摘要:
本文通过优化蛋白浓度测定方法和酶活力测定方法,并以酶解动力学曲线考察方法优化的合理性,以验证终点法测定碱性果胶酶比活的准确性。结果表明,果胶酶蛋白的最佳测定波长为550 nm,其线性和截距明显优于传统方法,可以忽略果胶酶稀释倍数对蛋白测定的影响。钙离子对该酶具有激活作用;聚半乳糖醛酸比果胶更适合作为酶活力测定底物,其最适pH为9.5,最佳浓度为2 mg/mL,果胶酶活力测定的最佳波长为232 nm,所测酶活力在吸光度值0~2范围内均保持零级反应状态,酶蛋白浓度与酶解速率线性相关且回归曲线截距更接近原点,因此,该测定条件不仅可用于终点法,酶解时间也可灵活掌握,而非拘泥于一个固定的时间段。以酶解30 min为例,其检测限(LOD)为0.15 mU/mL。由于DNS法的灵敏度较低,其半乳糖醛酸标准曲线不过原点,从而导致因酶液稀释倍数不同而产生测量误差,因此,紫外法测定碱性果胶酶活力为最佳选择。
关键词:
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碱性果胶酶
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蛋白浓度
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酶活力
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测定
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动力学
Abstract:
In this paper,the assay methods for protein and enzyme activity were optimized respectively. The rationality of the method optimization was investigated through the kinetic curve of the enzymolysis to confirm the accuracy of the endpoint determination of specific activity of alkaline pectinase. The results showed that optimal detection wavelength for pectinase protein was 550 nm,its linearity and intercept were significantly superior to the traditional method,and the effect of dilution factor on pectinase protein determination could be ignored. Calcium ion had an activating effect on this enzyme,polygalacturonic acid was more suitable as a substrate for enzyme activity measurement than pectin. The optimal substrate concentration and pH were 2 mg/mL and 9.5,respectively. The enzyme activity measured at a wavelength of 232 nm maintained a zero-order reaction state in the absorbance range within 0~2. The enzyme protein concentration was linearly related to the degradation rate and the intercept of the regression curve was closer to the origin. Therefore,this measurement condition could be used for the end point determination,and the enzymolysis time could be flexibly controlled,rather than being restricted to a fixed period of time.Taking 30 minutes as an example,the limit of detection(LOD)was 0.15 mU/mL due to the low sensitivity of the DNS method,the standard curve of galacturonic acid was not at the origin,resulting in measurement errors due to different dilutions of the enzyme solution. Therefore,the UV method was the best activity assay for alkaline pectinase.
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