唾液乳杆菌XH4B的α-半乳糖苷酶纯化及性质研究  

Purification and Catalyst Kinetics of α-galactosidase Derived from Lactobacillus salivarius XH4B

作　　者：杜新永[1] 刘同杰[1] 高世阳[1] 何国庆[1] 

机构地区：[1]浙江大学生物系统工程与食品科学学院,浙江省食品微生物技术重点实验室,浙江杭州310058

出　　处：《现代食品科技》2014年第1期137-142,184,共7页Modern Food Science and Technology

基　　金：国家自然科学基金项目(31130042);十二五国家科技支撑计划项目(2012BAD37B01)

摘　　要：本研究通过超滤、SephedaxG200凝胶过滤层析纯化来源于唾液乳杆菌XH4B（GeneBank索引号：JXl25456）的a-半乳糖苷酶，并对其酶学性质进行了研究。结果表明，超滤时采用100kDa的滤膜，分子量大于100kDa的组分有酶活。利用SephadexG200进行柱层析洗脱至370-400min时得到的组分表现出明显的a-半乳糖苷酶活力。SDS．PAGE电泳结果表明，该酶的蛋白质单体分子量为70-80kDa，未变性的酶蛋白应为多聚体，总分子量大于100kDa。利用酶比活力计算的结果，相对于粗酶，超滤纯化效率为149．80％，柱层析纯化效率为391．91％。经响应面优化，确定唾液乳杆菌XH4B来源的a-半乳糖苷酶最佳酶促反应条件为：柠檬酸缓冲液pH值5．5，离子强度0．15mol／L，反应温度52℃。该酶对pNPG的Km值为0．817。相对于纯水，各类金属离子中，仅有心和Na＋对酶活力有正向的激活作用，酶活力分别达到了102．50％和104．18％。EDTA（96．54％），Mg”（91．53％），ca2＋（82．51％），以及DTT（79．38％）能够较大限度保留酶活力，而Zn2＋、cu2＋、pb2＋、Fe3＋和vc则显著阻碍了酶促活力，仅能保留5-7％。In this study, ct-galactosidase was derived fi＇om Lactobacillus salivarius XH4B （GeneBank accession number： JX1125456）, and purified by super filtration and size exclusive chromatography of Sephedax G200. The results showed that the compounds with molecular weight （Mw） more than 100 kDa had enzymatic activity and those washing constituents of 370--400 min showed significant activity. The SDS-PAGE appeared that the Mw of the monomer of the enzyme was 70-80 kDa, while the native enzyme was a polymer with a Mw exceeded 100 kDa. Compared with the crude enzyme, the purification efficiencies of super filtration and column chromatography were 149.80% and 391.91%, respectively. The optimal catalyst conditions determined by responding surface method were citric buffer pH 5.5, ion strength 0.15 mol/L and reaction temperature 52 ℃ The Km value of the enzyme to pNPG was 0.817. Compared to pure water system, only K and Na＋ could improve the catalyst activity to 102.50% and 104.18% respectively. EDTA （96.54%）, Mg2＋ （91.53%）, Ca2＋ （82.51%）, and DTT （79.38%） could reserve most of the activity, while Zn2＋, CU2＋, Pb2＋, Fe3＋ and Vc could only remain 5-7% activity.

关 键 词：乳酸菌 唾液乳杆菌 a-半乳糖苷酶 超滤 凝胶过滤 酶挚性质 

分 类 号：TS201.25[轻工技术与工程—食品科学]