【作者】 陈玉娟；

【导师】 王正祥；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 硕士

【摘要】 β-1,3-1,4-葡聚糖酶（简称β-葡聚糖酶）是重要的工业酶制剂,在啤酒酿造及饲料加工等行业中有着广泛的应用。目前,国内外主要是利用微生物法生产β-葡聚糖酶。随着基因工程技术的快速发展,运用现代生物工程技术对原始菌株加以改造,通过对不同来源的β-葡聚糖酶基因进行克隆、表达以及酶学性质研究,从而获得能生产具有特殊性能的β-葡聚糖酶并且产酶量高的基因工程菌株。本研究通过基因工程手段构建了能高效分泌表达β-葡聚糖酶的重组解淀粉芽孢杆菌（Bacillus amyloliquefaciens）,并对重组酶表达情况及酶学性质进行了初步分析。主要研究结果如下:以解淀粉芽孢杆菌（CICIM B4801）染色体DNA为模板,经过PCR扩增获得β-葡聚糖酶基因（bglA）,并且以pUB110为载体,构建了含有PQ强启动子的重组表达质粒pUB-PQ-bglA,通过电转化的方法将其导入解淀粉芽孢杆菌（CICIM B4801）中,得到了能高效表达β-葡聚糖酶的重组菌B. amyloliquefaciens CICIM B4801（pUB-PQ-bglA）。该重组菌摇瓶发酵条件下的β-葡聚糖酶最高酶活为303 U/mL,是原始菌株酶活水平的11.84倍。15 L发酵罐发酵条件下胞外β-葡聚糖酶最高酶活为2023 U/mL,是摇瓶发酵条件下酶活水平的6.67倍。酶学性质研究表明,该酶的最适作用温度为55°C,最适反应pH值为6.5。温度在低于55°C条件下,酶活力保持稳定,经过70°C以上恒温处理30 min后失活率达到50%以上;β-葡聚糖酶在pH5.8-6.6条件下,酶活力保持在90%以上,pH低于5.0或高于7.0的条件下酶的失活率超过50%; Ca²⁺、K⁺、Zn²⁺和Li⁺对β-葡聚糖酶有激活作用,其中Li⁺的激活作用显著,Mn²⁺、Cu²⁺、Fe²⁺、以及EDTA都对β-葡聚糖酶的抑制作用显著,Fe³⁺、Mg²⁺和SDS对酶活力的抑制作用比较微弱。更多还原

【Abstract】 β-1,3-1,4-glucanase is an important industrial enzyme. It has been widely used in industry and agriculture, especially in brewing and feed industry. Presently,β-1,3-1,4-glucanase has mainly been produced by microbe in the country and abroad. The researches aboutβ-1,3-1,4-glucanase have been focused on constructing recombinant strains and improving the expression level and enzyme properties. The purpose of this research is to improve the expression level and properties ofβ-1,3-1,4-glucanase with the methods of molecular biology and gene engineering, and recombinant enzymes were characterized, respectively. The main studies and results in this research are as follows:A DNA fragment containingβ-1,3-1,4-glucanase structure gene bglA with its own signal peptide sequences was obtained by PCR from genomic DNA of Bacillus amyloliquefaciens CICIM B4801. The recombinant plasmid pUB-PQ-bglA, which contains promoter PQ, was constructed by inserting the amplified fragment into the cloning vector pUB110 directly. The recombinant plasmid pUB-PQ-bglA was transformed into Bacillus amyloliquefaciens （CICIM B4801） by electroporation. The bglA was overexpressed in the recombinant strain and the maximum enzyme activity was 303 U/mL, which was higher 11.84 times than the original strain. The highest extracellular engzym activity ofβ-1,3-1,4-glucanase reached up to 2023 U/mL with fermentator, which improved 6.67 times compared to flask fermentation for recombinant strain.The characterization researchs show that the optimal pH and temperature of the enzyme was 6.5 and 55°C, respectively. The isolate B. amyloliquefaciens CICIM B4801 （pUB-PQ-bglA） showed high stability ofβ-1,3-1,4-glucanase activity at the pH and temperature of 5.8-6.6 and 40-55°C, respectively. Theβ-1,3-1,4-glucanase activity was improved more or less by the addition of some ions such as Ca²⁺, K⁺, Zn²⁺ and Li⁺, and was significant inhibited by the addition of Mn²⁺, Cu²⁺, Fe²⁺ and EDTA.更多还原