Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2.
Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO4, 1.4% K2HPO4, 0.02% CaCl2 x 2H2O, 0.002% FeSO4 x 7H2O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F2 fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70 degrees C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90 degrees , the t(1/2) values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90 degrees C, the enzyme retained its activity up to 8 h in the presence of 1 mM MnCl2. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The K(m app) values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the Vmax values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F2 chitosanase at 24 h incubation (70 degrees C) were pentasaccharide (GlcN)5 and hexasaccharide (GlcN)6. The preliminary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium, Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.
Ekowati
Chasanah
C
Center for Marine and Fisheries Product Processing and Biotechnology, Jl. KS Tubun, Petamburan VI, Jakarta, Indonesia.
Hariyadi
Purwiyatno
P
Witarto
Arief B
AB
Hwang
Jae Kwan
JK
Suhartono
Maggy Thenawidjaja
MT
D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000254:growth & development / Q000302:isolation & purification; D002413:Cations, Divalent / Q000494:pharmacology; D049109:Cell Proliferation / Q000187:drug effects; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability / Q000187:drug effects; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D057927:Hydrophobic and Hydrophilic Interactions; D007214:Indonesia; D007700:Kinetics; D011489:Protein Denaturation / Q000187:drug effects; D012015:Reference Standards; D012997:Solvents; D013379:Substrate Specificity / Q000187:drug effects; D013997:Time Factors
D002413:Cations, Divalent; D012997:Solvents; D006026:Glycoside Hydrolases; C019868:chitosanase