低温β-半乳糖苷酶分离纯化及酶学性质研究  

Purification and characterization of a novel cold-adapted β-galactosidase

作　　者：张雪[1] 华霄[1] 许琪 杨瑞金[1] 范宇婷[1] 

机构地区：[1]江南大学食品学院,江苏无锡214122 [2]云南省中小企业服务中心,云南昆明650000

出　　处：《食品与机械》2014年第3期174-178,共5页Food and Machinery

基　　金：国家自然科学基金重点项目(编号:31230057);十二五国家科技支持计划课题(编号:2011BAD23B03)

摘　　要：对来自新疆天山冻土的Rahnella sp．R3所产的胞内低温β-半乳糖苷酶进行纯化，并对其酶学性质进行研究。采用硫酸铵分级沉淀、Phenyl Sepharose CL-4B疏水层析、QSepharose High Performance阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析，得到电泳纯酶。酶的活性回收率为21．3％，纯化倍数为35．6，比酶活由1．28U／mg提高到45．54U／mg。SDS—PAGE电泳显示其表观分子量为57．3kDa。酶学性质研究表明，该酶最适反应温度为45℃，在15℃时的酶活为最高酶活的40％，4℃时的酶活为最高酶活的23％。该酶对热敏感，45℃保温45min酶活全部丧失。纯酶的最适PH为7．0，在pH6．5～7．5时保持稳定。5mmol／LNa^＋、Ca^2＋、Cu^2＋、Al^3＋、Zn^2＋对酶活力有不同程度的抑制作用，其中Al^3＋抑制K^＋作用最强，Na^＋、Ca^2＋抑制作用不明显。5mmol／LMg^2＋、K^＋对酶活力具有促进作用，其中Mg^2＋促进作用较强，使酶活提高到1．19倍。25℃以ONPG为底物的Vmax，Km值分别为7．19mol／（min·mL）、4．64mmol／L。A novel cold-adapted β-galactosidase was isolated from Rahnella sp. R3, living permafrost of Tiansan Mount in Xinjiang province and the enzymatic properties were investigated. The enzyme was purified in sequence by ammonium sulfate precipitation, hydrophobic chromatography, Q Sepharose High Performance and Sephacryl S-200 High Resolution and proved by the single band in SDS-PAGE. The enzyme was purified by 35.6 folds with a yield of 21.3% and Mw of 57.3 kDa. This β-galactosidase was maximally active at 45 ℃ and pH 6.5-7.5. Enzyme recovery was 400/00 at 15 ℃ and 23% at 4 ℃, respectively. It was thermal-sensitive and undetect-able after incubation at 45 ℃ for 45 min. Some metal ions such as Na^＋ , Ca^2＋ , Cu^2＋ , and Znz＋ reduced enzyme activity. Al^3＋ strongly inhibited enzyme activity. Whereas, Mg^2＋ and K^＋ activated enzyme. Km and Vmax, for ONPG at 25 ℃ was found to be 4.64 mmol/L and 7.19 mol/（min · mL）, respectively.

关 键 词：低温β-半乳糖苷酶 拉恩氏菌 酶纯化 酶学性质 

分 类 号：TS201.2[轻工技术与工程—食品科学]