不同源自真菌材料之幾丁聚醣抑制痤瘡丙酸桿菌脂?之探討 Inhibition of lipase activity of Propionibacterium acnes by Chitosans isolated from various fungus
中文摘要
造成痤瘡的主要致病菌是痤瘡丙酸桿菌(Propionibacterium acnes)；菌體會 分泌脂解酵素(Lipase)，分解皮膚油脂產生脂肪酸，促進痤瘡發炎的形成。本研 究之目的為探討不同來源之幾丁聚醣(Chitosan)對於抑制脂解酵素活性及抑制 痤瘡丙酸桿菌生長之關連。第一部份以松杉靈芝(Ganoderma tsugae)之子實 體經萃取後之廢渣、雙紡錘包子蟲草(Cordyceps bifusispora)之菌絲體、匍枝 根黴菌(Rhizopus stolonifer)之菌絲體及蝦蟹殼等四種材料萃取所得之幾丁聚 醣為材料 (簡稱如下表所示)，進行其對痤瘡丙酸桿菌抑菌能力之篩選。試驗結 果顯示三種真菌來源之幾丁聚醣對痤瘡丙酸桿菌均有抑制效果，最小抑菌濃度 (minimum inhibitory concentration, MIC)為 16-32 μg/mL，其中以匍枝 根黴菌之抑菌效果最佳(16 μg/mL)；蝦蟹殼來源之幾丁聚醣之最小抑菌濃度
則為16 μg/mL。本研究之第二部份則探討上述四種不同來源之幾丁聚醣對於
痤瘡丙酸桿菌分泌之脂解酵素活性抑制之效果。試驗結果顯示在脂解酵素活性 90.75U/L 下，抑制活性效果隨材料濃度越高越好，其中以 CI 抑制效果最佳(16 μg/m 下仍有 32.7%抑制效果)，SAC 最差(64μg/m 以下即無抑制效果)。
本研究之第三部份為期望利用pET32a(+)質體轉殖痤瘡丙酸桿菌脂解酵素基因
(gehA)，藉誘導基因表現在大腸桿菌 BL21(DE3)中大量製備帶有 thioredoxin
之融合蛋白；結果顯示，轉殖之DNA 經序列比對為正確排列，亦可表現分子量
55kDa 之融合蛋白，但表現蛋白大部份出現在不可溶的包含體(inclusion body) 中；在嘗試取出可溶性蛋白或用強力介面活性劑取得不溶性蛋白，並經重新摺疊 蛋白步驟均無法呈現脂解酵素之活性，本部份研究得做為未來研究之基礎。
英文摘要
Propionibacterium acnes (P. acnes) which played an important role in the
pathogenesis of acne vulgaris. Extracellular lipases produced by P. acnes in vivo act as key enzyme in the hydrolyzation pathway of native sebum triacylglycerols to glycerol and free fatty acids (FFA) which can cause inflammation. The aim of this study is to investigate whether or not chitosan from different materials such as cell wall component from Ganoderma tsugae、Cordyceps bifusispora and Rhizopus stolonifer possessed the inhibitory potency on the growth of P. acnes and/or the activity of lipase. We first analysed the antimicrobial activity of chitosans from
Ganoderma tsugae、Cordyceps bifusispora、Rhizopus stolonifer and crustacean. The result showed that chitosans from three different fungi materials all works(minimum
inhibitory concentration, MIC value of 16~32 μg/ml).The chitosan from Rhizopus stolonifer, which showed markedly antimicrobial activity(MIC value of 16
μg/ml ).Chitosan showed an MIC value of 16 μg/ml toward crustacean. Moreover, analyzing the inhibitory of lipase activity of chitosans from different materials, we find all chitosans are work. Chitosan showed the markedly inhibitory of lipase activity value of 16 μg/ml toward crustacean (inhibitory rate about 32.7%). Furthermore, lipase gene (GehA) was constructed in the plasmid vector pET32a(+).The resultant plasmids were transformed into E. coli BL21(DE3) and over express of thioredoxin fusion lipase. The nucleotide sequence of fusion lipase is confirmed and a predicted molecular mass of 55kDa, but lipase was present in the in soluble fraction in the form of inclusion bodies. We tried express lipase in a soluble from or used detergent to purify protein and refold protein by several steps, but still not work. This part of study can be a foundation for further investigation
