华根霉胞内脂肪酶同功酶酶学性质研究

Purification and Characterization of Lipases from Rhizopus chinensis

摘要:

华根霉(Rhizopus chinensis CCTCCM201021)细胞破碎液经硫酸铵分级盐析、Phenyl- Sepharose FF疏水层析、DEAE-Sepharose FF离子交换层析和Sephadex G100凝胶层析得到两种脂肪酶同功酶: Lip1和Lip2。SDS-PAGE显示其亚基分子量分别为59.2kD和39.4kD。Lip1和Lip2的最适反应pH和最适反应温度相近，分别为8.0、8.5和40℃、35℃。但底物专一性差异明显：Lip1对pNP脂肪酸酯的长链脂肪酸有较高的专一性，Lip2对pNP脂肪酸酯的短链脂肪酸专一性较好；Lip1对三油酸甘油酯表现1,3-位置特异性，而Lip2没有位置选择性。1mmol/L的Ca²⁺、Mg²⁺对Lip1、Lip2有较好的激活作用；SDS强烈抑制酶活力。Lip1、Lip2在环己烷、正己烷、正庚烷和异辛烷(30% V/V)中稳定性良好。

Abstract:

Two lipase active fractions Lip1 and Lip2 were purified from the cell extract of Rhizopus chinensis CCTCCM201021.Both gave a single band on SDS-PAGE after using ammonium sulfate precipitation、Phenyl-Sepharose FF、DEAE-Sepharose FF and Sephadex G100 gel filtration chromatographies. The molecular masses of two lipases were 59.2kD and 39.4kD respectively. Lip1 and Lip2 showed optimal pH at 8.0 and 8.5 and their optimal temperatures were 40℃ and 35℃ respectively. The substrate specificity of the two lipases was obviously different. Lip1 was more specific to long chain fatty acid of p-nitrophenyl esters while Lip2 had a preference for the hydrolysis of short chain fatty acid of p-nitrophenyl esters. Lip1 had 1,3-position specificity for triacylglycerols hydrolysis while Lip2 had nonspecific position. Both lipases were stimulated by Ca²⁺、Mg²⁺ while SDS had strong inhibition on their activities. Lip1 and Lip2 had good stability in cyclohexane、hexane、heptane and isooctane(30% V/V).