Bacillus cereus TKU044 所生產幾丁聚醣酶之純化及定性

Purification and Characterization of Chitosanases from Bacillus cereus

余浩廷(Hao-Ting Yu)

指導教授 ： 王三郎

淡江大學/理學院/化學學系碩士班/碩士(2017年)

https://doi.org/10.6846/TKU.2017.00153

摘要

Bacillus cereus TKU044 係篩選自花蓮土壤之幾丁聚醣酶生產菌，以含有1%烏賊軟骨粉、0.1% K2HPO4、0.05% MgSO4．7H2O之100 mL液態培養基，於37℃進行搖瓶 (150 rpm)培養2天後，可得較高幾丁聚醣酶活性 (2.66U/mL)。
　　培養所得發酵上清液經硫酸銨沉澱、DEAE-Sepharose 及 Macro-Prep® DEAE Cartridge 陰離子交換層析等步驟，以285.9倍之純化度以及16%之回收率，純化出經 SDS-PAGE電泳測得分子量約為43 KDa 之一種幾丁聚醣酶。此幾丁聚醣酶的最適反應pH為7，pH安定性為6~7，最適反應溫度為40℃，而熱安定性則是低於50℃，其活性會受到Na+、Fe2+、Ba2+、Cu2+、Zn2+及EDTA所抑制。TKU044粗酵素液水解水溶性幾丁聚醣所得寡糖，具有抗氧化活性。

關鍵字

Bacillus cereus ； 幾丁聚醣酶 ； 烏賊軟骨

並列摘要

The chitosanase-producing strain TKU044 was isolated from the soils of Hualien with squid pen power (SPP) as the sole carbon/nitrogen source and identified as Bacillus cereus. A chitosanase was purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Macro-Prep® DEAE Cartridge. The molecular mass of chitosanase determined by SDS-PAGE was approximately 43 kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of the TKU044 chitosanase were pH 7, 40℃, pH 6~7 and under 50℃, respectively. The chitosanase activity was inhibited by Na+, Fe2+, Ba2+, Cu2+, Zn2+ and EDTA. The oligosaccharides prepared by hydrolyzing soluble chitosan with the crude chitosanase showed antioxidant activity.

並列關鍵字

Bacillus cereus ； chitosanase ； squid pens

參考文獻

Aranaz I, Mengibar M, Harris R, Panos I, Miralles B, Acosta N, Galed G, Heras A (2009) Functional characterization of chitin and chitosan. Current Chemical Biology, 3:203-230

Arslan S, Eyi A, Küçüksarı R (2014) Toxigenic genes, spoilage potential, and antimicrobial resistance of Bacillus cereus group strains from ice cream. Anaerobe, 25:42-46

Azuma K, Izumi R, Osaki T, Ifuku S, Morimoto M, Saimoto H, Minami S, Okamoto Y (2015) Chitin, chitosan, and its derivatives for wound healing: old and new materials. Journal of Functional Biomaterials, 6:104-142

Barikani M, Oliaei E, Seddiqi H, Honarkar H (2014) Preparation and application of chitin and its derivatives: a review. Iranian Polymer Journal, 23:307-326

Bernfeld P (1955) Amylase, α and β. Methods of Enzymology, 1:149-158