Purification and amino acid sequence of a bitter gourd inhibitor against an acidic amino acid-specific endopeptidase of Streptomyces griseus.

An inhibitor (BGIA) against an acidic amino acid-specific endopeptidase of Streptomyces griseus (Glu S. griseus protease) was isolated from seeds of the bitter gourd Momordica charantia L., and its amino acid sequence was determined. The molecular weight of BGIA based on the amino acid sequence was calculated to be 7419. BGIA competitively inhibited Glu S. griseus protease with an inhibition constant (Ki) of 70 nM, and gel filtration analyses suggested that BGIA forms a 1:1 complex with this protease. However, two other acidic amino acid-specific endopeptidases, protease V8 from Staphylococcus aureus and Bacillus subtilis proteinase (Glu B. subtilis protease), were not inhibited by BGIA. BGIA had no inhibitory activity against chymotrypsin, trypsin, porcine pancreatic elastase, and papain, although subtilisin Carlsberg was strongly inhibited. The amino acid sequence of BGIA shows similarity to potato chymotrypsin inhibitor, barley subtilisin-chymotrypsin inhibitor CI-1 and CI-2, and leech eglin C, especially around the reactive site. Although the residue at the putative reactive site of these inhibitors is leucine or methionine, the corresponding amino acid in BGIA is alanine.     

A high molecular weight glutamyl endopeptidase and its endogenous inhibitors from cucumber leaves

We purified a glutamyl endopeptidase that is a major foliar endopeptidase in cucumber. The endopeptidase had a molecular mass of 400 kDa, consisted of four subunits of 97 kDa, and was inactivated by SH-modifying reagents. Its optimum pH and optimum temperature were 8.0 and 30-37 degrees C, respectively. An internal amino acid sequence of the endopeptidase was highly homologous to a partial sequence of unidentified proteins deduced from genetic information for Arabidopsis thaliana, soybean and rice, but not to the sequences of bacterial glutamyl endopeptidases or animal proteases. Therefore, the unidentified proteins might be glutamyl endopeptidases and be widely distributed only among plant species. The activity of the cucumber glutamyl endopeptidase was inhibited by at least three inhibitors existing in cucumber leaves. One of the inhibitors was a competitive inhibitor of 25 kDa, which did not significantly inhibit commercial endopeptidases derived from animals and microorganisms. This suggests that the cucumber glutamyl endopeptidase might be controlled by endogenous inhibitors in vivo.     

Cloning and expression of subtilisin amylosacchariticus gene

The gene encoding subtilisin Amylosacchariticus from Bacillus subtilis var. amylosacchariticus was isolated and the entire nucleotide sequence of the coding sequence was determined. The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein comprising 275 residues. There were discrepancies in 10 amino acids between the sequence elucidated from the nucleotide sequence and the published protein sequence (Kurihara et al. (1972) J. Biol. Chem. 247, 5619-5631). The nucleotide sequence was highly homologous to that of subtilisin E gene from B. subtilis 168, with discrepancies at 12 nucleotides out of 1,426 nucleotides we sequenced. Ten of them were found in mature subtilisin coding sequence, which resulted in two amino acid changes and another one was in the putative promoter region between two genes. The productivity of subtilisin in culture broth of B. subtilis var. amylosacchariticus was much higher than that of B. subtilis 168. The enzyme gene was inserted in a shuttle vector pHY300PLK, with which B. subtilis ISW1214 was transformed. The proteolytic activity found in the culture broth of the transformed bacterium was 20- and 4-fold higher than those of the host strain and B. subtilis var. amylosacchariticus, respectively. Subtilisin Amylosacchariticus was easily purified to a crystalline form from culture filtrate of cloned B. subtilis, after a single step of chromatography on CM-cellulose.     

Degradation of streptomyces metalloprotease inhibitor (SMPI) by neutral protease from Bacillus subtilis var. amylosacchariticus.

The zinc-containing neutral endopeptidase (neutral protease: BANP) from Bacillus subtilis var. amylosacchariticus was inhibited by the proteinaceous metalloprotease inhibitor isolated from Streptomyces nigrescens (SMPI). The degree of inhibition was, however, significantly less than that for thermolysin (TLN). During incubation of BANP with SMPI, the inhibitor was proteolytically degraded and inactivated. Analysis of the digestion products suggested that a minor diversity in their substrate specificities between TLN and BANP affects the sensitivity to the proteinaceous metalloprotease inhibitor, SMPI.     

Directed evolution converts subtilisin E into a functional equivalent of thermitase

We used directed evolution to convert Bacillus subtilis subtilisin E into an enzyme functionally equivalent to its thermophilic homolog thermitase from Thermoactinomyces vulgaris. Five generations of random mutagenesis, recombination and screening created subtilisin E 5-3H5, whose half-life at 83 degrees C (3.5 min) and temperature optimum for activity (Topt, 76 degrees C) are identical with those of thermitase. The Topt of the evolved enzyme is 17 degrees C higher and its half-life at 65 degrees C is >200 times that of wild-type subtilisin E. In addition, 5-3H5 is more active towards the hydrolysis of succinyl-Ala-Ala-Pro-Phe-p-nitroanilide than wild-type at all temperatures from 10 to 90 degrees C. Thermitase differs from subtilisin E at 157 amino acid positions. However, only eight amino acid substitutions were sufficient to convert subtilisin E into an enzyme equally thermostable. The eight substitutions, which include known stabilizing mutations (N218S, N76D) and also several not previously reported, are distributed over the surface of the enzyme. Only two (N218S, N181D) are found in thermitase. Directed evolution provides a powerful tool to unveil mechanisms of thermal adaptation and is an effective and efficient approach to increasing thermostability without compromising enzyme activity.     

Bacillus intermedius glutamyl endopeptidase. Molecular cloning and nucleotide sequence of the structural gene

The glutamyl endopeptidase gene of Bacillus intermedius was cloned from a genomic library expressed in Bacillus subtilis and sequenced (EMBL accession number Y15136). The encoded preproenzyme contains 303 amino acid residues; the mature 23-kDa enzyme consists of 215 residues. The mature enzyme reveals 38% of identical residues when aligned with the glutamyl endopeptidase from Bacillus licheniformis, whereas only five invariant residues were found among all known glutamyl endopeptidases. The amino acid residues that form the catalytic triad (H47, D98, and S171) as well as H186 participating in the binding of the substrate carboxyl group were identified. It seems that the structural elements responsible for the function of glutamyl endopeptidases from various sources are highly variable.     

喀斯特地区不同土地利用方式对土壤有机碳、全氮以及微生物生物量碳和氮的影响

以广西环江大才为代表,选择亚热带典型喀斯特峰林谷地样区,通过对样区土壤进行密集采样和测定分析,研究了土地利用方式对土壤有机碳（O_C）和全氮（TN）含量及土壤微生物生物量碳（B_C）和氮（B_N）含量的影响.结果表明,3种土地利用方式下,土壤有机碳含量在稻田和林地中基本相同,而旱地显著低于稻田和林地.土壤全氮含量为稻田显著高于林地,而林地显著高于旱地.土壤微生物生物量碳含量为稻田显著高于林地,林地显著高于旱地.土壤微生物生物量氮含量在稻田和林地中基本相同,而旱地显著低于稻田和林地.旱地土壤pH值显著低于稻田和林地土壤.3种土地利用方式下,土壤微生物生物量碳与土壤有机碳、土壤微生物生物量氮与全氮含量之间均呈显著的正相关关系.土壤微生物生物量碳和氮含量可以作为评价喀斯特地区土壤质量和肥力的指标之一,对土地利用方式响应较为敏感.     

江苏省主要农田杂草种子库物种组成和多样性及其与环境因子的相关性分析

采用水洗镜检法对江苏省31个农田样点(包括旱田和水田)0～15 cm土层土壤杂草种子库的种类组成和物种多样性进行了调查研究;采用典范对应法分析了杂草种子库种类与环境因子(包括淹水天数、土壤有机质含量、土壤pH、年降水量、年均温、样点经度和样点纬度)的相关性并绘制了样点和种类与环境因子的二维排序散点图.调查结果表明:在31个样点的土壤杂草种子库中共检测到杂草种子15科54种,旱田和水田各有41和45种,二者共有种类占多数但优势种有差异,通泉草[ Mazus japonicus (Thunb.) Kuntze]、异型莎草(Cyperus difformis L.)、水苋菜(Ammannia baccifera L.)、千金子[Leptochloa chinensis (L.) Nees]、棒头革(Polypogon fugax Nees ex Steud.)和牛繁缕[ Malachium aquaticum (L.) Fries]等种类为二者的共有优势种;杂草种类最多的为禾本科(Gramineae)和莎草科(Cyperaceae),分别占种类总数的22.6％和20.8％.旱田不同样点杂草种子库的优势种类变化较大,而水田杂草种子库优势种较稳定;二者优势种频度差异较大,旱田中频度高于0.30的杂草有19种,水田中频度高于0.50的杂草有20种;旱田和水田中平均重要值大于0.03的杂草各有11和9种,这些频度高的种类重要值也较大.旱田和水田土壤种子库种子密度分别为21 015和37 847 m-2,平均为31 008 m-2;旱田3层土壤中种子密度差异不显著,而水田上、中层土壤的种子密度显著高于下层.旱田以夏熟和秋熟杂草为主,而水田则以水田和夏熟杂草为主.按形态类型划分,水田及旱田中杂草种类数和密度从大到小均依次排序为阔叶草类、禾草类、莎草类,且水田中这3类杂草的种类数和种子密度均高于旱田.与旱田相比,水田杂草种子库的物种丰富度指数(S)、Shannon-Wiener 指数(H')和Simpson指数(D)较高,但Pielou均匀度指数(E)较低;旱田杂草种子库的S、H'和D指数随土层深度增加而降低、E指数逐渐升高,水田各土层的物种多样性指数则无明显变化趋势.第1种类排序轴与淹水天数负相关性最高(R=-0.8143),第2种类排序轴与年降水量和经度也有较高的负相关性;相关性分析和CCA分析结果均表明淹水天数是影响杂草种子库群落构成的最大因素.通过二维排序可将31个样点分为旱田和水田2大类,旱田可划分为长期旱连作和水旱轮作2个亚类;水田可划分为淮河以北地区和淮河以南长江流域地区2个亚类.研究结果说明:杂草种子库潜群落优势种与地上部农田杂草显群落优势种具有一致性,通过土壤杂草种子库的研究可以预测地上杂草的发生和危害情况.     

BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification,biochemical and molecular characterization

A novel fibrinolytic enzyme, subtilisin BSF1, from a newly isolated Bacillus subtilis A26 was purified, characterized and the gene was isolated and sequenced. The subtilisin BSF1 was purified to homogeneity by five-step procedure with a 4.97-fold increase in specific activity and 6.28% recovery. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and gel filtration. The purified enzyme exhibited high fibrinolytic activity on fibrin agar plates.Interestingly, the enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 9.0. The relative activities at pH 10.0 and 11.0 were 97.8% and 85.2% of that at pH 9.0. The optimum temperature for enzyme activity was 60 °C. The activity of subtilisin BSF1 was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The N-terminal amino acid sequence of the first 11 amino acids (aa) of the purified fibrinolytic enzyme was AQSVPYGISQI.The bsf1 gene encoding the subtilisin BSF1 was isolated and its DNA sequence was determined. The bsf1 gene consisted of 1146 bp encoding a pre-pro-protein of 381 amino acids organized into a signal peptide (29 aa), a pro-peptide (77 aa) and a mature domain (275 aa). The deduced amino acids sequence of the mature enzyme (BSF1) differs from those of nattokinase from B. subtilis natto and subtilisin DFE from Bacillus amyloliquefaciens DC-4 by 5 and 39 amino acids, respectively.     

Pathogenesis of serratial infection: activation of the Hageman factor-prekallikrein cascade by serratial protease

A serratial protease with an apparent molecular weight of 56,000 (56K protease), which had been purified from the culture supernatant of a strain of Serratia marcescens isolated from a corneal lesion of a human eye [Matsumoto, K. et al. (1984) J. Bacteriol. 157, 225-232], greatly enhanced vascular permeability when injected into guinea pig skin. The 56K protease, which requires zinc ion for activity, was found to possess plasma kallikrein-like properties in vitro as judged by (i) preferential amidolysis of carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide and Pro-Phe-Arg-4-methylcoumaryl-7-amide, which are known substrates for plasma kallikrein; (ii) release of kinin from high-molecular-weight kininogen; and (iii) prompt activation of Hageman factor followed by generation of kallikrein from plasma prekallikrein. These results suggest that the 56K protease enhances vascular permeability through activation of a Hageman factor-kallikrein-kinin pathway in vivo, and this molecular process appears to be a rational mechanism of enhancement of permeability and serratial pathogenesis.     

Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.     

A novel endopeptidase from Xenopus that recognizes alpha-helical secondary structure

The magainin peptides of Xenopus laevis are broad-spectrum antimicrobial agents. Upon discharge from the skin glands, these basic, amphipathic peptides are each further processed at a single Xaa-Lys bond into half-peptides by a cosecreted protease. We describe the characterization and purification to homogeneity of this endopeptidase from Xenopus skin. The enzyme is a metalloprotease 110 kd in size. Analyses of substrate specificity revealed that the endopeptidase recognizes peptides that share the ability to adopt an amphipathic, alpha-helical motif composed of at least 12 residues, with one face strongly hydrophobic. Cleavage occurs on the amino side of a specific lysine that must be precisely positioned relative to the hydrophobic face of the alpha helix. This enzyme, which we propose to call "magaininase," represents a novel class of endopeptidases that hydrolyzes peptides on the basis of specific secondary structure rather than primary amino acid sequence.     

Subtilisin 72: a serine protease from Bac. subtilis strain 72 - an enzyme similar to subtilisin Carlsberg]

Subtilisin 72, a serine proteinase secreted by Bac. subtilis strain 72 was purified by covalent chromatography on Sepharose sorbent containing p-(omega-aminomethyl)phenylboronic acid as a ligand. The homogeneity of subtilisin 72 was confirmed by isoelectrofocusing in a thin layer of polyacrylamide gel (pl 8.6). The amino acid composition of this enzyme is different from that of other subtilisins, e. g. subtilisin Carlsberg. The N = terminal amino acid sequence of subtilisin 72 traced up to the 35th residue turned to be the same as that of subtilisin Carlsberg with the exception of the 21st (Tyr) and the 30th (Ile) residues. This very pronounced extent of homology shows that subtilisin 72 is very similar although not identical to subtilisin Carlsberg.     

Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule.

The N-terminal pro-peptide of 77 amino acid residues is essential for the folding of subtilisin, an alkaline serine protease from Bacillus subtilis. The synthetic pro-peptide has been shown to be capable of guiding the proper folding of denatured subtilisin to enzymatically active enzyme. Thus the pro-peptide serves as an intramolecular chaperone, which is removed by an autoprocessing reaction after the completion of the folding. With use of localized polymerase chain reaction random mutagenesis a total of 25 amino acid substitution mutations that affected subtilisin activities were isolated. These mutations occurred in a high frequency at the hydrophobic regions of the pro-peptide. For one of the mutations, M(-60)T, a second-site suppressor mutation, S(188)L, was isolated within the mature region. These results suggest that the pro-peptide consists of a few functional regions which interact with specific regions of the mature region of subtilisin during the folding process.     

红壤丘陵区水田和旱地土壤可溶性有机碳矿化对水分的响应

以亚热带红壤丘陵区典型水田和旱地土壤为研究对象,向土壤中添加¹⁴C标记稻草,培养30 d后,提取与原位土壤中结构相似的¹⁴C可溶性有机碳(DOC);将¹⁴C DOC加入水田和旱地土壤中,并设置45%、60%、75%、90%和105%田间持水量(WHC)5个水分梯度,在标准状态下(25 ℃)培养100 d,监测¹⁴C DOC在土壤中的矿化过程.结果表明: 培养100 d后,两种土壤中28.7%～61.4%的标记DOC被矿化为CO₂,且5个水分条件下,水田土壤DOC的矿化率均显著高于旱地,这主要是由于水田土壤DOC的结构组成比旱地土壤更简单.好气条件(56%～75%WHC)有利于两种土壤DOC的分解,淹水条件(105%WHC)则有利于DOC的积累.土壤处于好气条件(45%～90%WHC)时,DOC的生物可分解率及易分解态所占比例均随着含水量的增加而增加.100 d内,水田和旱地易分解态DOC分别占其累积矿化量的80.5%～91.1%和66.3%～72.4%,说明DOC的生物可分解率主要由其易分解态组分所占比例决定.     

三峡库区不同土地利用方式下土壤氧化亚氮排放及其影响因素

采用静态箱-气相色谱法对菜地、旱地、林地、果园、水改旱土壤N₂O排放特征及其相关影响因子进行研究.结果表明：不同土地利用方式下土壤N₂O的排放通量在-21～435 μg·m^-2·h^-1之间变化,N₂O年排放总量为菜地>果园>旱地>水改旱>林地,分别为447.14、313.57、167.00、124.87和7.24 mg·m^-2.土壤N₂O排放通量呈现明显的季节性变化,以春夏季最高,秋季次之,冬季最低,并与对应的大气及土壤温度的变化趋势基本一致.N₂O排放通量与5 cm土壤温度及土壤硝态氮含量呈显著或极显著正相关,与土壤水分及土壤铵态氮含量无明显相关关系.     

Proline-specific endopeptidases from microbial sources: isolation of an enzyme from a Xanthomonas sp.

An extensive screening among microorganisms for the presence of post-proline-specific endopeptidase activity was performed. This activity was found among ordinary bacteria from soil samples but not among fungi and actinomycetes. This result is in contrast to the previous notion that this activity is confined to the genus Flavobacterium. A proline endopeptidase was isolated from a Xanthomonas sp. and characterized with respect to physicochemical and enzymatic properties. The enzyme is composed of a single peptide chain with a molecular weight of 75,000. The isoelectric point is 6.2. It is inhibited by diisopropylfluorophosphate and may therefore be classified as a serine endopeptidase. The activity profile is bell shaped with an optimum at pH 7.5. By using synthetic peptide substrates and intramolecular fluorescence quenching it was possible to study the influence of substrate structure on the rate of hydrolysis. The enzyme specifically hydrolyzed Pro-X peptide bonds. With Glu at position X, low rates of hydrolysis were observed; otherwise the enzyme exhibited little preference for particular amino acid residues at position X. A similar substrate preference was observed with respect to the amino acid residue preceding the prolyl residue in the substrate. The enzyme required a minimum of two amino acid residues toward the N terminus from the scissile bond, but further elongation of the peptide chain by up to six amino acid residues caused only a threefold increase in the rate of hydrolysis. Attempts to cleave at the prolyl residues in oxidized RNase failed, indicating that the enzyme does not hydrolyze long peptides, a peculiar property it shares with other proline-specific endopeptidases.     

Activation of hageman factor and prekallikrein and generation of kinin by various microbial proteinases

Activation of the Hageman factor-kallikrein-kinin system by serratial 56-kDa proteinase was previously demonstrated (Matsumoto, K., Yamamoto, T., Kamata, T., and Maeda, H. (1984) J. Biochem. (Tokyo) 96, 739-749; Kamata, R., Yamamoto, T., Matsumoto, K., and Maeda, H. (1985) Infect. Immun. 48, 747-753). To investigate whether the activation of the system is specific for 56-kDa proteinase or is found similarly with other microbial proteinases, 11 proteinases of microbial origins were studied; the 56-kDa proteinase was the control. For in vitro studies, activation of guinea pig Hageman factor and prekallikrein was examined in purified systems as well as in plasma as a zymogen source. Specific antibodies and inhibitors confirmed the activation steps of the cascade. In the in vivo study the enhancement of vascular permeability in guinea pig skin and its sensitivity to inhibitors of activated Hageman factor, plasma kallikrein, or a kininase were examined. The results from the in vivo experiments were consistent with those in vitro. Taking all the data together, we classified the 11 microbial proteinases into three groups as follows: 1) Serratia marcescens 56-, 60-, and 73-kDa proteinases, Pseudomonas aeruginosa alkaline proteinase and elastase, and Aspergillus melleus proteinase (this group activated Hageman factor but not prekallikrein); 2) Vibrio vulnificus proteinase, subtilisin from Bacillus subtilis, and thermolysin from Bacillus stearothermophilus (this group activated both Hageman factor and prekallikrein); 3) Streptomyces caespitosus proteinase and V8 proteinase from Staphylococcus aureus (this group activated neither Hageman factor nor prekallikrein, but generated kinin from high molecular weight kininogen directly).     

上海市不同土地利用方式下的土壤碳氮特征

在野外采样和试验分析的基础上,研究了上海市土地利用方式及其变化对土壤有机碳、总氮含量及土壤有机碳密度的影响.结果表明： 上海不同土地利用方式下的土壤有机碳、总氮含量及有机碳密度均存在显著差异.不同土地利用方式下的土壤有机碳密度大小依次为：水稻田（3.86 kg·m^-2）＞旱地（3.17 kg·m^-2）＞林地（3.15 kg·m^-2）＞撂荒地（2.73 kg·m^-2）＞城市草坪（2.65 kg·m^-2）＞园地（2.13 kg·m^-2）＞滩涂（1.38 kg·m^-2）.通过相邻样地法,分析了水田转变为旱地、农田撂荒及水田转变为人工林地等3种土地利用变化对土壤有机碳、总氮的影响.由水田转化为旱地将导致土壤有机碳、总氮含量及有机碳密度显著降低;在水热充足、土壤肥沃、农田管理水平较高的长三角平原地区,农田撂荒并不是一种提高土壤有机碳储量的有效方式;水田转变为人工林地4～5年后,林地土壤有机碳、总氮含量及有机碳密度均低于相邻的水稻田,表明水田转变为林地并未引起土壤碳、氮的增加,从短期来看,人工林土壤有机碳的汇集效应因植被生产力水平的限制还处于较低水平.     

红壤丘陵景观单元土壤有机碳和微生物生物量碳含量特征

为了探讨我国亚热带红壤丘陵区不同利用方式下土壤有机碳(SOC)和土壤微生物生物量碳(SMB-C)含量的特征,在湖南省桃源县选取典型样区,通过密集取样,分析了红壤丘陵景观单元内水田、旱地、林地、果园4种典型利用方式下表层土壤(0～20 cm)SOC和SMB-C含量.结果表明,典型红壤丘陵景观单元中SOC含量高低的顺序为水田(16.0 g·kg^-1)＞旱地(11.2 g·kg^-1) ＞果园(9.5 g·kg^-1)＞林地(8.4 g·kg^-1),SMB-C含量则为水田(830 mg·kg^-1)＞旱地(361 mg·kg^-1)＞林地(200 mg·kg^-1)＞果园(186 mg·kg^-1),且在不同利用方式下SOC与SMB-C均呈极显著正相关(P＜0.01),说明本研究区内各土地利用类型的土壤SMB-C含量变化可以敏感地指示SOC的动态.研究结果还表明,将我国亚热带红壤丘陵林地开垦为果园或耕地后,表层土壤 SOC含量不可能降低.