The roles of the C-terminal region of Aeromonas hydrophila chitinase in chitin binding

• Main Authors: Yang, Jia-Shang, 楊嘉軒
• Other Authors: Chang Woei-Jer
• Format: Others
• Language: zh-TW
• Published: 1996
• Online Access: http://ndltd.ncl.edu.tw/handle/52403864118816183052

碩士 === 國立成功大學 === 生物化學研究所 === 84 === Chitinases (EC 3.2.1.14) hydrolyze the ?1,4 linkage of chitin that consistsof straight chains of N-acetyl-D-glucosamine residues linked ?1, 4. Variousorganisms including bacteria, fungi, plants, and some vertebrates producechitinases, and bacterial chitinases are thought to be important in thedigestion of chitin for utilization as carbon and energy sources and serve as animportant role in recycling chitin in nature. The C- terminal domain of AeromonasHydrophila chitinase has been found to have two 40 amino acid regions containingWXAXYWTXGXEP consensus sequence. On the basis of homology search, chitinases( U09139 and L42548), and cellulases (Z33876, P06565, and P06566) contain one ortwo regions with consensus sequence. The strong similarity of these amino acidsequences in chitinases and cellulases suggests that this sequence is organizedinto domain and performs specific function. In order to understand the role of this consensus sequence, the C-terminaldomain of Aeromonas Hydrophila chitinase with two WXAXYWTXGXEP consensussequences has been isolated. The structural gene of the 89 residues C-terminaldomain of Aeromonas Hydrophila chitinase (C89) was sequenced and expressed inE. coli BL21 from Dr. Ming- Cheng Chang's group. The engineering strain ofE. coli JM105 carries the vector pGEX-5X-3 for the C-terminal domain. Expressionof C89 was induced by addition of IPTG and was further purified by GlutathioneSepharose 4B chromatography. The separation of C89 from the glutathioneS-transferase was accomplished by site specific proteolysis using factor Xa andwas further purified by SP Sepharose chromatography. The proteolytic product ofC89 named C52 was purified by Biogel P10 gel filtration chromatography. Based onSDS-polyacryamide gel electrophoresis, the C89 and C52 of Aeromonas Hydrophilachitinase were purified to be homogeneous. The interaction of C89 with chitin and cellulose has been studied byfluorescence and circular dichroism (CD) spectroscopy. Chitin or cellulosebinding to C89 containing two WXAXYWTXGXEP consensus sequences is accompanied bya large quenching of tryptophan fluorescence, allowing the measurement ofequilibrium constants for chitin-C89 (Kd= 6.5-22.2 潒) and cellulose-C89complexes (Kd = 24.2-49 潒) with a 1:1 stoichiometry. On the contrary, C52 witha WXAXYWTXGXEP consensus sequence can not bind with chitin and cellulose. Theseresults indicate that C89 binds chitin tighter than cellulose, and an optimumsize of tetrameric saccharides is required for binding. This may explain why themain product of chitin cleavage is diacetylchitobiose. CD analysis of C89 alone in PBS buffer at pH 7.4 shows 25.2% ? helix, 6.5%?structure, and 68.3% coil, and the observed structure is similar to thecalculated structure by GGBSM method. N, N', N", N"'-Tetraacetylchitotetraosebinding increases the structure of C89, changing the CD analysis to 37.9%?helix, 8.1% ?structure, and 54% coil. The binding of chitin to C- terminaldomain of Aeromonas Hydrophila chitinase induces the helical formation from coilon C89, and the conformational change of C89 on chitin-binding may playimportant role for interaction between chitin and bacterial chitinasescontaining WXAXYWTXGXEP consensus sequence.