Purification and Characterization of a Major Extracellular Chitinase from a Biocontrol Bacterium, Paenibacillus elgii HOA73

Abstract

Chitinase-producing Paenibacillus elgii strain HOA73 has been used to control plant diseases. However, the antimicrobial activity of its extracellular chitinase has not been fully elucidated. The major extracellular chitinase gene (PeChi68) from strain HOA73 was cloned and expressed in Escherichia coli in this study. This gene had an open reading frame of 2,028 bp, encoding a protein of 675 amino acid residues containing a secretion signal peptide, a chitin-binding domain, two fibronectin type III domains, and a catalytic hydrolase domain. The chitinase (PeChi68) purified from recombinant E. coli exhibited a molecular mass of approximately 68 kDa on SDS-PAGE. Biochemical analysis indicated that optimum temperature for the actitvity of purified chitinase was 50ºC. However, it was inactivated with time when it was incubated at 40ºC and 50ºC. Its optimum activity was found at pH 7, although its activity was stable when incubated between pH 3 and pH 11. Heavy metals inhibited this chitinase. This purified chitinase completely inhibited spore germination of two Cladosporium isolates and partially inhibited germination of Botrytis cinerea spores. However, it had no effect on the spores of a Colletotricum isolate. These results indicate that the extracellular chitinase produced by P. elgii HOA73 might have function in limiting spore germination of certain fungal pathogens.

Reclamation of Marine Chitinous Materials for Chitosanase Production via Microbial Conversion by Paenibacillus macerans

“…1794 was pH 4.8 [ 24 ]; P. barengoltzii was pH 3.5 [ 28 ]; P. thermoaerophilus TC22-2b was pH 4 [ 25 ]; and Paenibacillus sp. M4 was pH 6.5, but it was consistent with P. elgii HOA73 (pH 7) [ 29 ]. These results also observed TKU029 chitosanase to have broad pH stability (pH 3–8).…”

“…Similar to most of the other chitinolytic enzymes-producing strains of Paenibacillus species, only one chitinase or chitosanase were purified from the culture supernatant [ 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 ]. However, the molecular mass of TKU029 chitosanase (63 kDa) estimated by SDS-PAGE ( Figure 3 ) differed from the chitinases or chitosanases of the other Paenibacillus strains, for instance, P. pasadenensis NCIM5434 chitinase (35 kDa) [ 23 ], Paenibacillus sp.…”

“…1794 chitosanase (40 kDa) [ 24 ], P. thermoaerophilus TC22-2b chitinase (48 kDa) [ 25 ], P. larvae ATCC9545 (49 kDa) [ 26 ], P. illinoisensis KJA-424 chitinase (54 kDa) [ 33 ], Paenibacillus sp. D1 chitinase (56 kDa) [ 27 ], P. barengoltzii CAU904 chitinase (67 kDa) [ 28 ], P. elgii HOA73 chitinase (68 kDa) [ 29 ], P. pasadenensis CS0611 chitinase (69 kDa) [ 30 ], Paenibacillus sp. TKU042 chitosanase (70 kDa) [ 22 ], Paenibacillus sp.…”

“…These results also observed TKU029 chitosanase to have broad pH stability (pH 3–8). Several Paenibacillus strains chitinases/chitosanases have broad pH stability close to that of TKU029 [ 28 , 29 , 34 ].…”

Differences in the chitinolytic activity of mammalian chitinases on soluble and crystalline substrates

“…Due to the high activation energy of effective binding to these structures, processive hydrolases are commonly employed by organisms to maximize the metabolism accomplished after each binding event (7,8) . In contrast to the complexity of polysaccharides in the environment, enzymatic hydrolysis is often quantified in vitro using small oligomer analogues, which release a chromophore or fluorophore when cleaved (9,10) . These simple substrates allow for high signal-to-noise and precise quantification, but activity measurement using these simple substrates is fundamentally limited in assessing enzyme activity on more complex substrates, due to their higher order, often crystalline, structures, variable polymer length, branching and other modifications.…”