【作者】 俞玲；

【导师】 王正祥；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 硕士

【摘要】 地衣芽孢杆菌(Bacillus licheniformis)是芽孢杆菌中最具应用潜力的菌种之一,具有酶系丰富,产酶量高等诸多优良特性。地衣芽孢杆菌基因组序列已于2004年被公布,为利用基因工程手段开发其应用潜力提供了基础。本文从地衣芽孢杆菌基因组DNA中分别克隆了被标注为pelB、amyX和yvdF的基因,在大肠杆菌等不同宿主中表达出有活性的重组酶并对重组酶表达情况及酶学性质进行了初步分析。对地衣芽孢杆菌基因组序列分析后发现有两条基因可能编码果胶酶,分别被标注为pelA和pelB,其中对pelB基因的研究迄今未见报道。本文从地衣芽孢杆菌CICIM B1699染色体DNA中扩增出可能编码果胶裂解酶的基因pelB,插入启动子PQ下游,分别构建了重组质粒pLa-PQ-pelB和pHY-PQ-pelB。重组大肠杆菌JM109 (pLa-PQ-pelB)表达出有活性的果胶裂解酶,酶活力为0.6 U/mL,上述实验证明了pelB基因编码的蛋白具有果胶裂解酶的功能。重组酶的酶学性质显示其最适反应温度为45℃,最适pH为10.5。金属离子Ca2+对重组酶具有激活作用,其中6 mmol/L Ca2+浓度激活作用最为明显,而Mg2+、Zn2+、Cu2+能强烈抑制酶活。利用HPLC-ESI-MS对重组酶酶解产物进行测定发现,酶解产物含有不饱和二聚半乳糖醛酸和不饱和三聚半乳糖醛酸。将重组质粒pHY-PQ-pelB导入地衣芽孢杆菌CICIM B1699,获得重组菌后进行发酵实验,发酵液中果胶裂解酶酶活达到4.2 U/mL,与原始菌株相比产酶水平提高了60%。对地衣芽孢杆菌14580基因组序列分析显示基因组中有两个基因amyX和yvdF,其编码的蛋白均具有典型的普鲁兰酶保守区,而有关其基因功能的研究均未见实验研究报道。以地衣芽孢杆菌CICIM B1699染色体DNA为模板通过PCR方法扩增获得amyX和yvdF编码区,分别构建了表达载体pHY-P43-amyX和pHY-P43-yvdF。含重组质粒pHY-P43-amyX的重组枯草芽孢杆菌1A717细胞内酶活有明显提高,但细胞外没有检测到明显酶活,该基因可能编码胞内普鲁兰酶。重组枯草芽孢杆菌1A717 (pHY-P43-yvdF)表达出有活性的胞外普鲁兰酶,发酵液中酶活力达到4.6 U/mL,对其重组酶水解性质分析显示该酶可能为I型普鲁兰水解酶。重组酶酶学性质分析显示该两种重组酶的最适反应温度都为45℃,最适pH为6.5,其中重组酶AmyX在30-80℃热稳定性较好,而重组酶YvdF在55℃以上时迅速失活。重组酶反应不需要金属离子的参与,但是很多离子如Mg2+、Mn2+、Li+、Na+对酶活性有不同程度的促进作用;Zn2+、Cu2+、Co2+、EDTA、SDS对该酶有不同程度的抑制作用。将重组质粒pHY-P43-yvdF导入地衣芽孢杆菌CICIM B1699,测得重组菌的发酵上清液酶活力约为6.8 U/mL,较原始对照菌株B. licheniformis CICIM B1699酶活力水平高出2倍。更多还原

【Abstract】 B. licheniformis was thought an industrial organism which has such good characters as anti-heat, nonpathogenic, abundant enzyme system. The genome sequence of B. licheniformis was published in 2004, which provided an important material to the development of its potential applications. In this paper, cloning and expression of the gene pelB, amyX and yvdF in different hosts were investigated and ecombinant enzymes were characterized, respectively.A complete structure gene pelB which probably encoded pectinase was cloned from B. licheniformis CICIM B1699 by polymerase chain reaction. The recombinant plasmids pLa-PQ-pelB and pHY-PQ-pelB were constructed with pelB under the control of PQ promoter. The pectinase was successfully expressed in Escherichia coli JM109 via the mediation of plasmid pLa-PQ-pelB. The optimal pH and temperature of recombinant pectinase were pH 10.5 and 45℃. The activity of enzyme was enhanced by Ca2+ and strongly inhibited by ions of Mg2+, Zn2+ and Cu2+. Under the different concentration of Ca2+, the optimum concentration was 6 mmol/L. The products from polygalacturonic acid degraded by enzyme were analyzed by electrospray ionization mass spectrometry (ESI-MS). Unsaturated bigalacturonic acid and unsaturated trigalacturonic acid were found. B. licheniformis CICIM B1699 carrying pHY-PQ-pelB produced 4.2 U/mL pectinase, which was about 60% higher than that of parent strain.Analysis of the complete genome sequence of B. licheniformis 14580 revealed that there were two genes amyX and yvdF, which probably encoded pullulanase. The recombinant plasmids of pHY-P43-amyX and pHY-P43-yvdF were constructed and expressed in Bacillus subtilis 1A717. The AmyX as intracellular enzyme were detected and the YvdF showed the enzyme activity of 4.6 U/mL in B. subtilis 1A717. The hydrolytic characters of YvdF were observed that the recombinant belonged to type I pullulan hydrolases. The enzymatic properties of the recombinant enzymes were analyzed systemically in the study. Pullulan hydrolysis activity was optimal at pH 6.5 and 45℃. Metal ions were needless in the enzymatic reaction, however, the pullulanase activity was improved more or less by the addition of some ions such as Mg2+、Mn2+、Li+ and Na+, and was inhibited by the addition of Zn2+、Cu2+、Co2+、EDTA and SDS. The recombinant B. licheniformis CICIM B1699 with pHY-P43-yvdF produced 6.8 U/mL, which was 2-fold higher than that of the control strain.更多还原