一株产卡拉胶酶南极菌Alteromonas sp.R11-5的筛

发布时间：2018-05-13 08:36

  本文选题：南极菌 + 卡拉胶酶　； 参考：《青岛大学》2017年硕士论文

【摘要】：本论文从85株南极细菌中筛选获得12株具有产卡拉胶酶活性的菌株,并对高活性菌株R11-5进行种属鉴定,优化其发酵条件;通过对该菌全基因组测序分析,克隆获得卡拉胶酶基因Car1853,并实现其异源高效表达;纯化重组卡拉胶酶,系统研究其酶学性质,分析该酶的降解特性,阐明其降解产物及其类型,以期为卡拉胶酶和卡拉寡糖的工业化生产提供理论和技术支持。本实验首先对筛选出的产高活性卡拉胶酶菌株R11-5进行种属鉴定,根据形态学和16S rDNA法的种属鉴定结果表明该南极菌属于交替单胞菌属(Alteromonas)。通过响应面分析法对菌株优化培养单因素实验的结果最佳培养条件为温度15.7℃、p H7.0、酵母粉0.6%、牛肉膏1.17%、卡拉胶1.05‰、CaCl25.78 mmol/L,接种量为2%,在此培养条件下,能够得到理论上最大酶活值为56.972 U·mL-1,经过优化培养后酶活提高了1.7倍。通过对该菌全基因组测序分析,克隆获得了卡拉胶酶基因Car1853,采用基因工程手段构建重组质粒pET30a-Car1853,,并成功转化大肠杆菌BL-21。采用Ni柱技术纯化获得单一条带的重组卡拉胶酶,SDS-PAGE显示其分子量为42 kDa;酶学性质研究表明,该酶最佳反应温度为55℃,最佳pH为7.0,且多种金属离子对其活性具有抑制作用。薄层层析法初步表明该卡拉胶酶裂解卡拉胶的终产物主要是卡拉胶二糖。
[Abstract]:In this paper, 12 strains with carrageenase-producing activity were obtained from 85 Antarctic bacteria, and the strain R11-5 with high activity was identified to optimize the fermentation conditions, and the whole genome of R11-5 was sequenced. The carrageenase gene Car1853 was cloned, and its heterologous expression was achieved. The recombinant carrageenase was purified, its enzymatic properties were systematically studied, the degradation characteristics of the enzyme were analyzed, and its degradation products and types were elucidated. In order to provide theoretical and technical support for the industrial production of carrageenase and carrageenase. In this experiment, the strain R11-5 producing high activity carrageenase was first identified. According to the morphological and 16s rDNA identification results, the Antarctic strain belongs to Alteromonas alternata. The results of single factor experiment with response surface analysis were as follows: temperature 15.7 鈩，

本文编号：1882436