Responsive Macroscopic Materials From Self‐Assembled Cross‐Linked SiO2‐PNIPAAm Core/Shell Structures

A way to obtain macroscopic responsive materials from silicon‐oxide polymer core/shell microstructures is presented. The microparticles are composed of a 60 nm SiO₂‐core with a random copolymer corona of the temperature responsive poly‐N‐isopropylacrylamide (PNIPAAm) and the UV‐cross‐linkable 2‐(dimethyl maleinimido)‐N‐ethyl‐acrylamide. The particles shrink upon heating and form a stable gel in both water and tetrahydrofuran (THF) at 3–5 wt% particle content. Cross‐linking the aqueous gel results in shrinkage when the temperature is increased above the lower critical solution temperature and it regains its original size upon cooling. By freeze drying with subsequent UV irradiation, thin stable layers are prepared. Stable fibers are produced by extruding a THF gel into water and subsequent UV irradiation, harnessing the cononsolvency effect of PNIPAAm in water/THF mixtures. The temperature responsiveness translates to the macroscopic materials as both films and fibers show the same collapsing behavior as the microcore/shell particle. The collapse and re‐swelling of the materials is related to the expelling and re‐uptake of water, which is used to incorporate gold nanoparticles into the materials by a simple heating/cooling cycle. This allows for future applications, as various functional particles (antibacterial, fluorescence, catalysis, etc.) can easily be incorporated in these systems.     

Catalytic mechanism of fungal glucoamylase as defined by mutagenesis of Asp176, Glu179 and Glu180 in the enzyme from Aspergillus awamori

Asp176, Glu179 and Glu180 of Aspergillus awamori glucoamylaseappeared by differential labeling to be in the active site.To test their functions, they were replaced by mutagenesis withAsn, Gln and Gln respectively, and kinetic parameters and pHdependencies of all enzyme forms were determined. Glu179 –Gln glucoamylase was not active on maltose or isomaltose, whilethe k_cat for maltoheptaose hydrolysis decreased almost 2000-foldand the K_M was essentially unchanged from wild-type glucoamylase.The Glu180 – Gln mutation drastically increased the K_Mand moderately decreased the k_cat with maltose and maltoheptaose,but affected isomaltose hydrolysis less. Differences in substrateactivation energies between Glu180 – Gln and wild-typeglucoamylases indicate that Glu180 binds D-glucosyl residuesin subsite 2. The Asp176 – Asn substitution gave moderateincreases and decreases in K_M and k_cat respectively, and thereforesimilar increases in activation energies for the three substrates.This and the differences in subsite binding energies betweenAsp176 – Asn and wild-type glucoamylases suggest thatAsp176 is near subsite 1, where it stabilizes the transitionstate and interacts with Trp120 at subsite 4. Glu179 and Asp176are thus proposed as the general catalytic acid and base ofpK_a 5.9 and 2.7 respectively. The charged Glu180 contributesto the high pK_a value of Glul79. Received May 25, 1989; accepted October 19, 1989.     

Biased mutagenesis in the N-terminal region by degenerate oligonucleotide gene shuffling enhances secretory expression of barley alpha-amylase 2 in yeast

Recombinant barley alpha-amylase 1 (rAMY1) and 2 (rAMY2), despite 80% sequence identity, are produced in very different amounts of 1.1 and <0.05 mg/l, respectively, by Saccharomyces cerevisiae strain S150-2B. The low yield of AMY2 practically excludes mutational analysis of structure-function relationships and protein engineering. Since different secretion levels of AMY1/AMY2 chimeras were previously ascribed to the N-terminal sequence, AMY1 residues were combinatorially introduced at the 10 non-conserved positions in His14-Gln49 of AMY2 using degenerate oligonucleotide gene shuffling (DOGS) coupled with homologous recombination in S.cerevisiae strain INVSc1. Activity screening of a partial library of 843 clones selected six having a large halo size on starch plates. Three mutants, F21M/Q44H, A42P/A47S and A42P rAMY2, also gave higher activity than wild-type in liquid culture. Only A42P showed wild-type stability and enzymatic properties. The replacement is located to a beta-->alpha loop 2 that interacts with domain B (beta-->alpha loop 3) protruding from the catalytic (beta/alpha)(8)-barrel. Most remarkably Pichia pastoris strain GS115 secreted 60 mg/l A42P compared with 3 mg/l of wild-type rAMY2. The crystal structure of A42P rAMY2 was solved and found to differ marginally from the AMY2 structure, suggesting that the high A42P yield stems from stabilization of the mature and/or intermediate form owing to the introduced proline residue. Moreover, the G to C substitution for the A42P mutation might have a positive impact on protein translation.     

Discourse Particles and Discourse Functions

Spoken language, especially spoken German, is rich in particles thatdo not contribute to the propositional content of utterances, but playimportant roles in steering the flow of the dialogue and inconveying various attitudes and expectations of the speaker. Languagesdiffer widely in their conventions on particle usage, and thereforethese words pose significant problems for translation. As a solution,we propose an inventory of discourse functions that characterizethe pragmatic impact of particles. These functions are to be assignedto particles in the analysis phase, so that the translation step canuse the abstract information to decide on the best way of renderingthe same effect in the target-language utterance.     

Sodium chloride enhances adherence and aggregation and strain variation influences invasiveness of Listeria monocytogenes strains

Some subtypes of Listeria monocytogenes can persist in the food-processing industry, but the reasons for such persistence are not known. In the present study, 10 strains of L. monocytogenes representing known persistent randomly amplified polymorphic DNA (RAPD) types from fish processing plants were compared to eight strains of different RAPD type and origin (clinical, food, and animal). All 18 strains of L. monocytogenes had similar growth patterns at different temperatures (5 or 37 degrees C) or different salinities (0.5 or 5% NaCl), and all strains formed a thin layer of adhered cells on a plastic surface when cultured in tryptone soya broth (TSB) with a total of 1% glucose. Many ready-to-eat foods, such as cold-smoked fish, contain NaCl at concentrations of 2 to 5%, and NaCl is present in the processing environment. Adding NaCl to TSB changed the adhesion patterns of all strains, and all adhered better when NaCl was added. Also, the addition of NaCl caused a marked aggregation of 13 of the strains; however, 5 of the 18 strains did not aggregate in the presence of up to 5% NaCl. The aggregates stuck to the plastic surface, and this occurred in all but one of the persistent RAPD types. Four strains represented one particular RAPD type that has been isolated as a persistent RAPD type in several fish processing plants for up to 10 years. Because this RAPD type often can contaminate fish products, it is important to address its potential virulence. The 18 strains differed markedly in their ability to invade Caco-2 cells, and the four strains representing the universal persistent RAPD type were the least invasive (10(2) to 10(3) CFU/ml), whereas other strains invaded Caco-2 cells at levels of 10(4) to 10(5) CFU/ml. Five of the 18 strains belonged to the genetic lineage 1 and were the most invasive. Although the most commonly isolated persistent RAPD type was low invasive, it is important to understand why moderate salinity facilitates aggregation and biofilm formation, for this understanding can be beneficial in developing procedures to reduce processing plant contamination.