蔗糖酶活性的测定(3,5-二硝基水杨酸比色法)
(关松萌主编,《土壤酶及其研究法》)
酶促反应试剂:
(1)pH5.5磷酸缓冲液:1/15M Na2HPO4(11.867克Na2HPO4·2H2O溶于1升蒸馏水中)0.5ml加1/15M KH2PO4(9.078克KH2PO4溶于1升蒸馏水中)9.5ml。
(2)8%蔗糖溶液(用pH5.5磷酸缓冲液配制)。
(3)甲苯。
测定试剂:
(1)3,5-二硝基水杨酸溶液:称0.5g二硝基水杨酸,溶于20ml 2N NaOH和50ml水中,再加30g酒石酸钾钠,用水稀释至100ml(不超过7天)。
(2)
标准
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葡萄糖溶液:将葡萄糖先在50-58℃条件下,真空干燥至恒重。取500mg葡萄糖溶于100ml苯甲酸饱和溶液中(5mg还原糖/ml),即成标准葡萄糖溶液。再由此溶液稀释成1ml含0.01-0.5mg葡萄糖工作溶液。
标准曲线绘制:分别取不同浓度葡萄糖工作溶液1ml于50ml容量瓶中,加入3ml二硝基水杨酸溶液,混匀后在沸水浴中加热5min,取出立即在冷水中冷却,用水稀释至50ml,于波长508nm处比色测定颜色深度。以光密度值为纵坐标,葡萄糖浓度为横坐标,绘制成标准曲线。
测定操作
称5g风干土,置于50ml三角瓶中,注入15ml 8%蔗糖溶液,5ml pH5.5磷酸缓冲液和5滴甲苯。摇匀混合物后,放入恒温箱,在37℃下培养24h。取出,迅速过滤。从中吸取滤液1ml,注入50ml容量瓶中,加3ml 3,5-二硝基水杨酸溶液,在沸水浴中加热5min,取出立即在冷水中冷却3min(溶液因生成3-氨基-5-硝基水杨酸而呈橙黄色),用水稀释至50ml,于波长508nm处比色。
为了消除土壤中原有的蔗糖、葡萄糖而引起的误差,每一土样需做无基质对照,整个试验需做无土壤对照。
结果计算
蔗糖酶活性以24h后1g土壤葡萄糖的毫克数表示:
葡萄糖(毫克)=a×4
式中a——从标准曲线查得的葡萄糖毫克数
4——换算成1g土的系数。
Assay of β-glucosidase activity
Assay of β-glucosidase activity was adapted from Tabatabai (1994). In brief, 5.0 g of moist soil was suspended in 12.5 mL of acetate buffer (0.2 M, pH 5.0), and 12.5 mL of 20 mM
p-nitrophenyl-β-D-glucopyranoside (Huich Biotech, Shanghai, China) was added as the reactive substrate. The suspension was reciprocally shaken at 200 r min?1 for 1 h at 25 ?C, and then 5 mL
of 0.5 mol L?1 CaCl2 and 20 mL of 0.1 M Tris buffer (pH 12.0) were added to cease substrate degradation. The solution was mixed and let stand for a few minutes to let soil particles settle. 1.5 ml of the solution was centrifuged at 13 000 × g for 1 min and the concentration of p-nitrophenol (PNP) in the supernatant was measured colorimetrically at 400 nm. The same procedure was applied for the control, except that the substrate was added after the incubation and addition of the CaCl2 and Tris buffer.
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