Cloning, purification, and characterization of chitosanase from Bacillus sp. DAU101.
A chitosanase-producing Bacillus sp. DAU101 was isolated from Korean traditional food. This strain was identified on the basis of phylogenetic analysis of the 16S rDNA sequence, gyrA gene, and phenotypic analysis. The gene encoding chitosanase (csn) was cloned and sequenced. The csn gene consisted of an open reading frame of 837 nucleotides and encodes 279 amino acids with a deduced molecular weight of 31,420 Da. The deduced amino acid sequence of the chitosanase from Bacillus sp. DAU101 exhibits 88 and 30 % similarity to those from Bacillus subtilis and Pseudomonas sp., respectively. The chitosanase was purified by glutathione S-transferase fusion purification system. The molecular weight of purified enzyme was about 27 kDa, which suggests the deletion of a signal peptide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pH and temperature optima of the enzyme were 7.5 and 50 degrees C, respectively. The enzyme activity was increased by about 1.6-fold by the addition of 5 or 10 mM Ca(2+). However, Hg(2+) and Ni(+) ions strongly inhibited the enzyme. The enzyme produced, GlcN(2-4), were the major products from a soluble chitosan.
Lee
Y S
YS
Division of Biotechnology, Faculty of Natural Resources and Life Science, Dong-a University, Busan, 604-714, South Korea.
Yoo
J S
JS
Chung
S Y
SY
Lee
Y C
YC
Cho
Y S
YS
Choi
Y L
YL
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D027081:DNA Gyrase; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D005516:Food Microbiology; D020459:Genes, rRNA; D005944:Glucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007723:Korea; D008628:Mercury / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009532:Nickel / Q000494:pharmacology; D010802:Phylogeny; D021382:Protein Sorting Signals; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA; D013696:Temperature
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D021382:Protein Sorting Signals; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D009532:Nickel; D006026:Glycoside Hydrolases; C019868:chitosanase; D027081:DNA Gyrase; D008628:Mercury; D005944:Glucosamine; D002118:Calcium