巴氏葡萄球菌TS-82类胡萝卜素裂解酶的分离纯化及其酶学特性

Isolation,Purification and Characterization of a Novel Carotenoid-Cleaving Enzyme from Staphylococcus pasteuri TS-82

巴氏葡萄球菌TS-82类胡萝卜素裂解酶经强阴离子柱、高效制备液相色谱和多肽分子筛纯化得到液相级纯酶(95.6%)。该酶比活力为125 U/g,纯化倍数为446,回收率为2.39%。纯化后的类胡萝卜素裂解酶经液相色谱-质谱联用测定,得其分子质量为655.093 D。关于酶学特性,研究发现该酶对C_(40)类胡萝卜素底物的最适温度为60℃,而作用于β-阿朴-8’-胡萝卜醛的最适温度是50℃,该酶的稳定温度为50℃以下;该酶对所测定底物的最适p H值为3.0;该酶与5种底物亲和力排列为:玉米黄质>虾青素>β-胡萝卜素>角黄质>β-阿朴-8’-胡萝卜醛;Al^(3+)和Fe^(3+)是该酶的强效催化剂,Fe^(2+)是该酶的强效抑制剂;H_2O_2在低浓度范围内(0~16 mmol/L)可促进酶活性;低体积分数乙醇(4%~16%)的添加对酶活性无明显抑制作用。结果表明该酶具有很好的耐酸性和热稳定性,能够适应果酒环境,为其工业化应用提供依据。

A carotenoid cleavage enzyme with HPLC grade purity of95.6%was obtained from Staphylococcus pasteuri(S.pasteuri)TS-82by using anion-exchange chromatography,preparative high performance liquid chromatography(PHPLC),and superdex peptide chromatography together.The purified enzyme had a specific activity of125U/g with466-fold purification and2.39%recovery.Its molecular weight was655.093D as identified by liquid chromatographyelectrosprayionization-tandem mass spectrometry(LC-ESI/MS).Enzymatic characterization indicated that the purifiedenzyme had optimal temperature of60℃for degrading C40carotenoids and50℃for degradingβ-apo-8’-carotenal,and itmaintained stable activity at50℃.The optimum pH value was3.0for degrading all investigated substrates.The Km andvmax values indicated that the enzyme showed the highest affinity towards zeaxanthin,followed by astaxanthin,β-carotene,canthaxanthin,andβ-apo-8’-carotenal in a decreasing order.The metal ions Al3+and Fe3+were identified as potent activatorsfor the purified enzyme,whereas Fe2+as a potent inhibitor.H2O2was able to increase the enzyme activity in degradingβ-carotene at levels of0-16mmol/L.Alcohol at low concentration(4%–16%)did not inhibit the activity of the enzyme.Inconclusion,the enzyme showed great acid resistance and thermostability,which makes it stable in wine environment andprovides a basis for industrial application.