Partial purification, characterization, and kinetic studies of a low-molecular-weight, alkali-tolerant chitinase enzyme from Bacillus subtilis JN032305, A potential biocontrol strain.
A new alkalophilic low-molecular-mass chitinase of 14 kD from the potent biocontrol agent Bacillus subtilis JN032305 was partially purified and enzymology of the chitinase was studied. The enzyme showed optimal pH of 9.0 and temperature of 50°C. The enzyme was found stable during the 60-min incubation at 50 °C. The chitinase was inhibited by group specific agents like IAA, DAN, TLCK, and SDS and metal ions Mg(2+), Ca(2+), Fe(2+), Mn(2+), Ba(2+), and Hg(2+), whereas Zn(2+) did not show significant inhibitory effect against the chitinase. PMSF partially inhibited the enzyme. Substrates specificity tests indicated that the enzyme showed 75% of relative activity on glycol chitin, 58% on carboxymethylcellulose (CMC), 33% on chitin flakes, and 166% laminarin compared to that on colloidal chitin. The enzyme also hydrolyzed 4-methylumbelliferyl-N-acetyl-D-glucosaminide, indicating its chitobiase activity. The chitinase of this study has broad specificity, which could hydrolyze not only the glycosidic bond in GlcNAc-GlcNAc but also that of related carbohydrates with glycosidic linkages. The partially purified chitinase not only showed antifungal activity against Rhizoctonia solani and Colletotrichum gloeosporioides, two potent phytopathogens of chilli, but also increased the germination of chilli seeds when infected with the two potent phytopathogenic fungi.
a Department of Microbiology , Jain University , Bangalore , India.
Srividya
S
Shivakumar
Anika Nayak
AN
Karmali
Charles
C
Ruhimbana
D000117:Acetylglucosamine / Q000031:analogs & derivatives / Q000737:chemistry; D000468:Alkalies / Q000737:chemistry; D000935:Antifungal Agents / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D001412:Bacillus subtilis / Q000737:chemistry* / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D001487:Basidiomycota / Q000187:drug effects / Q000254:growth & development; D061046:Biological Control Agents; D002212:Capsicum / Q000187:drug effects / Q000254:growth & development; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry; D002686:Chitin / Q000031:analogs & derivatives / Q000737:chemistry; D002688:Chitinases / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006923:Hymecromone / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D008970:Molecular Weight; D012639:Seeds / Q000187:drug effects / Q000254:growth & development; D013379:Substrate Specificity; D013696:Temperature
D000468:Alkalies; D000935:Antifungal Agents; D001426:Bacterial Proteins; D061046:Biological Control Agents; D002686:Chitin; C025158:4-methylumbelliferyl 2-acetamido-2-deoxy-beta-D-glucopyranoside; D006923:Hymecromone; C031610:glycolchitin; D002688:Chitinases; D002266:Carboxymethylcellulose Sodium; D000117:Acetylglucosamine