节杆菌烟碱脱氢酶性质及应用研究-食品科学与工程专业论文.docxVIP

摘 摘 要 烟碱脱氢酶可以广泛应用于烟草行业中。本论文筛选和鉴定了一株能高效降解烟碱的节杆 菌(Anthrobacter sp)，研究了烟碱脱氢酶的产酶条件，进而对烟碱脱氢酶进行了纯化和酶学性 质的研究。另外，还对烟碱脱氢酶在烟草薄片中的应用进行了初步研究。论文的主要内容和结 论如下： 通过平板筛选和摇瓶复筛。从土壤中筛选出一株能高效降解烟碱的菌株，根据16S rRNA 序列分析、培养特征及生理生化特征分析，该菌株鉴定为节杆菌(Arthrobacter sp)。经摇瓶实 验，Arthrobactersp．的较好的发酵产酶条件为：初始pH7．0，通气条件(摇床转速)220r／min， 接种量5％，30C下能进行良好发酵。其最优培养基组成为： (NH4)2S04 19／L；酵母粉19／L； Nicotine 4ml／L；无机盐：Mgs04．7H2 O．02％、MnS04．4H20 0．004％、CaCl2．2H20 0．002％、 K2HPO一．．3H20 13．39／L、KH2P04 49／L。 烟碱脱氢酶粗酶液经硫酸铵沉淀，离子交换柱层析分离和凝胶过滤层析分离纯化，得到了 电泳纯的烟碱脱氢酶。分离纯化过程中酶被提纯了44．67倍，酶活得率为6．57％。烟碱脱氢酶 的最适反应nH为6．85左右，在pH5．5-8．0范围内都很稳定。烟碱脱氢酶的最适温度为40(2。 各种金属离子中，Mn2+、c02+能提高酶活力，Cu2+、Fe“、ca2+对酶活力有抑制作用。Na+离子 对酶活力影响不丈。螫合剂EDTA能提高一定的酶活力，比对照样品提高了l 8％。 在温度30C、物水比1：10、pH7．0时用水溶液浸提烟末8小时后，烟末中的烟碱提取率 较高。在烟草薄片提取液中接入6％的节杆菌z3的菌液，306C培养48小时后，烟碱含量下降了 31．15％。在烟草薄片提取液中加入2％的烟碱脱氢酶酶液，40℃下反应16小时后，烟碱含量卜 降了50．58％。在烟草薄片中加入2％的经过烟碱脱氢酶处理的薄片提取液，可明显提高烟草薄片 的可用性。 关键词：节杆菌烟碱脱氢酶烟碱降解烟草薄片 ABSTRACTNicotine ABSTRACT Nicotine dehydrogenase have attracted considerable research interest in recent years because its potential application in the tobacco industry． In the paper，Nicotine dehydrogenase producing strains were isolated from soil by screening with nicotine as the sole carbon souFce，and among them a high nicotine degradation strain was selected by experiments in shake flasks．By the experimental results of the morphological，physiological and structural characteristics of bacterium s自rain and by the comparison of its whole sequence of 16s rRNA with that of the type strains of all knownArthrobacter in genebank，it was showed that the 16s rRNA of bacterium strain and that of Arthrobacter was 97．8％homogenous．Themfore bacterium strain was named asArthrobacter sp．． The effects of different factors on nicotine dehydrogenase production by Arthrobacter sp．were studied in shake flask condition．The better activity of nicotine dehydrogenase is reached after 48h of culturing in rotary shacker(220r／min)at 30(2，initial pH 7．0，4％inoculum culture． Nicotine dehydrogenase was purified fro