Research on dicyandiamide as a nitrification inhibitor and future outlook

Abstract

The action and decomposition of dicyandiamide (DCD), a nitrification inhibitor, is discussed.

DCD is especially efficient when used with animal manure slurries or potato starch waste water. As a consequence, nitrate leaching can be reduced, yields and N uptake increased. DCD‐amended mineral N fertilizers applied once can substitute for split N applications, thus reducing labor costs without any loss in crop yield and quality. With wheat and sugar beets, use of a DCD‐containing product ("Alzon 22") reduced the requirement of N for maximum yield. New formulations, such as DCD plus a reducing substance, ammonium thiosulfate (ATS), or new inhibitors, such as guanylthiourea (GTU), will receive more attention in the future.     

Nitrous oxide emissions from animal urine as affected by season and a nitrification inhibitor dicyandiamide

Purpose  

Nitrous oxide emissions from pasture soils account for one third of total agricultural greenhouse gas emissions in New Zealand. The aim of this study was to determine nitrous oxide (N₂O) emissions from animal urine patches under summer (with irrigation) and winter conditions as affected by dicyandiamide (DCD) in grazed grassland in New Zealand.     

Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.     

Kunitz-type serine protease inhibitor from potato (Solanum tuberosum L. cv. Jopung)

An antifungal protein, AFP-J, was purified from tubers of the potato (Solanum tuberosum cv. L Jopung) by various chromatographic columns. AFP-J strongly inhibited yeast fungal strains, including Candida albicans, Trichosporon beigelii, and Saccharomyces cerevisiae, whereas it exhibited no activity against crop fungal pathogens. Automated Edman degradation determined the partial N-terminal sequence of AFP-J to be NH2-Leu-Pro-Ser-Asp-Ala-Thr-Leu-Val-Leu-Asp-Gln-Thr-Gly-Lys-G lu-Leu-Asp-Ala-Arg-Leu-. The partially sequence had 83% homology with a serine protease inhibitor belonging to the Kunitz family, and the protein inhibited chymotrypsin, pepsin, and trypsin. Mass spectrometry showed that its molecular mass was 13 500.5 Da. This protease inhibitor suppressed over 50% the proteolytic activity at 400 microg/mL. These results suggest that AFP-J is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

脲酶抑制剂氢醌对土壤脲酶动力学行为的调控效应

研究了不同温度条件下脲酶抑制剂氢醌(HQ)对东北3种典型土壤(白浆土、棕壤、褐土)脲酶动力学参数的影响。结果表明,土壤类型、培养时间、培养温度及其相互作用均显著影响土壤脲酶动力学参数。与对照相比,加入HQ使土壤脲酶米氏常数(Km)增加,最大反应速率(Vmax)降低,表明HQ对土壤脲酶的作用机理属于混合型抑制。与白浆土相比,棕壤和褐土脲酶动力学参数受HQ的影响程度较大,表明高肥力土壤生物学活性较稳定。随着培养时间延长,土壤脲酶Km降低,Vmax和Vmax/Km增加。随着温度升高,土壤脲酶Km和Vmax增加,Vamx/Km无规律性变化。相关性分析表明,土壤脲酶动力学参数Km、Vmax和Vmax/Km与p H值、有机质、全氮、碱解氮和质地组成之间存在显著相关关系。     

Protein extraction from heat-stabilized defatted rice bran. 1. Physical processing and enzyme treatments

Physical processing with or without enzyme treatments on protein extraction from heat-stabilized defatted rice bran (HDRB) was evaluated. Freeze-thaw, sonication, high-speed blending, and high-pressure methods extracted 12%, 15%, 16%, and 11% protein, respectively. Sonication (0-100%, 750 W), followed by amylase and combined amylase and protease treatments, extracted 25.6-33.9% and 54.0-57.8% protein, respectively. Blending followed by amylase and protease treatment extracted 5.0% more protein than the nonblended enzymatic treatments. High-pressure treatments, 0-800 MPa, with water or amylase-protease combinations, extracted 10.5-11.1% or 61.8-66.6% protein, respectively. These results suggest that physical processing in combination with enzyme treatments can be effective in extracting protein from HDRB.     

Minor diterpenoids from cascarilla (Croton eluteria Bennet) and evaluation of the cascarilla extract and cascarillin effects on gastric acid secretion

Three new diterpenoids belonging to the clerodane (2-3) and halimane (4) structural types have been isolated from the bark of Croton eluteria Bennet, commonly known as cascarilla. Their structures have been fully characterized by spectroscopic means. Cascarilla extract and its major component, cascarillin, were found to significantly increase histamine-induced gastric acid secretion in the mouse stomach. These preliminary results provide the first rationale for the use of cascarilla in bitter preparations aimed at improving digestion.     

不同生境薇菜根际土壤养分与酶活力研究

为研究薇菜根际土壤养分与酶活力对薇菜生长性状指标的影响,调查了4种生境薇菜生长性状指标,并利用土壤学方法研究根际土壤养分与酶活力。结果表明,两种野生生境的薇菜生长性状指标极显著的优于栽培的薇菜,野生薇菜平均株高比栽培的高11.30%~19.54%,平均地径比栽培的高7.63%~20.21%,平均发叶数比栽培的高5.13%~23.08%;土壤有机质、碱解氮、脲酶活力及蛋白酶活力对薇菜生长性状指标有显著影响,薇菜产量最高的铜盆水的这几项指标均最高,分别为栽培的3.7、4.5、3.4、12.4倍。     

Angiotensin I converting enzyme inhibitory peptides purified from bovine skin gelatin hydrolysate.

Bovine skin gelatin was hydrolyzed with sequential protease treatments in the order of Alcalase, Pronase E, and collagenase using a three-step ultrafiltration membrane reactor. The molecular weight distributions of the first, second, and third hydrolysates were 4.8-6.6, 3.4-6.6, and 0.9-1.9 kDa, respectively. The angiotensin I converting enzyme (ACE) inhibitory activity of the third hydrolysate (IC(50) = 0.689 mg/mL) was higher than that of the first and second hydrolysates. Two different peptides showing strong ACE inhibitory activity were isolated from the hydrolysate using consecutive chromatographic methods including gel filtration chromatography, ion-exchange chromatography, and reversed-phase high-performance liquid chromatography. The isolated peptides were composed of Gly-Pro-Leu and Gly-Pro-Val and showed IC(50) values of 2.55 and 4.67 microM, respectively.     

The most abundant protease inhibitor in potato tuber (cv. Elkana) is a serine protease inhibitor from the Kunitz family

The gene of the most abundant protease inhibitor in potato cv. Elkana was isolated and sequenced. The deduced amino acid sequence of this gene showed 98% identity with potato serine protease inhibitor (PSPI), a member of the Kunitz family. Therefore, the most abundant protease inhibitor was considered to be one of the isoforms of PSPI. The PSPI group represents approximately 22% of the total amount of proteins in potato cv. Elkana and is composed of seven different isoforms that slightly differ in isoelectric point. Antibodies were raised against the two most abundant isoforms of PSPI. The binding of these antibodies to PSPI isoforms and protease inhibitors from different groups of protease inhibitor in potato showed that approximately 70% of the protease inhibitors present in potato juice belong to the Kunitz family.     

Influence of soil parameters on the effect of 3,4-dimethylpyrazole-phosphate as a nitrification inhibitor

Nitrification inhibitors specifically retard the oxidation of NH₄⁺ to NO₂^- during the nitrification process in soil. In this study, the influence of soil properties on the nitrification-inhibiting effect of 3,4-dimethylpyrazole-phosphate (DMPP), a newly developed nitrification inhibitor, has been investigated. Based on short-term incubation experiments, where the degradation of DMPP could be largely disregarded, the oxidation of the applied NH₄⁺ was more inhibited in sandy soils compared with loamy soils. The influence of soil parameters on the relative NO₂^- formation could be described by a multiple regression model including the sand fraction, soil H⁺ concentration and soil catalase activity (R²=0.62). Adsorption studies showed that the binding behaviour of DMPP was influenced markedly by soil textural properties, viz. the clay fraction (r²=0.61). The adsorption of DMPP was found to be an important factor for the inhibitory effect on NH₄⁺ oxidation in a short-term incubation (r²=0.57). It is concluded that the evaluated soil properties can be used to predict the short-term inhibitory effect of DMPP in different soils. The significance of these results for long-term experiments under laboratory and field conditions needs further investigation.     

Novel protein from Labramia bojeri A. DC. seeds homologue to Kunitz-type trypsin inhibitor with lectin-like properties

This study starts by isolating and characterizing the first protein from Labramia bojeri seeds, which belong to the Sapotaceae family. The purified lectin analyzed by SDS-PAGE with and without beta-mercaptoethanol shows two protein bands (M(r) = 19 and 20 kDa), which cannot be resolved. Protein bands have shown similar characteristics as molecular masses, determined by gel filtration and native gel; N-terminal sequences presented a difference in their isoelectric points. We have suggested that those protein bands might be variants of the protein named Labramin. The sequence database search has shown that the N-terminal sequence of Labramin presented a high degree of homology to Kunitz-type trypsin inhibitor (82-52%) despite no trypsin inhibition activity detection. The lectin-like form from Labramin was better inhibited by glycoproteins and has also presented growth inhibition of the fungus Colletotrichum lindemuthianum and the yeast Saccharomyces cerevisiae, but it has not presented an apparent effect on Fusarium oxysporum.     

A Kunitz-type inhibitor of coleopteran proteases, isolated from Adenanthera pavonina L. seeds and its effect on Callosobruchus maculatus

The cowpea weevil Callosobruchus maculatus is one of the major pests of Vigna unguiculata cowpea. Digestion in the cowpea weevil is facilitated by high levels of cysteine and aspartic acid proteinases. Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves against attack by insects. In this work, a trypsin inhibitor (ApTI) isolated from Adenanthera pavonina seeds showed activity against papain. The inhibition of papain by ApTI was of the noncompetitive type, with a K(i) of 1 microM. ApTI was highly effective against digestive proteinases from C. maculatus, Acanthoscelides obtectus (bean weevil), and Zabrotes subfasciatus (Mexican bean weevil) and was moderately active against midgut proteinases from the boll weevil Anthonomus grandis and the mealworm Tenebrio molitor. In C. maculates fed an artificial diet containing 0.25% and 0.5% ApTI (w/w), the latter concentration caused 50% mortality and reduced larval weight gain by approximately 40%. The action of ApTI on C. maculatus larvae may involve the inhibition of ApTI-sensitive cysteine proteinases and binding to chitin components of the peritrophic membrane (or equivalent structures) in the weevil midgut.     

Soil moisture pre-treatment effects on enzyme activities as indicators of heavy metal-contaminated and reclaimed soils

Heavy metal contamination can inhibit soil functions but it is often difficult to determine the degree of pollution or when soil reclamation is complete. Enzyme assays offer potential as indicators of biological functioning of soils. However, antecedent water content of soil samples may affect the outcome of biological measurements. In Mediterranean regions, for much of the year ‘field moist’ surface soil can have water content similar to that of air-dry samples. The objectives of this study were to: (1) determine the sensitivity of a range of enzyme assays to detect the degree of pollution from a heavy metal mine spill; (2) evaluate rewetting field-dry soil as a pre-treatment for enzyme assays; and (3) test multivariate analysis for improving discrimination between polluted, reclaimed and non-polluted soils. The Aznalcóllar mining effluent spill provided a unique opportunity to address these objectives. This accident released toxic, heavy metal-contaminated (As, Bi, Cd, Cu, Pb, Tl, Zn…) and acid tailings into the Guadiamar watershed (SW Spain) in 1998, severely affecting the riparian zone along more than 4000 ha. Contaminated soils were collected from the highly polluted upper watershed and less polluted lower watershed along with reclaimed soil at both sites. Enzyme activities (phosphatases, arylsulfatase, β-glucosidase, urease and dehydrogenase) were assessed on both field-moist samples and soils rewetted to 80% of water-holding capacity and then incubated at 21 °C for 7 d prior to the assay. The reclaimed soils had higher activities than polluted soils but, typically, 1.5-3 times lower levels of activity than the non-polluted soil. Regardless of the moisture pre-treatment, all enzymes showed significant effects due to pollution, with urease and β-glucosidase showing the greatest discrimination between degrees of contamination. In general, rewetting field-dried soils increased activities on non-polluted and reclaimed soils which improved discrimination with polluted soils. Another method to increase the potential of soil enzyme activities to detect soil contamination could be to combine them in multivariate analysis, which provides a more holistic representation of the biochemical and microbial functionality of a soil.     

Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian traditional fermented shrimp paste

Bacillus sp. nov. SK006 producing four extracellular fibrinolytic enzymes was isolated from fermented shrimp paste, a traditional and popular Asian seasoning. One fibrinolytic enzyme was purified to homogeneity with a molecular mass of 43-46 kDa by SDS-PAGE and gel filtration chromatography. The specific activity was determined to be 11.2 units/mg using plasmin as a standard. The enzyme displayed optimal activity at 30 degrees C and pH 7.2. It was stable below 40 degrees C for 4 h between pH 5.0 and pH 11.0. Zinc ion stimulated the enzyme activity whereas Cu2+, Ca2+, Fe3+, and Hg2+ caused its inhibition. The fibrinolytic activity was strongly inhibited by PMSF and moderately inhibited by EDTA as well as PCMB. The enzyme exhibited a higher affinity toward N-Succ-Ala-Ala-Pro-Phe-pNA and was able to degrade fibrin clots either by forming active plasmin from plasminogen or by direct fibrinolysis. The N-terminal amino acid sequence was found to be AQSVPYEQPHLSQ, which is different from that of other known fibrinolytic enzymes.     

Characterization of "lettucine", a serine-like protease from Lactuca sativa leaves, as a novel enzyme for milk clotting

In this work we focused on the characterization of a novel plant rennet purified from lettuce leaves (Lactuca sativa L. cv Romana). The lettuce protease, lettucine, showed trypsin-like, SV8-like, and caseinolytic activities. Although the enzyme did not recognize peptides having hydrophobic amino acid residues in the P(1) position of the target bond, it did show milk-clotting activity, suggesting that different bonds rather than the Phe(105)-Met(106) of the kappa-casein might be cleaved, still inducing milk-clotting. The enzyme exhibited proteolytic activity toward alpha-casein, beta-casein, kappa-casein, and milks with different fat contents, with the highest activity observed with partially skimmed milk, total casein, and alpha- and kappa-casein. SDS-PAGE studies showed that lettucine cleaved alpha-casein, beta-casein, and kappa-casein. In particular, we showed that alpha-casein breakdown occurred even though total casein or milks were supplied, suggesting that the lettuce enzyme is able to operate a significant disorganization of the casein's micellar structure. Moreover, the proteolytic activity of the enzyme analyzed under various technological parameters, such as temperature and pH, indicated that the lettuce enzyme is highly consistent with the milk-clotting process.     

Pectin methylesterase from kiwi and kaki fruits: purification, characterization, and role of pH in the enzyme regulation and interaction with the kiwi proteinaceous inhibitor

Pectin methylesterase was purified from kiwi (Actinidia chinensis) and kaki fruit (Diospyros kaki). The pH values of the fruit homogenates were 3.5 and 6.2, respectively. The kiwi enzyme is localized in the cell wall and has a neutral-alkaline pI, whereas the kaki enzyme is localized in the soluble fraction and has a neutral-acidic pI. The molecular weights of the kiwi and kaki enzymes were 50 and 37 kDa, respectively. The two enzymes showed a similar salt and pH dependence of activity, and a different pH dependence of the inhibition by the kiwi proteinaceous inhibitor.