摘要
从黑曲霉发酵液中经硫酸铵分级沉淀,Phenyl-Sepharose疏水柱层析,DEAE-Sepharose 4B阴离子交换柱得到电泳纯的脂肪酶,纯化倍数达10倍,回收率50%。对脂肪酶的性质分析表明:该酶分子质量约为35 kDa,最适温度和最适pH分别为37℃和9.5,50℃以下和pH 6.0～11.0之间保持稳定,属于碱性脂肪酶。Mg^(2+)、Ca^(2+)、Cu^(2+)、Zn^(2+)、Co^(2+)、Mn^(2+)对该酶有激活作用,而Al^(3+)、Fe^(2+)、Fe^(3+)对酶有严重抑制作用。变性剂盐酸胍和脲对其未见显著影响,而SDS强烈抑制其酶活。用不同氨基酸修饰剂对酶进行修饰,其中NBS和PMSF强烈抑制该酶活性,NBSF和DTT在低浓度下对酶活影响不大,2,3-丁二酮在高浓度下影响其活性。外加稳定剂如NaCl、PEG、甘油、山梨醇、海藻酸钠,均可不同程度的延长脂肪酶的半衰期。在一定质量比条件下,该酶有良好的抗蛋白酶性质。
Lipase from Aspergillus niger was purified approximately 10-fold with 50 % final yield using ammonium sulfate graded precipitation, hydrophobic interaction chromatography （phenyl-sepharose）, anion-exchange chromatography chromatography （DEAE-Sepharose 4B）. Molecular weight was approximately 35 kD. Optimum pH and temperature were 37 ~C and 9.5 respectively. Lipase was stable within the pH range 6.0-- 11.0 and temperature below 50 ℃, belonging to alkaline lipase. Metal ions Mg2＋ , Ca2＋ , Cu2＋ , Zn2＋ , Co2＋ , Mn＋ stimulated its activity, whereas Al3＋ , Fe2＋ , Fe3＋ caused inhibition. Denaturant SDS was a strong inhibitor while urea and guanidine hydrochloride had no much affect. Aminophenol modifier NBS and PMSF inhibited lipase activity intensively, NBSF and DTT had no effect at low concentration while 2,3-butanedione affected it at high concentration. Half-life of lipase was enhanced by stabilizer NaCl, PEG, glycerol, sorbitol and sodium alginate. Lipase was resistant to trypsin under certain conditions.
出处
《工业微生物》
CAS
CSCD
2012年第1期68-73,共6页
Industrial Microbiology
基金
国家科技支撑计划2006BAF07B00
关键词
碱性脂肪酶
纯化
性质分析
氨基酸修饰
抗蛋白酶
alkaline lipase
purification
characterization
aminophenol modifier
protease resistance
