Purification, characterization and comparison of reptile lysozymes.
Cation exchange column chromatography and gel filtration chromatography were used to purify four reptile lysozymes from egg white: SSTL A and SSTL B from soft shelled turtle (Trionyx sinensis), ASTL from Asiatic soft shelled turtle (Amyda cartilagenea) and GSTL from green sea turtle (Chelonia mydas). The molecular masses of the purified reptile lysozymes were estimated to be 14 kDa by SDS-PAGE. Enzyme activity of the four lysozymes could be confirmed by gel zymograms and showed charge differences on native-PAGE. SSTL A, SSTL B and ASTL had sharp pH optima of about pH 6.0, which contrasts with that of GSTL, which showed dual pH optima at about pH 6.0 and pH 8.0. The activities of the reptile lysozymes rapidly decreased within 30 min of incubation at 90 degrees C except for ASTL, which was more stable. Partial N-terminal amino acid sequencing and peptide mapping strongly suggested that the enzymes were C-type lysozymes. Interestingly, the mature SSTL lysozymes show an extra Gly residue at the N-terminus, which was previously found in soft-shelled turtle lysozyme. The reptile lysozymes showed lytic activity against several species of bacteria, such as Micrococcus luteus and Vibrio cholerae, but showed only weak activity to Pseudomonas aeruginosa and lacked activity towards Aeromonas hydrophila.
Department of Biochemistry, Faculty of Science, Khon Kaen University, Thailand. somkly@kku.ac.th
Sompong
S
Thammasirirak
Pornpimol
P
Ponkham
Sutthidech
S
Preecharram
Rathakarn
R
Khanchanuan
Phalakorn
P
Phonyothee
Sakda
S
Daduang
Chantragan
C
Srisomsap
Tomohiro
T
Araki
Jisnuson
J
Svasti
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D004527:Egg Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D016982:Micrococcus luteus / Q000187:drug effects / Q000254:growth & development; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D017510:Protein Folding; D030162:Reptilian Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D016415:Sequence Alignment; D013696:Temperature; D014426:Turtles / Q000378:metabolism*; D014734:Vibrio cholerae / Q000187:drug effects / Q000254:growth & development
D000900:Anti-Bacterial Agents; D004527:Egg Proteins; D030162:Reptilian Proteins; D009113:Muramidase