Characterization of humus-protease complexes extracted from soil
Characterization of humus-protease complexes extracted from soil
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DOI：
10.1016/j.soilbio.2009.02.032
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发表时间：
2009-06-01
影响因子：
9.7
通讯作者：
Valero, Jordi
中科院分区：
文献类型：
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作者：
Bonmati, Manuel;Ceccanti, Brunello;Valero, Jordi
对两种耕作土壤和一种牧场土壤的焦磷酸盐(140 mM，pH 7.1)浸提液进行超滤，将萃取物分成三个组分：A组分，名义分子量(NMW)为100kD，均为10~100kD，R组分为10kD。用三种不同的底物测定了它们的酶活力：胰酶专一性底物N-苯甲酰-L-精氨酰胺；羧肽酶专一性底物N-苯甲酰基-L-苯丙氨基L亮氨酸；以及酪蛋白。并对其氨基酸N、腐植酸(HA)和黄腐酸(FA)的含量进行了分析。用等电聚焦(IEF)和裂解气相色谱(Py-GC)分析了草地土壤提取物和衍生组分的有机质。测定了萃取物的热稳定性以及萃取物及其衍生部分的最佳pH。由于焦磷酸钠在分离过程中对腐殖质的机械分解作用，萃取物和衍生部分中的总有机碳和脂肪酸的含量依次为R>；A(II)>；A(I)。氨基酸N/有机C以R组分最低，而所有组分均富含腐植酸、碳水化合物和氨基酸N、A，碳水化合物含量最低。至少70%的BAA-和ZPL-水解酶活性与NMW>10kD的颗粒有关，其中至少30%的活性与NMW>100kD的颗粒有关。酪蛋白水解酶活性在三个组分(A(I)、A(II)和R)之间分布相当均匀。由于酶分子周围腐殖质的多阴离子特性和碱性蛋白酶的存在，所提取的酶-有机复合体具有耐热变性，其中一些在pH高于10时表现出最高活性。将Py-GC分析的数据与蛋白酶活性进行比较，表明BAA的水解性与高度浓缩的腐殖质有关，而ZPL的水解性与抗性较低的腐殖质有关，而至少部分提取的酪蛋白水解性是以糖蛋白的形式存在的，与腐殖质无关。BAA-水解酶活性可能被碳水化合物来源的新鲜有机物抑制，而木质素来源的有机物可能抑制ZPL-和酪蛋白水解酶活性。(C)2009爱思唯尔有限公司。保留所有权利。
Pyrophosphate (140 mM, pH 7.1) extracts of two arable soils and one pasture soil were ultrafiltrated separating the extracted material into three fractions: A, with nominal molecular weight (nmw) > 100 kD, All with nmw between 10 kD and 100 kD and R with nmw < 10 kD. Protease activity was determined in the fractions by using three different substrates: N-benzoyl-L-argininamide (BAA), specific for trypsin; N-benzyloxy-carbonyl-L-phenylalanyl L-leucine (ZPL), specific for carboxypeptidases; and casein, essentially a non-specific substrate. The derivative fractions were also analysed for their amino acid N and humic (HA) and fulvic (FA) acid contents. The organic matter of extracts and derivative fractions obtained from the pasture soil was analysed by isoelectric focusing (IEF) and that of fractions analysed by pyrolysis gas chromatography (Py-GC). Activities of the extract were monitored for their thermal stability and those of the extract and derivative fractions for their optimal pH.Due to the mechanical disintegrating action of sodium pyrophosphate over the humic substances during the fractionation process the amount of total organic C and FA in the fractions was ranked as R > A(II) > A(I). The lowest amino acid N/organic C was found in the R fraction, whereas All fraction was rich in humic acids, carbohydrates and amino acid N and A, fraction showed the lowest carbohydrate content. At least 70% of the total BAA- and ZPL-hydrolysing activity was associated to particles with nmw higher than 10 kD and at least 30% of these activities were present in particles with nmw higher 100 kD. Casein-hydrolysing activity was quite evenly distributed among the three fractions (A(I), A(II) and R). The extracted protease-organic complexes were resistant to thermal denaturation and some of them showed optimal activity at pH values higher than 10 as a result of the polyanionic characteristics of the humic material surrounding enzyme molecules and of the presence of alkaline protease. Comparison of data obtained in Py-GC analyses and in protease activity suggests that BAA-hydrolysing activity was associated to a highly condensed humic matter and ZPL-hydrolysing activity to less resistant humic substances, while at least some of the extracted casein-hydrolysing activity was present as glyco-proteins not associated to humus. BAA-hydrolysing activity was probably inhibited by fresh organic matter of carbohydrate origin whereas lignin derived organic matter probably inhibited ZPL- and casein-hydrolysing activity. (C) 2009 Elsevier Ltd. All rights reserved.