Characterization of chito-oligosaccharides prepared by chitosanolysis with the aid of papain and Pronase, and their bactericidal action against Bacillus cereus and Escherichia coli

Abstract

Papain (from papaya latex; EC 3.4.22.2) and Pronase (from Streptomyces griseus; EC 3.4.24.31) caused optimum depolymerization of chitosan at pH 3.5 and 37 degrees C, resulting in LMMC (low molecular mass chitosan) and chito-oligomeric-monomeric mixture. The yield of the latter was 14-16% and 14-19% respectively for papain- and Pronase-catalysed reactions, depending on the reaction time (1-5 h). HPLC revealed the presence of monomer(s) and oligomers of DP (degree of polymerization) 2-6, which was also confirmed by matrix-assisted laser-desorption ionization-time-of-flight MS. Along with the chito-oligomers, the appearance of only GlcNAc (N-acetylglucosamine) in Pronase-catalysed chitosanolysis was indicative of its different action pattern compared with papain. Fourier-transform infrared, liquid-state 13C-NMR spectra and CD analyses of chito-oligomeric-monomeric mixture indicated the release of GlcNAc/GlcNAc-rich oligomers. The monomeric sequence at the non-reducing ends of chito-oligomers was elucidated using N-acetylglucosaminidase. The chito-oligomeric-monomeric mixture showed better growth inhibitory activity towards Bacillus cereus and Escherichia coli compared with native chitosan. Optimum growth inhibition was observed with chito-oligomers of higher DP having low degree of acetylation. The latter caused pore formation and permeabilization of the cell wall of B. cereus, whereas blockage of nutrient flow due to the aggregation of chito-oligomers-monomers was responsible for the growth inhibition and lysis of E. coli, which were evidenced by scanning electron microscopy analysis. The spillage of cytoplasmic enzymes and native PAGE of the cell-free supernatant of B. cereus treated with chito-oligomeric-monomeric mixture further confirmed bactericidal activity of the latter. Use of papain and Pronase, which are inexpensive and easily available, for chitosanolysis, is of commercial importance, as the products released are of considerable biomedical value.

Figure 1. RP-HPLC profiles of (A) papain and (B) Pronase

Figure 2. SEM of (A) native chitosan (×500), and chito-oligomeric–monomeric mixture (×2000) obtained using papain (B) and (C) Pronase

Figure 3. FTIR spectra of (A) native chitosan, and chito-oligomeric–monomeric mixture obtained using papain (B) and (C) Pronase

Figure 4. CD spectra of (■) native chitosan, and chito-oligomeric–monomeric mixture obtained using papain (●) and (▲) Pronase

Figure 5. Liquid-state ¹³C-NMR spectra of (A) native chitosan, and chito-oligomeric–monomeric mixture obtained using papain (B) and (C) Pronase

Figure 6. HPLC profiles of chito-oligomeric–monomeric mixture obtained after depolymerization (5 h) of chitosan using (A) papain and (B) Pronase

Figure 7. MALDI–TOF-MS of the chito-oligomers obtained after depolymerization of chitosan using (A) papain and (B) Pronase

Figure 8. Growth inhibitory effect of individual sugars towards B. cereus (10⁶ CFU·ml⁻¹) and E. coli (10⁴ CFU·ml⁻¹)

(A) and (B) represent chitosanolytic products of papain- and Pronase-catalysed reactions respectively.

Figure 9. SEM of B. cereus and E. coli before (A, C respectively) and after (B, D respectively) treatment with chito-oligomeric–monomeric mixture

Figure 10. Native PAGE of cell-free supernatants of B. cereus (A) and E. coli (B)

Lanes I and IV, cells subjected to sonication; lanes II and V, control; lanes III and VI, cells treated with chito-oligomeric–monomeric mixture.

Figure 11. Mechanism of bactericidal action of chito-oligomeric–monomeric mixture towards B. cereus and E. coli: a hypothetical model

LPS, lipopolysaccharide. The large arrows indicate the sequence of bactericidal actions.