Optimized extraction and partial purification of S. Cerevisiae invertase from peach puree

Abstract The extraction was performed using an autolysis process where enzymes degrade cell structures and release the cytoplasmic contents to the extracellular medium. The autolysis process was carried out in shake flasks with different agitation speeds (50 - 350 rpm) and the concentration of sodium bicarbonate (50 -350 mM NaHCO3) at 40 ° C for 24 hours, using a central composite design adapted for two variables and five levels and response surface methodology (RSM) and canonical analysis to define the optimal conditions of extraction of invertase S. cerevisiae from peach puree (ISc). In great condition extraction, we studied the effect of the liofilization and purified in the invertase activity using colorimetric method of 3,5-dinitrosalicílico (DNS) at 490 nm to monitor their activity. One unit (U) defined as 1 mg glicose.min-1. Protein concentrations were determined by Lowry and invertase comercial yeast (ICY). The influence of concentration NaHCO3 and stirring speed to extract the enzyme invertase showed linear effect, quadratic and interactive. The polynomial model of second order could explain the extraction phenomenon of invertase with R2 of 0.80, with significantly dependent on both variables activity. The maximum extraction was observed in the experimental center point (200 mM NaHCO3 and 200 rpm) with an activity of 14.7 U.mg-1. The stationary point is a point of maximum extraction, differing by at least 10% of the experimental center point. Based on these characteristics, the optimal conditions for extraction of S. cerevisiae invertase from peach puree (ISc) are 200 mM NaHCO3 as autolysis agent, 200 rpm orbital shaking at 40ºC for 24 hours and freeze-dried using biomass. Ethanol is more effective than acetone for recovery of specific activity.

摘要：本研究采用自溶工艺实现酶提取，即通过内源酶降解细胞结构，将胞质内容物释放至胞外培养基。自溶实验于摇瓶中开展，设置梯度搅拌转速（50~350 rpm）与碳酸氢钠浓度（50~350 mM NaHCO₃），于40℃下反应24小时；实验采用适配双变量五水平的中心复合设计（Central Composite Design, CCD），结合响应面法（Response Surface Methodology, RSM）与典范分析，确定从桃泥中提取酿酒酵母蔗糖酶（Invertase from *Saccharomyces cerevisiae*, ISc）的最优条件。在提取优化阶段，本研究借助490 nm波长下的3,5-二硝基水杨酸（3,5-Dinitrosalicylic Acid, DNS）比色法监测蔗糖酶活性，考察了冷冻干燥与纯化步骤对酶活性的影响。1个酶活力单位（U）定义为每分钟催化生成1 mg葡萄糖所需的酶量。蛋白浓度通过洛里（Lowry）法测定，实验同时以商业酵母蔗糖酶（Commercial Yeast Invertase, ICY）作为对照。结果表明，碳酸氢钠浓度与搅拌转速对蔗糖酶提取过程均存在线性、二次及交互效应；二阶多项式模型可较好解释该蔗糖酶提取过程，决定系数R²为0.80，酶活力与两个变量均存在显著相关性。在实验中心点（200 mM NaHCO₃、200 rpm）处可获得最大提取酶活，达14.7 U·mg⁻¹。该驻点为最大提取驻点，与实验中心点的差异至少为10%。基于上述结果，从桃泥中提取酿酒酵母蔗糖酶（ISc）的最优条件为：以200 mM碳酸氢钠作为自溶助剂，40℃下以200 rpm转速振荡培养24小时，菌体经冷冻干燥处理。在回收比活性方面，乙醇的效果优于丙酮。