来源于类芽孢杆菌属碱性甲壳素酶的分离纯化及其性质（英文）

发布时间：2018-06-07 23:18

  本文选题：甲壳素酶 + 分离纯化　； 参考：《催化学报》2017年04期

【摘要】：甲壳素,又名几丁质(chitin),是自然界中含量仅次于纤维素的第二大天然多糖,有第六生命要素之美称.其主要存在于甲壳类动物的外壳、真菌细胞的细胞壁以及一些昆虫的外壳中,每年自然界中约有100多亿吨甲壳素生成.甲壳素是由2-乙酰氨基-2-脱氧-D-吡喃葡萄糖和2-氨基-2-脱氧-D-吡喃葡萄糖通过β-1,4糖苷键连接而成的二元线性聚合物,分子链中分布许多羟基、氨基及乙酰氨基,形成大量分子间及分子内氢键,致使其结晶度较高,化学性质十分稳定,直接利用较为困难.甲壳素不溶于稀酸、稀碱以及一般有机溶剂,工业上常用强酸强碱法处理甲壳素,以制备壳寡糖类产品,但该方法具有产品结构不单一,环境污染较为严重等缺点.甲壳素酶可特异性水解甲壳素链中β-1,4糖苷键,得到甲壳寡糖和乙酰氨基葡萄糖.酶解法降解甲壳素工艺简单、反应条件温和、环境友好,有很好的应用前景.我们以Paenibacillus pasadenensis CS0611为出发菌株,以蟹壳粉末为培养基唯一碳源及氮源,在适宜条件下培养48h.发酵液经离心、硫酸铵(80%饱和度)盐析、透析除盐后得到粗酶液.再利用HiTrap DEAE FF离子交换层析和HiLoad 26/600Superdex 200pg凝胶过滤层析对该粗酶液进行分离纯化,以得到电泳纯甲壳素酶.所制备甲壳素酶比活力为10.28U/mg,最终纯化倍数为5.3,酶活得率为15.7%.SDS-PAGE结果表明,该甲壳素酶相对分子质量约为69 kDa.后经MALDI-TOF-MS鉴定,该酶部分肽段和来源于另一株Paenibacillus pasadenenss的甲壳素酶(accession No:gi655151624)具有较高的同源性,进一步证实所纯化蛋白为甲壳素酶.对上述纯化的甲壳素酶的酶学性质进行研究,结果发现:其最适反应温度为50℃,在20-35℃内有较好的稳定性,50℃及以上热稳定性较差;最适pH为5.0,在pH4.0-11.0间具有较高稳定性,表明该酶具有很好的耐碱性;金属离子对该酶催化活性没有明显的激活作用,表明该甲壳素酶是非金属酶.同时,对该酶的底物特异性进行研究,发现该酶对胶体甲壳素和甲壳素水解能力较强,对淀粉和纤维素无水解能力,对不同脱乙酰度的壳聚糖的水解程度随脱乙酰度不同而变化,表明该酶只能特异性识别并降解GlcNAc-GlcNAc之间的糖苷键;以胶体甲壳素为底物时,米氏常数K_m为4.41 mg/mL,最大反应初速度为1.08 mg/min.利用薄板层析和高效液相色谱对酶解产物进行分析,结果表明该甲壳素酶对胶体甲壳素的降解产物主要是(G1cNAc)_2.综上所述,本研究所涉甲壳素酶在甲壳二糖的酶法制备方面具有较好的应用前景.
[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，

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