基于响应面法对黑木耳栽培料中纤维素酶提取及测定条件的优化

Optimization of extraction and determination conditions of cellulases from Auricularia heimuer cultivated substrate by response surface methodology

为了准确测定黑木耳菌包中纤维素酶的酶活性,为黑木耳菌种活力检测、栽培生产提供科学的理论依据,运用3种不同的方法对菌料进行处理,并根据单因素试验,利用响应面Box-Behnken设计方法,以纤维素酶活性为测定指标,选择出黑木耳菌料的最佳处理方法及纤维素酶的最优浸提条件与测定条件。结果表明,将黑木耳菌料经过筛处理后培养3 d纤维素酶有最大的活性;响应面结果显示,黑木耳纤维素酶的最佳浸提条件为:浸提时间2.51 h、料液比1:15.64、浸提温度50.87℃,纤维素酶活性为12.7031 U/mL。纤维素酶的最佳测定条件为反应温度52.31℃,酶量0.92 mL,pH 4.98,纤维素酶活性为14.8565 U/mL。本实验通过对测定中损伤的菌丝进行再培养,使其恢复产酶活性后,运用响应面的方法对纤维素酶的提取条件及测定条件进行优化,系统地测定出黑木耳纤维素酶的最优活性检测条件,为黑木耳菌株的筛选和品种鉴定提供了准确、系统的理论依据。

In order to accurately determine the enzymatic activity of cellulase in Auricularia heimuer packaged substrate and provide a scientific and theoretical basis for the detection of the viability of A.heimuer strains under cultivation condition,three different methods were applied to treat the cultivated substrate for achieving the optimum method and conditions of cellulase extraction and determination.The results showed that the cellulase activity of the substrate siftings reached 12.7031 U/mL after incubation for 3 d.The Box-Behnken response surface test showed that the optimal conditions for cellulase extraction were:extraction time of 2.51 h,material-liquid ratio of 1:15.64,extraction temperature of 50.87℃,and cellulase activity of 12.7031 U/mL.The optimal conditions for cellulase determination were:reaction temperature of 52.31℃,enzyme volume of 0.92 mL,pH value of 4.98 and cellulase activity of 14.8565 U/mL.In this study,the mycelium damaged during the assay process was recultivated to restore the enzyme production activity,and then the cellulase extraction and assay conditions by using response surface methodology were optimized.This method was proved to be accurate for identification of A.heimuer strains.