Stabilized variant of Streptomyces subtilisin inhibitor and its use in stabilizing subtilisin BPN'

Protein protease inhibitors could potentially be used to stabilize proteases in commercial products such as liquid laundry detergents. However, many protein protease inhibitors are susceptible to hydrolysis inflicted by the protease. We have engineered Streptomyces subtilisin inhibitor (SSI) to resist proteolysis by adding an interchain disulfide bond and removing a subtilisin cleavage site at leucine 63. When these stabilizing changes were combined with changes to optimize the affinity for subtilisin, the resulting inhibitor provided complete protease stability for at least 5 months at 31 degrees C in a subtilisin-containing liquid laundry detergent and allowed full recovery of the subtilisin activity upon the dilution that occurs in a North American washing machine.     

A Stingless Bee (Melipona seminigra) Marks Food Sources with a Pheromone from Its Claw Retractor Tendons

By depositing scent marks on flowers, bees reduce both the search time and the time spent with the handling of nonrewarding flowers. They thereby improve the efficiency of foraging. Whereas in honey bees the source of these scent marks is unknown, it is assumed to be the tarsal glands in bumble bees. According to histological studies, however, the tarsal glands lack any openings to the outside. Foragers of the stingless bee Melipona seminigra have previously been shown to deposit an attractant pheromone at sugar solution feeders, which is secreted at the tips of their tarsi. Here we show that the claw retractor tendons have specialized glandular epithelia within the femur and tibia of all legs that produce this pheromone. The secretion accumulates within the hollow tendon, which also serves as the duct to the outside, and is released from an opening at the base of the unguitractor plate. In choice experiments, M. seminigra was attracted by feeders baited with pentane extracts of the claw retractor tendons in the same way as it was attracted by feeders previously scent marked by foragers. Our results resolve the seeming contradiction between the importance of foot print secretions and the lack of openings of the tarsal glands.     

Synthesis of new boron-rich building blocks for boron neutron capture therapy or energy-filtering transmission electron microscopy

The synthesis of a new ortho-carborane derivative, tetracarboranylketone 4, is reported here. Ketone 4 was prepared from a tetraalkynylated ketone by the addition of decaborane. The keto group was then easily modified to yield the glycosides 17alpha and 18beta, which contain glucose or galactose, respectively, and the nucleotide 13b. In addition to ketone 4, which is acyclic, cyclic ketone 8 was also synthesised. X-ray diffraction analysis of compound 4 indicated the presence of two toluene guest molecules per molecule of the host compound. Furthermore, compound 4 displays a rather low cytotoxicity. These novel products can be used as building blocks to create a new class of biomolecules containing high-density carborane clusters. Such molecules may constitute powerful tools for applications like Boron Neutron Capture Therapy or Energy-Filtering Transmission Electron Microscopy.     

Identification of Two Sex Pheromone Components of the Potato Aphid, Macrosiphum euphorbiae (Thomas)

Females of the potato aphid Macrosiphum euphorbiae exhibit typical calling behavior, with virgin female oviparae raising their back legs off the substrate to release sex pheromone from glands on the tibia. Airborne collections from calling oviparae were analyzed by GC and GC-MS to determine if, like the majority of aphids examined to date, they produced (1R,4aS,7S,7aR)-nepetalactol (1) and (4aS,7S,7aR)-nepetalactone (2). Both components were present and produced in ratios that varied with age from 4:1 to 2:1. The relative stereochemical configurations of these components were determined by GC-coinjection of the aphid-derived sample with synthetic standards on both HP-1 and DB-Wax GC columns. The absolute stereochemical configuration of the nepetalactol (determined from approximately 15 microg of material in an air entrainment sample) was determined as (1R,4aS,7S,7aR)-1 by derivatization of the aphid sample with (S)-(+)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride (Mosher's acid chloride) to generate a diastereoisomer that was compared to synthetic samples by NMR spectroscopy and GC. In bioassays in the wind tunnel, M. euphorbiae males responded to potato plants with oviparae but not to unattacked plants or those infested with parthenogenetically reproducing apterae. In no-choice laboratory bioassays, the same level of male response was observed to virgins and to the 3:1-5:1 synthetic blends of nepetalactol (1):nepetalactone (2). However, the time taken to reach the source was significantly less to virgin females than to the synthetic pheromone blends. In all cases, males walked rather than flew to the source. Males showed lower responses to a 1:1 synthetic mixture and did not respond to either of the components when presented alone. Under field conditions, few M. euphorbiae males were captured in traps baited with different ratios of the synthetic pheromone. Possible reasons for the different responses under laboratory and field conditions are discussed.     

Improving the design of depth filters: A model‐based method using optimal control theory

Because there is no general design method for depth filters, especially for layered configurations, this methodological gap is addressed here. Using optimal control theory, paths of the filter coefficient, a measure for local filtration performance, are determined along the filter depth. An analytical optimal control solution is derived and used to validate the numerical algorithm. Two optimal control scenarios are solved numerically: In the first scenario, the goal of constant deposition along the filter depth is addressed. The second scenario aims at maximizing the time until some maximal pressure drop is reached. Furthermore, a computational strategy is presented to derive discrete layers suitable for practical design from the continuous optimal control solutions. All optimized scenarios are compared to one‐layered filter designs and significant improvements are found. As this work is based on strongly validated and widely used filtration models, the presented methods are expected to have broad applicability. © 2017 American Institute of Chemical Engineers AIChE J, 63: 68–76, 2018     

Adhesion between oppositely charged polyelectrolytes

The adhesion between a grafted polyelectrolyte layer (brush) and a gel of an oppositely charged polyelectrolyte has been measured as a function of applied pressure, and the interface has been traced using neutron reflectometry. The interface (in aqueous medium at pH 6) between the (polycationic) brush and the (polyanionic) gel has a limited pressure dependence, with a small amount of deformation of the interface at the brush–gel contact. Brushes with a dry thickness of up to 13 nm exhibit weak adhesion (measured using a mechanical force tester) with an adhesive failure when the gel is detached. Thicker brushes result in the gel exhibiting cohesive failure. Reversing the geometry, whereby a polycationic brush is replaced with a polyanion and the polyanionic gel is replaced with a polycation, reveals that the pH dependence of the adhesion is moderately symmetric about pH 6, but that the maximum force required to separate the polycation gel from the polyanion brush over the range of pH is greater than that for the polycation brush and polyanion gel. The polyanion used is poly(methacrylic acid) (PMAA) and polycations of poly[2-(diethylamino)ethyl methacrylate] (PDEAEMA) and poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) were used.     

Enhanced gas migration through permeable bubble networks within consolidated soft sediments

Many consolidated sediments experience in situ gas generation from methanogenesis, corrosion, or radiolysis reactions and can retain bubbles for long periods. Particular interest is motivated by the retention and acute release of flammable hydrogen from nuclear legacy waste sludge. X‐ray computed tomography was employed to observe 0.07–10 mm bubble populations within 30–1112 Pa yield strength Mg(OH)₂ sediments. High rates of partial coalescence were observed among sub‐millimeter microvoids, forming extensive bubble networks which spanned the 32 mm field of view. Lattice Boltzmann and Monte Carlo modeling demonstrated these networks to be highly pervious to gas, with effective diffusivities for hydrogen of 3.7–12.5 × 10^?5 m² s^?1. Continuous vessel‐spanning bubble networks, dynamic connectivity between ganglia of coalesced bubbles, Haines jumps, and composite diffusion through the gas and aqueous phase can account for enhanced gas migration over length‐scales of several meters, thus enabling chronic gas release from low‐intermediate strength sediments ( kPa) too strong for buoyant bubble ebullition and too weak for vertical channel formation. © 2018 The Authors. AIChE Journal published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers. AIChE J, 64: 4131–4147, 2018     

Identification of Sulfur‐Containing Impurities in Biodiesel Produced From Brown Grease

Fatty acid methyl esters (FAME) from waste grease usually contain higher concentrations of sulfur (S) than allowed to meet the specified quality standard for biodiesel (<15 ppm). Brown grease lipid‐derived FAME was produced and fractionated by two passes through a wiped‐film evaporator (WFE) to produce three fractions: (1) a 120 °C pass distillate, (2) a 170 °C pass distillate, and (3) a heavy residue. Solid phase extraction (SPE) was used to concentrate the S species from the distillate fractions so that they could be detected by a gas chromatography–pulsed flame photometric detector (GC–PFPD) and GC–mass spectrometry (MS). The ethyl acetate and methanol (MeOH) fractions obtained by SPE of the 120 °C WFE distillate and methyl tert‐butyl ether and acetone fractions obtained by SPE of the 170 °C WFE distillate had the highest concentration of S and were, therefore, the best candidates for GC–PFPD analysis. GC–PFPD methods were developed to separate the S species adequately enough for those peaks to be analyzed by GC–MS which matched fragmentation patterns identified by the MS chemical library as tetrahydrothiophenes, dithiolanes, and thiophenes. MS fragmentation patterns were used to identify other, larger, S‐bearing species as sulfides and disulfides cross‐linking between two FAME molecules. The results obtained from this study provide a foundation for developing effective purification methods to remove S‐containing impurities from waste grease‐derived biodiesel.     

Unravelling the Carbohydrate‐Binding Preferences of the Carbohydrate‐Binding Modules of AMP‐Activated Protein Kinase

The β subunit of adenosine monophosphate (AMP)‐activated protein kinase (AMPK), which exists as two isoforms (β1 and β2) in humans, has a carbohydrate‐binding module (CBM) that interacts with glycogen. Although the β1‐ and β2‐CBMs are structurally similar, with strictly conserved ligand‐contact residues, they show different carbohydrate affinities. β2‐CBM shows the strongest affinity for both branched and unbranched oligosaccharides and it has recently been shown that a Thr insertion into β2‐CBM (Thr101) forms a pocket to accommodate branches. This insertion does not explain why β2‐CBM binds all carbohydrates with stronger affinity. Herein, it is shown that residue 134 (Val for β2 and Thr for β1), which does not come into contact with a carbohydrate, appears to account for the affinity difference. Characterisation by NMR spectroscopy, however, suggests that mutant β2‐Thr101Δ/Val134Thr differs from that of β1‐CBM, and mutant β1‐Thr101ins/Thr134Val differs from that of β2‐CBM. Furthermore, these mutants are less stable to chemical denaturation, relative to that of wild‐type β‐CBMs, which confounds the affinity analyses. To support the importance of Thr101 and Val134, the ancestral CBM has been constructed. This CBM retains Thr101 and Val134, which suggests that the extant β1‐CBM has a modest loss of function in carbohydrate binding. Because the ancestor bound carbohydrate with equal affinity to that of β2‐CBM, it is concluded that residue 134 plays an indirect role in carbohydrate binding.     

Exploiting the Catalytic Diversity of Short‐Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope

Numerous short‐chain dehydrogenases/reductases (SDRs) have found biocatalytic applications in C=O and C=C (enone) reduction. For NADPH‐dependent C=N reduction, imine reductases (IREDs) have primarily been investigated for extension of the substrate range. Here, we show that SDRs are also suitable for a broad range of imine reductions. The SDR noroxomaritidine reductase (NR) is involved in Amaryllidaceae alkaloid biosynthesis, serving as an enone reductase. We have characterized NR by using a set of typical imine substrates and established that the enzyme is active with all four tested imine compounds (up to 99 % conversion, up to 92 % ee). Remarkably, NR reduced two keto compounds as well, thus highlighting this enzyme family's versatility. Using NR as a template, we have identified an as yet unexplored SDR from the Amaryllidacea Zephyranthes treatiae with imine‐reducing activity (≤95 % ee). Our results encourage the future characterization of SDR family members as a means of discovering new imine‐reducing enzymes.