刺梨果实转化酶提取方法比较及酶学特性分析  

Comparison of extraction methods and analysis of enzymatic characteristics of invertase from the fruit of Rosa roxburghii

作　　者：黄亚成 何斌 胡晓莎 苏龙兴 罗炳 刘林娅 HUANG Yacheng;HE Bin;HU Xiaosha;SU Longxing;LUO Bing;LIU Linya(School of Biological Science and Technology,Liupanshui Normal University,Liupanshui,Guizhou,553004,China)

机构地区：[1]六盘水师范学院生物科学与技术学院,贵州六盘水553004

出　　处：《中国南方果树》2024年第4期157-163,共7页South China Fruits

基　　金：六盘水师范学院科研培育项目(LPSSY2023KJZDPY07);贵州省教育厅2022年度自然科学研究项目(黔教技[2022]008号);六盘水市科技计划项目(52020-2022-PT-03);贵州省大学生创新训练项目(S202210977126)资助。

摘　　要：刺梨(Rosa roxburghii Tratt.)果实转化酶(蔗糖酶)通过调控蔗糖分解影响果实的生长发育和品质形成。提取方法能够影响植物转化酶的提取效率和活性。以“贵农7号”刺梨花后45 d的果实为材料,比较了液氮研磨样品和提取液冰上研磨样品下6种提取转化酶方法的效果,探究了可溶性转化酶的酶学特性。结果表明,液氮研磨法对刺梨果实转化酶活性影响较大,其中可溶性酸性转化酶(soluble acid invertase,SAI)活性丧失严重,而细胞壁酸性转化酶(cell wall-bound invertase,CWI)和中碱性转化酶(neutral or alkaline invertaes,N/A-INV)均无活性。采用提取液冰上研磨样品,用Hepes-NaOH法提取刺梨果实转化酶的效果最好,SAI、CWI和N/A-INV的活性最高,分别为3.16、2.09和2.09μmol/(g·h),磷酸钠法次之,且Hepes-NaOH法提取的可溶性转化酶(SAI和N/A-INV)的比活性最高。酶学特性分析表明,SAI的最适温度为60℃,最适pH值为4.5~5.0,Km值为87.15 mmol/L;N/A-INV的最适温度为45~50℃,最适pH值为8.5,K m值为3.46 mmol/L。研究结果为刺梨果实转化酶的提取和转化酶生物学功能的深入研究奠定了基础。Convertase(sucrase)of Rosa roxburghii fruit affects the fruit growth,development and quality formation by regulating sucrose decomposition.Different extraction methods could affect the extraction efficiency of plant invertase and its activity.Effect of six methods for extracting invertase from the fruit of Guinong No.7 R.roxburghii by two ways of sample preparation,grinding fruits in liquid nitrogen and in extraction buffer on ice,were compared.The enzymatic characteristics of soluble invertase were explored.The results showed that the activities of invertases were seriously destroyed by grinding fruit in liquid nitrogen,especially those of cell wall-bound invertase(CWI)and neutral/alkaline invertase(N/A-INV),which were totally inactivated.When fruits were ground on ice,the activity of crude soluble invertase extracted by Hepes-NaOH method revealed to be the best.The enzyme activities of SAI,CWI and N/A-INV were the highest,which were 3.16,2.09 and 2.09μmol(glucose,fructose)/[g(FW)·h],respectively,followed by phosphate-Na method.Regarding to specific enzymatic activity,the Hepes-NaOH method was the most appropriate.The results of enzymatic characteristics showed that SAI had the highest enzymatic activity at pH value of 4.5-5.0 with suitable temperature of 60℃,and a Km of 87.15 mmol/L for its substrate of sucrose.N/A-INV showed the highest enzymatic activity at pH value of 8.5 with suitable temperature of 45-50℃,and had a Km of 3.46 mmol/L for its substrate of sucrose.The results provide a foundation for the extraction of sucrose invertase and the further study of the biological function of sucrose invertase.

关 键 词：刺梨果实 转化酶 蔗糖酶 提取方法 酶学特性 

分 类 号：S661.9[农业科学—果树学]