Characterization of Thermotolerant Chitinase from the Strain Cohnella sp. IB P-192 and Its Application for the Production of Bioactive Chitosan Oligomers

• Published in: Applied biochemistry and microbiology 2022-04, Vol.58 (2), p.143-154
• Main Authors: Gilvanova, E. A., Aktuganov, G. E., Safina, V. R., Milman, P. Yu, Lopatin, S. A., Melentiev, A. I., Galimzianova, N. F., Kuzmina, L. Yu, Boyko, T. F.
• Format: Article
• Language: English
• Subjects: Biochemistry; Biomedical and Life Sciences; Calcium ions; Cations; Chitin; Chitinase; Chitosan; Colloid chitin; Copper; Deacetylation; Enzymes; Fungicides; Gel electrophoresis; Glucosaminidase; Hydrolysis; Hydrophobic chromatography; Hydrophobicity; Life Sciences; Manganese; Medical Microbiology; Microbiology; Molecular weight; Oligomers; pH effects; Polymers; Reaction products; Sodium lauryl sulfate; Substrates; Ultrafiltration
• ISSN: 0003-6838; 1608-3024
• DOI: 10.1134/S0003683822020077

Thermostable exochitinase was purified from a culture medium of a moderately thermophilic strain Cohnella sp. IB-P192 via ultrafiltration, affinity sorption, and hydrophobic chromatography and was then characterized. Enzyme synthesis was induced by colloidal chitin from carb shells. It reached the highest level at 50°C in 72 h of submerged cultivation. The molecular weight of the purified chitinase as determined with SDS-PAGE was 69 kDa. The enzyme had pH and temperature optima of 7.5 and 70°C, respectively. It retained 100% activity under 65°C and was stable at a pH of 5–10.5. The Michaelis–Menten constant and specific V max of the purified chitinase were 0.83 mg × mL –1 and 116.75 μM-eqv × mL –1 × min –1 × mg –1 , respectively. The enzyme was inhibited by Ag + and Hg +2 cations and insignificantly inhibited by 1 mM Cu +2 and Ni +2 , while 1 mM Mn +2 , Ca +2 and Co +2 cations and Tween-80 increased its activity. The chitinase hydrolyzed specific substrate according to the exomechanism of substrate hydrolysis, forming (GlcNAc) 2 as main reaction product and it functioned as N -acetyl-β-D-glucosaminidase at a later stage of hydrolysis (3–4 h). The highest rate of chitosan hydrolysis by the enzyme was recorded at a deacetylation degree (DD) of 50% at 70°C and an [E]/[S] ration of 1 : 60. The fungicidal effect of produced chitosan oligomers depended on the DD of the original polymer and most strongly increased under the destruction of the chitosan with a DD of 50%.