Proteolytic enzyme activity of organic and mineral soil core samples collected near Toolik Lake field station, Alaska, July 2001

The original focus of this study was an analysis of proteolytic enzyme activity of Alaskan arctic tundra soils, however initial results raised questions regarding the method (Watanabe and Hayano, 1995). Thus, the goals of the study changed to 1) an investigation of the method, and 2) a comparison of enzyme activities of two different soil layers from the arctic tundra. Methodological examination included the impact of toluene, used to prevent immobilization of the product, and blank correction of enzyme activity, and a search for a true 6-h linear rate of activity during a 48-hour incubation. We measured native and potential, using casein as an artificial substrate, activities as net amino acid production in mineral and organic soil layer samples. Varying toluene concentration had no clear effect on activity; omitting toluene resulted in zero native activity and reduced potential for the organic samples, but not for the mineral. Comparison of activities with and without blank correction indicated, particularly for potential activity of samples with low native rates, that correction was required for accuracy. Native and potential activity of the organic samples, and native of the mineral were linear for the first 6 h of incubation; linearity was observed during the 6 to 24 h incubation for potential activity of the mineral. Soil layer activity data indicated that native activity was higher in organic soils as compared with mineral. The organic layer potential activity was ten-fold greater than the native, suggesting substrate limitation; potential and native activities did not differ in the mineral layer, indicating substrate sufficiency. Casein addition changed the kinetic pattern for both layers from hyperbolic to sigmoidal for the mineral and linear for the organic, implying different enzyme pools or behavioral changes of existing pools. Native activity based on total soluble protein was higher for the mineral samples relative to the organic, reiterating substrate capacity differences and variations in enzyme/substrate interactions.

本研究最初的核心目标为分析阿拉斯加北极苔原土壤的蛋白水解酶活性，但初步结果引发了针对实验方法的质疑（Watanabe与Hayano，1995）。因此本研究的目标调整为两项：一是对该实验方法开展验证，二是对比北极苔原两种不同土层的酶活性差异。方法学验证环节涵盖：用于抑制产物固定化的甲苯的作用影响、酶活性的空白校正，以及探寻48小时培养过程中真正的6小时线性活性速率。本研究以酪蛋白作为人工底物，以矿质土层与有机土层样品中的氨基酸净生成量为指标，测定了样品的固有酶活性与潜在酶活性。不同浓度的甲苯对酶活性未产生显著影响；但去除甲苯后，有机土层样品的固有活性完全消失，且其潜在活性有所降低，而矿质土层样品则未出现此类变化。对是否进行空白校正的两组活性结果进行对比后发现，尤其是针对固有活性较低的样品的潜在活性而言，空白校正对保证结果准确性必不可少。有机土层样品的固有与潜在活性、以及矿质土层样品的固有活性，在培养初始6小时内均呈线性变化；而矿质土层样品的潜在活性则在6至24小时的培养阶段内保持线性。土层活性数据显示，有机土层的固有酶活性高于矿质土层。有机土层的潜在活性是固有活性的10倍，提示存在底物限制现象；而矿质土层的潜在活性与固有活性无显著差异，表明底物供应充足。添加酪蛋白后，两种土层的酶促动力学模式均发生改变：矿质土层的动力学模式从双曲线型转为S型，有机土层则转为线性模式，这暗示两类土层的酶库存在差异，或是原有酶库的行为模式发生了改变。以总可溶性蛋白为基准的固有活性测定结果显示，矿质土层样品的活性高于有机土层，进一步印证了底物容纳能力的差异，以及酶-底物相互作用模式的不同。