shown previously in other model investigations [33][34][35]66]. For this, 48-h old biofilms were incubated for either 4 or 24 h in the presence of Longidaza ® (750 IU) with antimicrobials (Ciprofloxacine and Cefuroxime) at their respective 1×, 4× and 16× MBCs (minimal bactericidal concentrations, see Table S1 for values), followed by the evaluation of the biofilm-embedded cells' viability with an MTT-assay (Figure 7).…”

“…In urinary patients, both implanted catheters and the epithelium of the urinary tract are subjected to bacterial biofilm fouling, in turn leading to the development of long-term diseases [6,17,18] such as chronic bacterial prostatitis and pyelonephritis [20,21]. While the enzymatic destruction of biofilms in general seems to be an attractive approach [31][32][33][34][35]43,47,50,51], relatively few enzymes have been proposed for the treatment of urinary biofilms. In clinical practice, most enzymes are used for wound healing, and are not suitable for internal use due to the low stability of the preparations, as well as the high allergic potential [72][73][74].…”

“…While various strategies have been offered to date for the targeting of topical biofilms for their destruction or the prevention of their formation [31][32][33][34], very few options are available for the treatment of urinary and urogenital biofilm-associated infections. One of the promising strategies for the destruction of already-formed biofilms seems to be their enzymatic treatment.…”

“…To date, various strategies have been offered for targeting topical biofilms, focusing either on their destruction or on the prevention of their formation, or both [2,[31][32][33][34][35][36][37][38][39]. Nevertheless, very few options are available for the treatment of urinary biofilm-associated infections.…”

“…In numerous in vivo and in vitro studies, an efficient disruption of mature biofilms by various enzymes like DNase [42,43] and proteases (Ficin [35], Proteinase K [44] and aureolysin [45,46]), glycoside hydrolases (Pel, Psl [47], dispersin B [48], alginate lyase [49], cellulase [50] and extracellular levanase from Bacillus subtilis [51]) has been reported. Biofilm matrix lysis leads to increased bacterial susceptibility to antimicrobials, and thus also a considerable improvement of the latter [33,35,51,52]. Unfortunately, there is no universal enzyme efficiently targeting arbitrary biofilms due to the considerable differences in the composition of the proteins, polysaccharides, and extracellular DNA in the biofilm matrix depending on the bacterial species and their growth conditions [53].…”