| 摘要: | TKU024，係以烏賊軟骨粉末為唯一碳/氮源，篩選自台灣中部土壤之一株幾丁聚醣酶生產菌，經鑑定為Acinetobacter calcoaceticus。TKU024 幾丁聚醣酶較適生產條件為1.5% 烏賊軟骨粉末、0.1% K2HPO4、0.05% MgSO4．7H2O之液態培養基(pH 8)於37℃振盪培養4 天。將發酵所得上清液離心，經硫酸銨沉澱、DEAE-Sepharose、Mcaro-Prap DEAE 及Phenyl Sepharose 管柱層析後，可純化出兩個幾丁聚醣酶(C1/C2)，經SDS-PAGE 測得分子量分別為27 kDa及66 kDa。經胜肽質譜鑑定，C1 分別與Cellvibrio japonicus Ueda107 之幾丁質酶(GenBank 編號為gi1192361966)、Pseudomonas aeruginosa PA01 之琥珀脫氫酶鐵硫亞基(GenBank 編號為gi115596781)相似，相似度分別為4%及10%；C2與 Arthrobacter sp. 之幾丁質酶(GenBank 編號為gi127065204)相似，相似度為7%。於最適反應pH、最適反應溫度、pH 安定性、熱安定性方面，C1為pH 6、50℃、pH 8-9 及<70℃，C2 為pH 7、60℃、pH 6 及<60℃。C1 活性受5 mM Cu2+、Zn2+、Mn2+及2% (v/v) SDS 抑制，且於4~5 mM Mn2+完全抑制；C2 活性受5 mM Ca2+、Urea、EDTA、Zn2+、Mn2+抑制，但在1 mM Mn2+則有少許促進效果，於2% (v/v) SDS、Triton X-100、Tween 20 和0.5% (v/v)Tween 20 有輕微抑制效果。
Strain TKU024 was isolated from the soil in the middle Taiwan by using squid pen powder(SPP) as the sole carbon/nitrogen source and identified as Acinetobacter calcoaceticus. The optimized culture condition for chitosanase production was composed of 1.5% squid pen power(SPP), 0.1% K2HPO4 and 0.05% MgSO4．7H2O at pH 8 and incubated in 50 mL of liquid media in shaking flasks at 37℃ for 4 days.
Two chitosanases(C1/C2) were purified from the culture supernatant by using ammonium sulfate precipitation, chromatography procedures of DEAE-Sepharose, Macro-Prep DEAE, and Phenyl Sepharose. The molecular mass of C1 and C2 determined by SDS-PAGE were approximately 27 kDa and 66 kDa, respectively. The results of peptide mass mapping
indicated that C1 matched to chitinase from Cellvibrio japonicus Ueda107(GenBank accession number gi1192361966) with 4% sequence coverage and Pseudomonas aeruginosa PA01(GenBank accession number gi115596781) with 10% sequence coverage, and C2 matched to chitinase from Arthrobacter sp.(GenBank accession number gi127065204) with 7% sequence coverage. The optimum temperature, optimum pH, thermal stability, and pH stability of TKU024 chitosanase C1 and C2 were 50℃, pH 6,<70℃, pH 8~9 and 60℃, pH 7,<60℃, pH 6, respectively. C1 was inhibited by 5 mM Cu2+, Zn2+, Mn2+ and 2% (v/v) SDS, and was inhibited completely by 4~5 mM Mn2+ ; C2 was inhibited by 5 mM Ca2+, Urea, EDTA, Zn2+, Mn2+, 2% (v/v) SDS, Triton X-100, Tween 20 and 0.5% (v/v) Tween 20, but activated by 1 mM Mn2+. |
