高通量MBTH法测定β-葡聚糖酶的活力  

High Throughput Determination of β-Glucanase Activity Using the 3-Methyl-2-benzothiazole Hydrazine(MBTH) Method

作　　者：张永勤[1] 刘征东[1] 程袁芬 常海燕[1] 吴国耀 韩美芳[1] 

机构地区：[1]青岛科技大学化工学院,山东青岛266042

出　　处：《现代食品科技》2015年第1期221-225,共5页Modern Food Science and Technology

基　　金：山东省自然科学基金(ZR2013CM027)

摘　　要：本文建立了一种以3-甲基-2-苯并噻唑酮腙（MBTH）为氧化剂,以微孔板测定β-葡聚糖酶活力的新方法,该法特别适合于成分复杂的材料中痕量β-葡聚糖酶活力的测定。根据MBTH法测定β-葡聚糖酶解产物还原端基的特点,设计并优化了酶活力测定的关键参数。结果表明,在最适p H 5.0和30℃条件下测定β-葡聚糖酶活力,所得酶的工作浓度范围为0~12.3 m U/m L,饲料中酶的工作浓度范围均为0~2.94 U/g,本法测β-葡聚糖酶和四种饲料样品中酶的平均回收率分别为93.1%、97.5%、104.3%、97.0%、106.4%,β-葡聚糖酶活力检测限为0.37 m U/m L,定量限为1.24 m U/m L,四种饲料酶活力检测限分别为0.10 U/g、0.09 U/g、0.14 U/g、0.09 U/g,定量限分别为0.33 U/g、0.30 U/g、0.46 U/g、0.31 U/g。该法准确、低成本、省时、省力,特别是其灵敏度远高于DNS法,可极大程度上避开饲料中其它成分对测定的干扰。In this paper, a novel microplate-based method, using 3-methyl-2-benzothiazole hydrazine （MBTH） as the oxidant, for assaying β-glucanase activity was established. This method was suitable for the determination of trace levels of β-glucanase activity, especially in materials with complex composition. The parameters of the activity assay were designed and optimized based on the characteristics of the reducing end groups in the products of β-glucanase-mediated enzymatic hydrolysis. The results showed that, for enzymatic activity determination carried out under optimal pH （pH = 5.0） and temperature （30 ℃） conditions, the activity of pure β-glucanase was linear in the 0-12.3 mU/mL range, while the β-glucanase activities in the four types of feeds were linear within the 0-2.94 U/g range. The average recoveries using pure β-glucanase and the enzymes in the four feeds were 93.1%, 97.5%, 104.3%, 97.0%, and 106.4%, respectively. The limits of detection for pure β-glucanase and the enzymes in the four feeds determined using this method were 0.37 mU/mL, 0.10 U/g, 0.09 U/g, 0.14 U/g, and 0.09 U/g, respectively. The limits of quantitation for pure β-glucanase and the enzymes in the four feeds determined using this method were 1.24 mU/mL, 0.33 U/g, 0.30 U/g, 0.46 U/g, and 0.31 U/g, respectively. This method was accurate, low-cost, as well as time and labor saving. In particular, the sensitivity of this method was much higher than that of the 3,5-dinitrosalicylic acid （DNS） method. Moreover, interference from other components present in the feeds could largely be avoided.

关 键 词：高通量法 MBTH Β-葡聚糖酶 活力 饲料 测定 

分 类 号：O657[理学—分析化学] TS201.25[理学—化学]