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