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.

本研究最初的核心研究方向为分析阿拉斯加北极冻土带土壤的蛋白水解酶（proteolytic enzyme）活性，但初步结果引发了对所采用方法（Watanabe与Hayano，1995）的质疑。因此，本研究的研究目标调整为两项：1）对该方法开展验证性考察；2）对比北极冻土带两种不同土层的酶活性差异。方法学验证环节涵盖以下内容：考察甲苯（toluene，用于防止产物固定化）的影响、酶活性的空白校正（blank correction），以及探索48小时孵育过程中真正的6小时线性活性速率。本研究以酪蛋白（casein）作为人工底物，以净氨基酸生成量作为活性表征指标，测定了矿质土层（mineral soil layer）与有机土层（organic soil layer）样品的本底活性与潜在活性。实验结果显示：不同甲苯浓度对酶活性未产生明确影响；省略甲苯时，有机样品的本底活性归零且潜在活性降低，但矿质样品未受该操作影响。对比有无空白校正的活性结果可见，尤其针对本底速率较低的样品的潜在活性，为保证结果准确性需开展空白校正。有机样品的本底活性与潜在活性，以及矿质样品的本底活性，在孵育最初6小时内均呈线性关系；矿质样品的潜在活性在6至24小时的孵育期间同样呈现线性特征。土层活性对比数据表明：有机土层的本底活性高于矿质土层。有机土层的潜在活性较本底活性高出一个数量级，提示存在底物限制；而矿质土层的潜在活性与本底活性无显著差异，表明底物供应充足。添加酪蛋白后，两层土壤的动力学模式（kinetic pattern）均发生改变：矿质土层由双曲线型（hyperbolic）动力学模式转变为S型（sigmoidal），有机土层则变为线性动力学模式，这暗示存在不同的酶库（enzyme pools），或是现有酶库的行为模式发生了变化。以总可溶性蛋白为基准的本底活性，矿质样品高于有机样品，再次印证了底物容量的差异以及酶-底物相互作用的变化。