Enzymatic Properties of Antifungal Endochitinase and Its Function in the Preparation of Chitosan Oligosaccharides

In order to develop chitin and chitosan bioconversion of active oligosaccharides and achieve the application of chitinase in food preservation and biocontrol, chitinase (ChiA-Sa62) was isolated and purified from Streptomyces aureofaciens XZ-Sa62, and its bioconversion and antibacterial function were studied. ChiA-Sa62 was purified by Q-Sepharose Fast Flow, Sephadex G-100 chromatography, and reverse phase high performance liquid chromatography (RP-HPLC), with a purification factor of 41.3 and a specific activity of 1119.8 U/mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) showed that ChiA-Sa62 was a single subunit protein with a molecular weight of 62.55 kDa. ChiA-Sa62 exhibited maximum activity at 50 ℃ and pH 5, and exhibited good stability below 60 ℃ and within pH range of 3.0-9.0. Ca²⁺, Mn²⁺, Mg²⁺, and Co²⁺ could activate enzyme activity. ChiA-Sa62 had a specific endonucleating hydrolysis activity on chitin and chitosan with a degree of deacetylation of 75%. Thin layer chromatography (TLC) showed that the hydrolysis products were 2-5 N-acetylglucosamine (GlcNAc) units of chitosan oligosaccharides and 2-4 D-glucosamine (GlcN) units of chitosan oligosaccharides, respectively. The colloidal chitin was used as the substrate, with ChiA-Sa62 K_m and V_max values of 2.75 mg/mL and 64.52 U/mg, respectively. ChiA-Sa62 strongly inhibited the tested pathogenic fungi, and propidium iodide (PI) staining showed that ChiA-Sa62 disrupted the integrity of the cell membrane of Botrytis cinerea. The hydrolyzed chitosan oligosaccharide had antibacterial activity against pathogenic G^- bacteria (Escherichia coli and Pseudomonas aeruginosa) and G⁺ bacteria (Staphylococcus aureus and Bacillus cereus). LIVE/DEAD staining showed that chitosan oligosaccharide could cause the death of S. aureus. ChiA-Sa62 chitin and chitosan have great potentials for application in biotransformation, food preservation, and biological control.