本文選題：甲殼素酶 + 分離純化　； 參考：《催化學報》2017年04期

【摘要】：甲殼素,又名幾丁質(zhì)(chitin),是自然界中含量僅次于纖維素的第二大天然多糖,有第六生命要素之美稱.其主要存在于甲殼類動物的外殼、真菌細胞的細胞壁以及一些昆蟲的外殼中,每年自然界中約有100多億噸甲殼素生成.甲殼素是由2-乙酰氨基-2-脫氧-D-吡喃葡萄糖和2-氨基-2-脫氧-D-吡喃葡萄糖通過β-1,4糖苷鍵連接而成的二元線性聚合物,分子鏈中分布許多羥基、氨基及乙酰氨基,形成大量分子間及分子內(nèi)氫鍵,致使其結晶度較高,化學性質(zhì)十分穩(wěn)定,直接利用較為困難.甲殼素不溶于稀酸、稀堿以及一般有機溶劑,工業(yè)上常用強酸強堿法處理甲殼素,以制備殼寡糖類產(chǎn)品,但該方法具有產(chǎn)品結構不單一,環(huán)境污染較為嚴重等缺點.甲殼素酶可特異性水解甲殼素鏈中β-1,4糖苷鍵,得到甲殼寡糖和乙酰氨基葡萄糖.酶解法降解甲殼素工藝簡單、反應條件溫和、環(huán)境友好,有很好的應用前景.我們以Paenibacillus pasadenensis CS0611為出發(fā)菌株,以蟹殼粉末為培養(yǎng)基唯一碳源及氮源,在適宜條件下培養(yǎng)48h.發(fā)酵液經(jīng)離心、硫酸銨(80%飽和度)鹽析、透析除鹽后得到粗酶液.再利用HiTrap DEAE FF離子交換層析和HiLoad 26/600Superdex 200pg凝膠過濾層析對該粗酶液進行分離純化,以得到電泳純甲殼素酶.所制備甲殼素酶比活力為10.28U/mg,最終純化倍數(shù)為5.3,酶活得率為15.7%.SDS-PAGE結果表明,該甲殼素酶相對分子質(zhì)量約為69 kDa.后經(jīng)MALDI-TOF-MS鑒定,該酶部分肽段和來源于另一株Paenibacillus pasadenenss的甲殼素酶(accession No:gi655151624)具有較高的同源性,進一步證實所純化蛋白為甲殼素酶.對上述純化的甲殼素酶的酶學性質(zhì)進行研究,結果發(fā)現(xiàn):其最適反應溫度為50℃,在20-35℃內(nèi)有較好的穩(wěn)定性,50℃及以上熱穩(wěn)定性較差;最適pH為5.0,在pH4.0-11.0間具有較高穩(wěn)定性,表明該酶具有很好的耐堿性;金屬離子對該酶催化活性沒有明顯的激活作用,表明該甲殼素酶是非金屬酶.同時,對該酶的底物特異性進行研究,發(fā)現(xiàn)該酶對膠體甲殼素和甲殼素水解能力較強,對淀粉和纖維素無水解能力,對不同脫乙酰度的殼聚糖的水解程度隨脫乙酰度不同而變化,表明該酶只能特異性識別并降解GlcNAc-GlcNAc之間的糖苷鍵;以膠體甲殼素為底物時,米氏常數(shù)K_m為4.41 mg/mL,最大反應初速度為1.08 mg/min.利用薄板層析和高效液相色譜對酶解產(chǎn)物進行分析,結果表明該甲殼素酶對膠體甲殼素的降解產(chǎn)物主要是(G1cNAc)_2.綜上所述,本研究所涉甲殼素酶在甲殼二糖的酶法制備方面具有較好的應用前景.
[Abstract]:Chitin, also known as chitin, is the second largest natural polysaccharide in nature after cellulose. It mainly exists in the shell of crustaceans, the cell walls of fungal cells and the shells of some insects. About 10 billion tons of chitin is produced in nature every year. Chitin is a binary linear polymer composed of 2-acetylamino-2-deoxy-Dpyranose and 2-amino-2-deoxy--Dpyranose, which is linked by 尾 -1t4 glucoside bond. Many hydroxyl, amino and acetyl amino groups are distributed in the molecular chain. A large number of intermolecular and intramolecular hydrogen bonds were formed, which resulted in high crystallinity, stable chemical properties and difficult direct utilization. Chitin is insoluble in dilute acid, dilute alkali and general organic solvent. Chitin is usually treated with strong acid and strong base in industry to prepare chitosan oligosaccharides. However, this method has many disadvantages, such as product structure is not single, environment pollution is serious, etc. Chitosan oligosaccharide and acetyl glucosamine could be obtained by hydrolyzing 尾-1 / 4 glycoside bond in chitin chain by chitin enzyme. The degradation of chitin by enzymatic hydrolysis is simple, the reaction conditions are mild, and the environment is friendly, so it has a good prospect of application. Paenibacillus pasadenensis CS0611 was used as the starting strain and crab shell powder as the sole carbon and nitrogen source for 48h. After centrifugation, ammonium sulfate 80% saturation) salting out, dialysis desalination to obtain crude enzyme liquid. The crude enzyme was purified by HiTrap DEAE FF ion exchange chromatography and HiLoad 26 / 600 Superdex 200pg gel filtration chromatography. The specific activity of chitin was 10.28 U / mg, the final purification multiple was 5.3, and the yield of the enzyme was 15.7.SDS-PAGE. The results showed that the relative molecular weight of the chitin enzyme was about 69 kDa. By MALDI-TOF-MS, some peptides of the enzyme and a chitin enzyme from another strain of Paenibacillus pasadenenss were identified by MALDI-TOF-MS with high homology. It was further confirmed that the purified protein was chitin enzyme. The enzymatic properties of the purified chitin enzyme were studied. The results showed that the optimum reaction temperature was 50 鈩，

本文編號：1993243