酒曲中羧甲基纤维素酶活力测定影响因素分析

Analysis of affecting factors on determination of carboxymethyl cellulose activity in starter

对还原糖法测定酒曲中羧甲基纤维素酶活力的影响因素进行了探讨,建立了适合酒曲中羧甲基纤维素酶活的测定方法。研究了酒曲中酶的提取方法(缓冲液与酒曲提取比例、酒曲中酶的溶出方式)、酶解反应条件(酶促反应pH、反应温度、酶液与底物添加量)及空白的选择。结果表明,最优条件为酒曲经-20℃冷冻过夜处理,用15倍质量的缓冲液振荡提取30 min,5 000 r/min离心5 min,上清为原始酶液。酶解反应条件为pH值4.8,样品为0.50mL酶液和2.00mL底物,空白为0.50mL酶液和2.00mL缓冲液,同时50℃反应30 min后添加3.0 mL DNS试剂,沸水浴显色10 min,冷却后空白补加2.00 mL底物,样品管补2.00 mL缓冲液,分别用水定容至25 mL,测定波长540nm。

The influence factor of reducing sugar method on carboxyl methyl cellulose activity（CMCA） of starter was discussed and a testing method suitable for CMCA determination was established. The research focused on the effect of enzyme extraction method in starter（extraction ratio of buffer and starter, enzyme extraction methods of starter）, enzymolysis reaction conditions（enzymatic reaction pH, reaction temperature, enzyme and substrate addition） and blank choice. Experimental results showed that the optimal determination conditions were overnight cooling at-20℃,extracted through oscillation by 15 times quality buffer for 30 min, centrifuged with 5 000 r/min for 5 min. The supernatant was the crude enzyme solution. The reaction conditions of enzyme hydrolysis were as follows： 0.50 ml enzyme with 2.00 ml substrate as sample and 0.50 ml enzyme with2.00 ml buffer solution as blank, reaction time 30 min, temperature 50℃ and pH 4.8. Then 3.0 ml DNS reagent was added and water bathed for 10 min for chromogenic reaction. After cooling, the blank was supplemented with 2.00 ml substrate and the sample was supplemented with 2.00 ml buffer solution, and then water were added to the solution to 25 ml. Finally the absorbance was determined at 540 nm.