Which enzyme is fastest at turning chitosan into antifungal oligomers?

Chitosan, a derivative of chitin, can become a powerful antifungal when broken into short chains called oligomers. Two enzymes produced by the bacterium Bacillus thuringiensis B‑387 – chitinase (73 kDa) and chitosanase (40 kDa) – were compared to see which works best. After laboratory purification, scientists added each enzyme to a solution of chitosan with 85 % deacetylation. Chitosanase acted faster, creating short oligomers (two to four glucosamine units) and, with less time and less enzyme, also generated larger molecules (2.4 to 66.5 kDa). Chitinase, in contrast, needed more time and a higher enzyme dose for similar results. The chitosanase products were more water‑soluble and showed stronger antifungal activity against plant‑pathogenic fungi in vitro. Moreover, the chitosanase production process is simpler and yields more enzyme. For these reasons, chitosanase B‑387 looks better suited to produce chitosan oligomers with potential uses in sustainable agriculture, cutting down chemical pesticide use.

Chitosanase B‑387 belongs to the GH‑46 family of glycoside hydrolases, featuring a catalytic site that specifically recognizes β‑1,4 linkages between glucosamine (GlcN) and N‑acetyl‑glucosamine (GlcNAc) units. Its single‑domain structure, with a molecular weight of 40 kDa, provides greater conformational flexibility compared to the GH‑18 chitinase (73 kDa), which has a more rigid catalytic domain and a larger binding region. TLC and LC‑ESI‑MS analyses showed that chitosanase yields a narrower oligomer distribution, mainly DP 2‑4, but can also generate higher DP oligomers (up to DP 10) when substrate concentration is increased. Kinetic studies using Michaelis‑Menten revealed a Km of 0.12 mg mL⁻¹ for chitosanase, significantly lower than the chitinase Km of 0.35 mg mL⁻¹, indicating higher affinity for chitosan. Moreover, chitosanase remains active over a broad pH range (5.5‑8.0) and temperature window (25‑45 °C), making it suitable for moderate industrial conditions. ¹³C NMR confirmed the presence of β‑1,4 and β‑1,3 bonds in the products, suggesting that chitosanase can perform both endo‑ and exo‑hydrolysis, favoring the formation of more soluble oligomers. From a biotechnological perspective, B. thuringiensis produces high levels of chitosanase (≈ 150 U mL⁻¹) and purification requires only a single ion‑exchange chromatography step, lowering downstream costs. These traits, combined with the enhanced antifungal activity of the resulting COS—attributed to their ability to interact with fungal cell membranes and disrupt permeability—make chitosanase B‑387 an ideal candidate for developing chitosan‑oligomer‑based biopesticides.