【作者】 刘文玉；

【导师】 娄恺；

【作者基本信息】 石河子大学 ， 农产品加工及贮藏工程， 2008， 硕士

【摘要】 从新疆低温环境中筛选到一株可产低温乳糖酶的菌株14-1,利用形态学方法,生理生化实验、16S rDNA基因序列同源性比对及系统发育树构建对菌株进行鉴定,结果显示该菌属于水生拉恩氏菌（Rahnella aquatilis）,命名为Rahnella aquatilis sp.14-1。对低温乳糖酶产生菌Rahnella aquatilis sp.14-1进行了摇瓶条件下产低温乳糖酶的研究,确定了摇瓶最佳发酵工艺:乳糖含量为2.5%,接种量3%,培养基初始pH为6.5,装液量为在500 mL三角瓶装液体培养基100 mL。20℃,200 r·min^-1摇床培养48 h,14^-1菌产低温乳糖酶活性4.51 U·mL^-1。Rahnella aquatilis14-1 sp.乳糖酶为胞内酶,将菌体用超声波破壁提取粗酶液,用硫酸铵分级沉淀,Sephadex G-25和DEAE-Cellulose柱层析进一步纯化。经过纯化的酶用SDS-PAGE检验其纯度,结果为电泳单条带,该酶的单亚基表观分子量约为60 KDa。酶的纯化倍数为4.19,回收率25%。Rahnella aquatilis sp.14-1乳糖酶的酶学性质研究表明,以ONPG为底物,酶的最适反应温度为35℃,对热敏感,45℃保温2 h,酶完全失去活力;最适反应pH值为6.5～7.0,在pH值低于5和高于9酶不稳定。。金属离子Mn2+对酶有激活作用,活力提高了19.8%。而Cu²⁺、Zn²⁺、Fe³⁺、Al³⁺离子对酶活力有明显的抑制作用,其活力分别减少了94.35%、92.76%、71.98%和88.93%。K⁺、Li⁺、Mg²⁺、Na⁺和Ca²⁺对酶活力的影响很小。Rahnella aquatilis sp.14-1乳糖酶水解牛乳中的乳糖,35℃水解2 h,乳糖水解率达到80%以上。更多还原

【Abstract】 One cold-adapted lactase producing strain 14-1 was isolated from its low temperature environment in XINJINAG province. The strain was identified by studying its characteristics of morphology, cultivation, physiology and biochemistry and 16S rDNA sequence alignment. The results showed that the strain belongs to Rahnella aquatilis.Shake-flask culture conditions of cold-adapted lactase were studied with psychrotrophic strain14-1. The fermentation conditions were optimized: lactose concentration 2.5%, initial pH 6.5, seed inoculation volume 3%, 500 mL flask contained 100mL medium. The maximumβ-Galactosidase activity, about 4.51 U·mL^-1 was obtained at 20℃when cultured for 48 hours.The lactase was intracellular enzyme,so the cell pellets were disrupted with a sonfier sonicator.The crude enzyme was purified by column chromatography and a single band was obtained on SDS-PAGE. Apparent molecular weight of subunits of the purified enzyme estimated by SDS-PAGE were about 60 KDa.The specific activity of crude enzyme was increase up to 321.7 U·mg^-1 with purifying fold of 4.19 and the yield was 25%.Research about the properties of the enzyme showed that the optimal temperature was 35℃, and the optimal pH value was 6.5 to 7.0 for the enzyme with ONPG as a substrate. The enzyme activity was sensitive to temperatures above 40℃and was undetectable after 2h incubation at 45℃. The metal ions Mn²⁺, K⁺ activated the enzyme activity while Cu²⁺, Zn²⁺ , Fe³⁺ and Al³⁺ inhibited activity.When milk was treated with the lactase at 35℃for 2 hours, the degree of hydrolysis for lactose can reached 80%.更多还原