Extracellular prolyl endoprotease from Aspergillus niger and its use in the debittering of protein hydrolysates

The observation that the bitterest peptides from casein hydrolysates contain several proline residues led us to hypothesize that a proline-specific protease would be instrumental in debittering such peptides. To identify the desired proline-specific activity, a microbiological screening was carried out in which the chromogenic peptide benzyloxycarbonyl-glycine-proline-p-nitroanilide (Z-Gly-Pro-pNA) was used as the substrate. An Aspergillus niger (A. niger) strain was identified that produces an extracellular proline-specific protease with an acidic pH optimum. On the basis of sequence similarities, we conclude that the A. niger-derived enzyme probably belongs to the S28 family of clan SC of serine proteases rather than the S9 family to which prolyl oligopeptidases belong. Incubating the overexpressed and purified enzyme with bitter casein hydrolysates showed a major debittering effect. Reversed phase HPLC analysis revealed that this debittering effect is accompanied by a significant reduction of the number of hydrophobic peptides present.     

Organic acid concentrations in soil solution: effects of young coniferous trees and ectomycorrhizal fungi

Organic acids may play a key role in rhizosphere and pedogenic processes. The effects of young trees and ectomycorrhizas on the soil solution concentrations of low molecular weight organic acids (LMWOAs) were studied in soil columns (E horizon) in the presence or absence of Pinus sylvestris and Picea abies with or without three ectomycorrhizal fungi. Several LMWOAs were identified at concentrations ranging from <0.1 to 11 μM. Compared to soil columns without tree seedlings, the presence of non-mycorrhizal or mycorrhizal tree seedlings sometimes resulted in small but statistically significant increases in citrate, formate, malonate and oxalate concentration. The general nutrient concentration and low P had little short-term effect on soil solution organic acid concentrations. The results suggest that biodegradation rather than production may be the major factor regulating soil solution organic acid concentrations.     

Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting

 We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns obtained with target DNA extracted from a soil by direct and indirect methods. For this purpose, two direct extraction methods, i.e. cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, i.e. cell extraction followed by DNA extraction, and combined RNA/DNA extraction from the bacterial cell fraction, were performed. Crude extracts were purified and amplified using universal bacterial primers. PCR products were then analysed by DGGE, and similarity between the profiles obtained was determined by unweighted pair group with mathematical averages clustering. The results showed clear profiles that presumably represented the dominant bacterial fractions in the samples. The profiles generated by all four methods were similar, indicating that the methods were of approximately equal efficiency in the extraction of target DNA representative of the soil bacterial community. However, the patterns of clustering also indicated that different populations of bacteria could be detected in the same soil using different soil DNA extraction methods. The application of two dilution levels of DNA in PCR-DGGE showed that the most stable profile of the soil bacterial community could be generated by the direct methods. The indirect methods gave clustered profiles at both dilution levels. It is likely that these methods extracted DNA from a major, easily desorbed, bacterial fraction, consisting of low-density populations. PCR-DGGE was found to be a suitable technique with which to assess differences in methods for DNA extraction from soil, which can be further used for the determination of microbial community diversity at the molecular level. Received: 22 June 1999     

Arbuscular mycorrhizae in a long-term field trial comparing low-input (organic, biological) and high-input (conventional) farming systems in a crop rotation

 Arbuscular mycorrhizal (AM) root colonization was studied in a long-term field trial in which four farming systems currently in use in Switzerland were continuously applied to a randomized set of plots at a single field site from 1978 till 1993. There were two low-input farming systems (organic and bio-dynamic) and two high-input (conventional) farming systems (according to Swiss guidelines of integrated plant production with and without farmyard manure). The systems had an identical 7-year crop rotation and tillage scheme and differed essentially only in the amount and type of fertilizer supplied and in plant protection management. The percentage of root colonization by AM fungi was determined in field samples 2–3 times over the growing season in crops in the rotation, namely in winter wheat (Triticum aestivum L. cv. Sardona), vetch-rye and grass-clover. We found the percentage of root length colonized by AM fungi to be 30–60% higher (P≤0.05) in the plants grown in soils from the low-input farming systems than in those grown in conventionally farmed soils. Approximately 50% of the variation of AM root colonization was explained by chemical properties of the soils (pH, soluble P and K, exchangeable Mg), the effect of soluble soil P being most pronounced. The potential of the field soils from the differently managed plots to cause symbiosis with AM fungi was tested in a glasshouse experiment, using wheat as a host plant. Soils from the low-input farming systems had a greatly enhanced capacity to initiate AM symbiosis. The relative differences in this capacity remained similar when propagules of the AM fungus Glomus mosseae were experimentally added to the soils, although overall root colonization by AM fungi was 2.8 times higher. Received: 27 August 1999     

Catechin contents of foods commonly consumed in The Netherlands. 1. Fruits, vegetables, staple foods, and processed foods

Catechins, compounds that belong to the flavonoid class, are potentially beneficial to human health. To enable epidemiological evaluation of these compounds, data on their contents in foods are required. HPLC with UV and fluorescence detection was used to determine the levels of (+)-catechin, (-)-epicatechin, (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) in 24 types of fruits, 27 types of vegetables and legumes, some staple foods, and processed foods commonly consumed in The Netherlands. Most fruits, chocolate, and some legumes contained catechins. Levels varied to a large extent: from 4.5 mg/kg in kiwi fruit to 610 mg/kg in black chocolate. (+)-Catechin and (-)-epicatechin were the predominant catechins; GC, EGC, and ECg were detected in some foods, but none of the foods contained EGCg. The data reported here provide a base for the epidemiological evaluation of the effect of catechins on the risk for chronic diseases.     

Influence of reduced tillage on earthworm and microbial communities under organic arable farming

Although reduced tillage is an agricultural practice reported to decrease soil erosion and external inputs while enhancing soil fertility, it has still rarely been adopted by European organic farmers. The objective of this study was to assess the long-term interactive effects of tillage (conventional (CT) vs. reduced (RT)) and fertilization (slurry (S) vs. composted manure/slurry (MCS)) on earthworms and microbial communities in a clay soil under spelt in an organic 6-year crop rotation. Earthworm populations (species, density and biomass, cocoons) were investigated by handsorting the soil nine years after initial implementation of the treatments. Soil microbial carbon (C_mic) and nitrogen (N_mic) were measured by chloroform-fumigation extraction and a simplified phospholipid fatty acid (PLFA) analysis was used to separate for populations of bacteria, fungi and protozoa. Significantly increased total earthworm density in RT plots was mainly attributed to increased numbers of juveniles. Moreover, we found five times more cocoons with RT. Species richness was not affected by the treatments, but tillage treatments had differentially affected populations at the species-level. In addition, cluster analysis at the community level revealed two distinct groups of plots in relation to tillage treatments. In RT plots C_mic increased in the 0–10 cm and 10–20 cm soil layers, while PLFA concentrations indicative of Gram-negative bacteria, fungi and protozoa only increased in the topsoil. Lower bacteria-to-fungi ratios in the upper soil layer of RT plots indicated a shift to fungal-based decomposition of organic matter whereas a higher C_mic-to-C_org ratio pointed towards enhanced substrate availability. Slurry application decreased microbial biomass and enhanced density of juvenile anecic earthworms but overall fertilization effect was weak and no interactions with tillage were found. In conclusion, tillage is a major driver in altering communities of earthworms and microorganisms in arable soils. The use of reduced tillage provides an approach for eco-intensification by enhancing inherent soil biota functions under organic arable farming.     

Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.     

Colonization of wheat seedling (Triticum aestivum) roots by Pseudomonas fluorescens and Bacillus subtilis

Summary Colonization patterns of Pseudomonas fluorescens and Bacillus subtilis on roots of wheat seedlings growing on water agar were studied qualitatively by replica printing and quantitatively by the plate count method. The results indicated a stronger colonization potential for P. fluorescens (up to 10⁷ cfu/cm root) than for B. subtilis (up to 10⁵ cfu/cm root). Although the numbers of both species were lower when inoculated together, the observed colonization patterns on the roots were comparable to those found with single inoculations. For none of these bacteria was active migration along the root surface in any direction observed, indicating that distal positions are reached mainly by a passive displacement on the root tip and elongating cells. Ecological implications of the observed phenomena are discussed.