|Abstract: ||摘要 本研究以化學法或生物法處理不同來源的蝦蟹殼及魷魚鞘以去除蛋白質製備幾丁質，分析其殘留物的成分，剖析魷魚鞘含幾丁質的外層結構，及不同幾丁質影響Cellulomonas flavigena NTOU 1 幾丁質酶活性的情形。 以2 N HCl去鈣及以2 N NaOH去蛋白質處理後，大頭紅蝦殼、紅蟳外殼、龍蝦外殼及魷魚鞘殘留物的蛋白質及幾丁質含量分別為0.75及99.05﹪、5.86及88.5﹪、9.83及87.39﹪、3.60及97.15﹪。以2 N HCl去鈣及以Pseudomonas maltophilia 1-1發酵去蛋白質處理後，大頭紅蝦殼、紅蟳外殼、龍蝦外殼及魷魚鞘殘留物的蛋白質及幾丁質含量分別為3.25及91.40﹪、3.53及88.51﹪、13.60及81.63﹪、53.45及 43.92﹪。以P. maltophilia 1-1發酵可去除甲殼類外殼的蛋白質但無法有效去除魷魚鞘的蛋白質。以鹼處理去除甲殼類外殼的蛋白質以製備幾丁質比以微生物法有效。 為瞭解魷魚鞘的外層構造，魷魚鞘以酵素處理、胺基酸分析、以掃描式電子顯微鏡觀察外層結構、及蛋白質電泳分析。結果發現在魷魚鞘的外表似包覆一層類似膠原蛋白的物質，約佔魷魚鞘總蛋白質的22﹪。在此層似膠原蛋白質之內，有幾丁質的層狀物質（每一層厚度約1.7 μm）。一些無法被膠原蛋白酶水解但可被鹼性蛋白酶水解的楔形蛋白質(0.1 × 0.3 μm) 結合在幾丁質的層狀構造上。在幾丁質的層狀構造之間（約0.5 μm）則充滿可被鹼性蛋白酶水解及無法被鹼性蛋白酶水解也無法溶於磷酸緩衝液的蛋白質。 以化學法或生物法分離的大頭紅蝦殼及魷魚鞘幾丁質誘導C. flavigena NTOU 1 幾丁質酶活性之表現及其水解產物的情形。以 2 N NaOH在80℃ 作用去鈣蝦殼或魷魚鞘2小時後，殘留物中幾丁質含量均比以P. maltophilia 1-1發酵5天或以酵素處理分離的幾丁質含量高，但其黏度平均分子量及乙醯度則較低。未經脫色處理的大頭紅蝦幾丁質誘導C. flavigena NTOU 1 的幾丁質酶活性均較脫色處理的為高。而魷魚鞘幾丁質在誘導酵素的活性表現均比以大頭紅蝦的為低。C. flavigena NTOU 1水解大頭紅蝦幾丁質的水解產物主要以N-乙醯葡萄糖胺 (GlcNAc) 為主，其次為N-乙醯幾丁三糖〔 (GlcNAc)3〕或 N-乙醯幾丁二糖〔(GlcNAc)2〕；而水解魷魚鞘幾丁質的水解產物則以(GlcNAc)3為主，GlcNAc的量極微。以膠原蛋白酶及鹼性蛋白酶處理分離的魷魚鞘幾丁質的水解產物中，含有N-乙醯幾丁六糖〔(GlcNAc)6〕，在水解第5天時產量最高（62 mg/100 mL）。大頭紅蝦不論以鹼處理或細菌發酵去除蛋白質（SSN及SSP），以及魷魚鞘以鹼處理或酵素去除蛋白質（SPN及SPCA），再經幾丁質酶水解後產物均以GlcNAc為主，其次為 (GlcNAc)5。|
Abstract The investigation was conducted to compare the compositions in the chitin residues of crustacean shells and squid pens isolated using chemical or biological methods for deproteinization. The research also included in the dissection of chitin-containing cuticle structure of squid (Illex argentinus) pen and the activity inductions of chitinase produced by Cellulomonas flavigena NTOU 1. The protein and chitin contents in the residues of shrimp (Solenocera prominentis) shell, crab (Scylla serrata) shell, lobster (Panulirus japonicus) shell and squid pen were decalcified using 2 N HCl and deproteinized using 2 N NaOH, were 0.75 and 99.05﹪(dry base), 5.86 and 88.58﹪, 9.83 and 87.39﹪, and 3.60 and 97.15﹪, respectively. The protein and chitin contents in the residues of the shrimp shell, the crab shell, the lobster shell and the squid pen decalcified by 2 N HCl and deproteinized by the fermentation of Pseudomonas maltophilia 1-1 were 3.25 and 91.40﹪, 3.53 and 88.51﹪, 13.60 and 81.63﹪, and 53.45 and 43.92﹪, respectively. Fermentation by P. maltophilia 1-1 can deproteinize decalcified crustacean shells but is ineffective in deproteinizing decalcified squid pen. Deproteinization using alkali to isolate the chitin from crustacean shells is more effective than using enzymes or microorganisms. The cuticle structure of squid pen was investigated by enzymatic hydrolysis, amino acid analysis, scanning electron micrography observation, and electrophoresis of proteins in each enzyme(s)-treated squid pen. It is suggested that a thin layer of collagen-like substance was coated outside the pen, which comprised 22﹪of total protein in the pen. Under this layer, there was a row of chitinous lamella (1.7 μm for each lamellae). A number of cuneiform protein particles (0.1 × 0.3 μm) which could not be hydrolyzed by collagenase but could be hydrolyzed by alcalase were bound to the lamella. The spaces (0.5 μm) between each two lamella were filled with proteins. These proteins included those that could be hydrolyzed by alcalase and those that could not be hydrolyzed by alcalase nor be dissolved in phosphate buffer solution. These chitins were conducted to induce chitinases produced from C. flavigena NTOU 1 and to analysis their degraded products. Chitin content in chemically prepared residues was higher than that in biologically prepared residues, but the molecular weights and degrees of acetylation were higher in the former. Undecolorized shrimp-shell chitin could induce higher chitinase activity of C. flavigena NTOU 1 than that from decolorized chitin, and the activity induced from squid-pen chitin was lower than that from shrimp-shell chitin. By C. flavigena NTOU 1 the main hydrolytic products of shrimp-shell chitin was N-acetylglucosamine (GlcNAc), and then N-acetylchitotriose〔(GlcNAc)3〕or N-acetylchitobiose〔(GlcNAc)2〕. When squid-pen chitin was hydrolysed by C. flavigena NTOU 1, the main product was (GlcNAc)3 with trace of GlcNAc. When collagenase and alcalase deproteinized squid-pen chitin was hydrolyzed by C. flavigena NTOU 1, N-acetylchitohexaose was detected in the hydrolysate. After the treatment of commercial chitinase, the main hydrolytic products of shrimp shell or squid pen chitin was N-acetylglucosamine (GlcNAc) and (GlcNAc)5.