产β-木糖苷酶Enterobacter cloacae LY-62的筛选分离、发酵优化及其木糖苷酶基因的克隆表达

发布时间：2018-02-01 18:56

  本文关键词： β-木糖苷酶 Enterobacter cloacae 发酵优化 克隆表达　出处：《华南理工大学》2016年硕士论文　论文类型：学位论文

【摘要】：半纤维素作为应用潜力巨大的可再生资源,其主要成分是木聚糖。木聚糖的完全降解需要组成复杂的木聚糖降解酶系。β-木糖苷酶是木聚糖降解酶系的关键酶,其酶催化功能是从低聚木糖和木二糖的非还原性末端开始水解而产生木糖单糖。研究β-木糖苷酶对于半纤维素的降解应用意义重大。本文以从森林土壤样品中筛选出的一株高产β-木糖苷酶菌株为基础,测定发酵产酶的最高酶活力值和相应比酶活力值分别为1.29 U/mL和1.05 U/mg,并通过测定胞外液和胞内液确定该菌所产β-木糖苷酶为胞内酶。经16S rDNA系统进化树分析、形态学观察和生理生化实验鉴定,确定该菌株为Enterobacter cloacae,并命名为Enterobacter cloacae LY-62。以初始发酵培养条件为基础,对Enterobacter cloacae LY-62进行发酵产酶条件的初步优化,考察不同碳源及其浓度、不同氮源及其浓度、培养基起始pH值和培养温度对发酵产酶的影响。经优化发酵条件后,Enterobacter cloacae LY-62发酵产酶水平最高可达到的酶活力值和相应比酶活力值分别为2.2 U/mL和2.87 U/mg,产酶水平提高了170%左右。研究优化发酵条件下该菌的产酶曲线和生长曲线,发现该菌株具有生长快速、木聚糖利用效率高、产酶快和酶活力高的特点。为进一步研究Enterobacter cloacae LY-62的β-木糖苷酶,设计引物成功对其β-木糖苷酶基因进行了克隆表达以及功能基因的分子鉴定。以该酶基因序列和氨基酸序列为基础,应用生物信息学方法获得了其蛋白质氨基酸组成、疏水性和相对分子量等一级结构的相关信息,并对其二级结构元件和蛋白质的三级空间立体结构进行预测。随后,对Enterobacter cloacae LY-62的β-木糖苷酶进行表达,重组蛋白的N端携带组氨酸标签,重组蛋白His-Xyl62经镍柱亲和层析纯化后进行酶活性测定并研究其酶学特性:该β-木糖苷酶的最适pH值为7.0,酶活性在弱酸性和弱碱性的条件下较稳定;最适酶反应温度为40℃,热稳定性相对较差;以PNPX为底物测定的His-Xyl62的米氏常数Km、最大反应速率Vmax和催化速度常数Kcat分别为1.55 mmoL/L、38.61 umoL/(min·mg)和8.51 s~(-1)。
[Abstract]:Hemicellulose is a renewable resource with great application potential. The main component is xylan. The complete degradation of xylan requires the formation of complex xylanase system. 尾 -xylosidase is the key enzyme of xylan degradation enzyme system. The catalytic function of 尾 -xylosidase is to hydrolyze xylose and xylose from the non-reducing end of xylose and to produce xylose monosaccharide. It is important to study the application of 尾 -xylosidase in hemicellulose degradation. A strain with high 尾 -xylosidase production was screened. The highest enzyme activity and the corresponding specific enzyme activity were 1.29 U / mL and 1.05 U / mg, respectively. The 尾 -xylosidase produced by the strain was identified as intracellular enzyme by measuring the extracellular fluid and intracellular fluid. The phylogenetic tree analysis of 16s rDNA, morphological observation and physiological and biochemical experiments were performed to identify the 尾 -xylosidase. The strain was identified as Enterobacter cloacae. And named Enterobacter cloacae LY-62.Based on the initial fermentation conditions. The fermentation conditions of Enterobacter cloacae LY-62 were optimized, and different carbon sources and their concentrations, different nitrogen sources and their concentrations were investigated. The effects of the initial pH value and the culture temperature on the fermentation enzyme production were studied and the fermentation conditions were optimized. The highest enzyme activity and corresponding specific enzyme activity of Enterobacter cloacae LY-62 were 2.2 U / mL and 2.87 U / mL, respectively. U/mg. The enzyme production level was increased by about 170%. The enzyme production curve and growth curve of the strain were studied under optimized fermentation conditions. It was found that the strain had rapid growth and high Xylan utilization efficiency. In order to further study the 尾 -xylosidase of Enterobacter cloacae LY-62. The 尾 -xylosidase gene was cloned and expressed successfully with primers, and the functional gene was identified based on the sequence and amino acid sequence of the 尾 -xylosidase gene. Bioinformatics was used to obtain the information about the amino acid composition, hydrophobicity and relative molecular weight of the protein. The third order spatial stereoscopic structure of its secondary structure element and protein was predicted. Then, the 尾 -xylosidase of Enterobacter cloacae LY-62 was expressed. The N-terminal of the recombinant protein carries a histidine label. The recombinant protein His-Xyl62 was purified by nickel column affinity chromatography and its enzymatic properties were studied. The optimum pH value of the 尾 -xylosidase was 7.0. The enzyme activity was stable under the condition of weak acidity and weak alkalinity. The optimum reaction temperature is 40 鈩，

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