雨生红球藻胡萝卜素酮基化酶体外固定化催化研究

Research on in vitro immobilization ofβ-carotene ketolase from Haematococcus pluvialis

【目的】虾青素是具有广阔市场前景的抗氧化剂原料,本研究旨在利用探索虾青素合成关键酶胡萝卜素酮基化酶体外固定化后体外合成虾青素的可行性,为开发环境友好的虾青素合成新方法提供依据。【方法】利用源于雨生红球藻的胡萝卜素酮基化酶mRNA序列,通过密码子优化、质粒构建和感受态大肠杆菌转染、IPTG诱导来表达含组氨酸标签的胡萝卜素酮基化酶融合蛋白,通过Ni-NAT镍柱来纯化,继而采用琼脂糖凝胶多孔性微珠固定装载有酶蛋白的脂质体,以胡萝卜素为底物在体外开展固定化酶催化反应研究,并探讨多种表面活性剂对酶活性的影响。【结果】成功表达和制备了组氨酸标签化的胡萝卜素酮基化酶在体外固定化酶反应中,有脱氧胆酸钠存在时,该酶显示了最高活性,其表观反应速率常数为1.9μg_(胡萝卜素)·mg_(酶)^(-1)·h^(-1)。【结论】雨生红球藻胡萝卜素酮基化酶体外固定化转化胡萝卜素具备可行性。

【Objective】Astaxanthin is a popotent antioxidant with broad market prospects.The present study aims to explore the practicability of synthesis astaxanthin by in vitro immobilization of carotene ketoase,the key enzyme in astaxanthin biosynthesis thus to provide more information for exploring an environmentally friendly new method for astaxanthin synthesis.【Method】The expression plasmid was constructed using optimized mRNA sequence of carotene ketonease derived from Haematococcus pluvialis and pET-32a plasmid.After transfection,the expression of the enzyme fused with a His-tag peptide was induced by IPTG.The enzyme was purified by Ni-NAT nickel column.The enzyme was first loaded on liposome and then immobilized in the porous microbeads based on agarose gel.The enzyme-containing beads were used to catalyze carotene in vitro.The effects of several surfactants on the activity of the immobilized enzyme were also discussed.【Result】The His-tagged carotene ketoase is active in the in vitro reaction after immobilization.The activity of the immobilized enzyme reached its maximum in the presence of sodium deoxycholate,with an apparent reaction rate constant of 1.9μg carotene per mg enzyme per h.【Conclusion】Carotene ketoase from H.pluvialis is feasible for in vitro immobilization and conversion of carotene.