Enhanced Thermostability of Pseudomonas nitroreducens Isoeugenol Monooxygenase by the Combinatorial Strategy of Surface Residue Replacement and Consensus Mutagenesis

Vanillin has many applications in industries. Isoeugenol monooxygenase (IEM) can catalyze the oxidation of isoeugenol to vanillin in the presence of oxygen under mild conditions. However, the low thermal stability of IEM limits its practical application in the biosynthesis of natural vanillin. Herein, two rational strategies were combined to improve the thermostability of IEM from Pseudomonas nitroreducens Jin1. Two variants (K83R and K95R) with better thermostability and one mutant (G398A) with higher activity were identified from twenty candidates based on the Surface Residue Replacement method. According to the Consensus Mutagenesis method, one mutant (I352R) with better thermostability and another mutant (L273F) with higher activity were also identified from nine candidates. After combinatorial mutation, a triple mutant K83R/K95R/L273F with the best thermostability and catalytic efficiency was generated. Compared with the wild-type IEM, the thermal inactivation half-lives (t1/2) of K83R/K95R/L273F at 25 °C, 30 °C, and 35 °C increased 2.9-fold, 11.9-fold, and 24.7-fold, respectively. Simultaneously, it also exhibited a 4.8-fold increase in kcat, leading to a 1.2-fold increase in catalytic efficiency (kcat/Km). When the whole cell of K83R/K95R/L273F was applied to the biotransformation of isoeugenol on preparative scale, the vanillin concentration reached 240.1 mM with space-time yield of 109.6 g/L/d, and vanillin was achieved in 77.6% isolated yield and >99% purity.

Soil compaction and cover with black oat on soybean grain yield in lowland under no-tillage system

ABSTRACT: Brazil is the largest exporter and second largest producer of soybean grains. Most of this production is from plants grown under no-tillage system (NT). This research evaluated the effect of soil compaction, and different amounts of black oat residues on the soil surface on soybean growth and grain yield in lowland under NT. The experiment was conducted in a completely randomized design with seven treatments and four replications, in the 2016/2017 and 2017/2018 crop seasons. The treatments consisted of: 1) winter fallow without soil compaction (WF); 2) winter fallow with soil compaction (WF-C); 3) black oats and complete removal of surface residues, with soil compaction (0R-C); 4) black oats and removal half of surface residues, with soil compaction (0.5R-C); 5) black oats without surface residue removal, with soil compaction (1R-C); 6) black oats without surface residue removal, plus the residues from treatment 3, with soil compaction (2R-C); 7) black oats without surface residue removal, without soil compaction (1R). When the soybean plants were at the phenological stage R2, they were evaluated nodule, root and shoot dry matter, nitrogen contents, plant height, and grain yield. The soil physical properties were evaluated in the 0.0-0.05, 0.10-0.15 and 0.20-0.25 m layers. The soybean aerial dry matter is > 38% in non-compacted soil in year with soil water excess, regardless of the amount of surface oat straw. In year with small water deficit, soil with more surface oat straw produced > 5% shoot dry matter and > 4% of soybean grain, regardless of compaction. The plant growth and grain yield soybean in lowland varied according to the water conditions, and were affected by soil compaction and amounts of black oats residues on soil surface.

A conserved evolutionary mechanism permits Δ9 desaturation of very-long-chain fatty acyl lipids

Δ9 fatty acyl desaturases introduce a cis–double bond between C9 and C10 of saturated fatty acyl chains. From the crystal structure of the mouse stearoyl-CoA desaturase (mSCD1) it was proposed that Tyr-104, a surface residue located at the distal end of the fatty acyl binding pocket plays a key role in specifying 18C selectivity. We created mSCD1-Y104G to test the hypothesis that eliminating this bulky side chain would create an opening and permit the substrate's methyl end to protrude through the enzyme into the lipid bilayer, facilitating the desaturation of very-long-chain (VLC) substrates. Consistent with this hypothesis, Y104G acquired the ability to desaturate 24C and 26C acyl-CoAs while maintaining its Δ9-regioselectivity. We also investigated two distantly related very-long-chain fatty acyl (VLCFA) desaturases from Arabidopsis, ADS1.2 and ADS1.4, which have Ala and Gly, respectively, in place of the gatekeeping Tyr found in mSCD1. Substitution of Tyr for Ala and Gly in ADS1.2 and ADS1.4, respectively, blocked their ability to desaturate VLCFAs. Further, we identified a pair of fungal desaturase homologs which contained either an Ile or a Gly at this location and showed that only the Gly-containing desaturase was capable of very-long-chain desaturation. The conserved desaturase architecture wherein a surface residue with a single bulky side chain forms the end of the substrate binding cavity predisposes them to single amino acid substitutions that enable a switch between long- and very-long-chain selectivity. The data presented here show that such changes have independently occurred multiple times during evolution.

Effects of the surface polarity of nanomaterials on their interaction with complement protein gC1q

The dependence of the recognition mode of gC1q on the surface polarity of nanomaterials is mainly attributed to its distinct surface residue composition.

Fuzzy Object-Based Image Analysis Methods Using Sentinel-2A and Landsat-8 Data to Map and Characterize Soil Surface Residue

Soil degradation, defined as the lowering and loss of soil functions, is becoming a serious problem worldwide and threatens agricultural production and terrestrial ecosystems. The surface residue of crops is one of the most effective erosion control measures and it increases the soil moisture content. In some areas of the world, the management of soil surface residue (SSR) is crucial for increasing soil fertility, maintaining high soil carbon levels, and reducing the degradation of soil due to rain and wind erosion. Standard methods of measuring the residue cover are time and labor intensive, but remote sensing can support the monitoring of conservation tillage practices applied to large fields. We investigated the potential of per-pixel and object-based image analysis (OBIA) for detecting and estimating the coverage of SSRs after tillage and planting practices for agricultural research fields in Iran using tillage indices for Landsat-8 and novel indices for Sentinel-2A. For validation, SSR was measured in the field through line transects at the beginning of the agricultural season (prior to autumn crop planting). Per-pixel approaches for Landsat-8 satellite images using normalized difference tillage index (NDTI) and simple tillage index (STI) yielded coefficient of determination (R2) values of 0.727 and 0.722, respectively. We developed comparable novel indices for Sentinel-2A satellite data that yielded R2 values of 0.760 and 0.759 for NDTI and STI, respectively, which means that the Sentinel data better matched the ground truth data. We tested several OBIA methods and achieved very high overall accuracies of up to 0.948 for Sentinel-2A and 0.891 for Landsat-8 with a membership function method. The OBIA methods clearly outperformed per-pixel approaches in estimating SSR and bear the potential to substitute or complement ground-based techniques.