Serratia marcescens TKU011係一株以蝦頭殼粉末作為唯一碳/氮源之蛋白酶及幾丁聚醣酶生產菌。本研究發現以烏賊軟骨粉末(SPP)作為碳/氮源時，TKU011生產蛋白酶及幾丁聚醣酶之較適條件為1%烏賊軟骨粉末、0.1% K2HPO4及0.05% MgSO4．7H2O，在25℃、pH 7之100 mL液態培養基振盪培養5天。延長培養基滅菌時間(在121℃下滅菌120分鐘)可得較佳之蛋白酶及幾丁聚醣酶活性。在培養基之不同滅菌時間條件下，所得發酵上清液經SDS-PAGE分析發現，酵素濃度隨滅菌時間延長而增加。自發酵物回收之烏賊粉末乾重，會隨著培養天數增加而逐漸減少，上清液可測得含有胜肽、寡糖。發酵上清液經清除DPPH自由基能力、還原力、螯合亞鐵離子能力及總酚量等測定抗氧化能力試驗，TKU011發酵SPP可生產抗氧化物質，這些抗氧化物質可能為胜肽、幾丁質及幾丁聚醣等化合物。將較適培養條件下發酵所得上清液經硫酸銨沉澱、DEAE-Sepharose及Phenyl Sepharose等層析步驟，可純化出二種幾丁質酶/幾丁聚醣酶(C1及C2)，經SDS-PAGE測得分子量分別為49 kDa及66 kDa，將進一步探討酵素生化特性分析及基質特異性。 A protease- and chitosanase-producing strain, Serratia marcescens TKU011, was cultivated by using shrimp head shell powder (SHP) as the sole carbon/nitrogen source. In this study, the optimized condition for protease and chitosanase production using squid pen powder (SPP) as the carbon/nitrogen source was found to be when the culture was shaken at 25℃ for five days in 100 mL of medium (pH 7) containing 1% SPP, 0.1 % K2HPO4, 0.05% MgSO4．7H2O. In a prolonged autoclave (at 121℃ for 120 min), the protease and chitosanase activity were measured were better than others. The culture supernatants from various time of the medium in an autoclave by SDS-PAGE analysis was found that the enzyme concentration increased with an increase in heating time. The dry weight of the recovered squid pen decreased with an increase in cultivation. The peptides and oligosaccharides in the culture supernatants was determined. The antioxidative activity of the supernatant was derermind by DPPH radical-scavenging activity, reducing activity, total phenolic, Fe2+-Chelating activity. Two chitosanase( or chitinase) (C1,C2) were purified from the culture supernatant by chromatography procedures of DEAE-Sepharose and Phenyl Sepharose. The molecular mass of C1 and C2 determined by SDS-PAGE was approximately 49 kDa and 66 kDa, respectively. The substrate specificity and biochemical characterization of the enzymes were studied.
