A New Family of Biuret Hydrolases Involved in S-Triazine Ring Metabolism

Biuret is an intermediate in the bacterial metabolism of s-triazine ring compounds and is occasionally used as a ruminant feed supplement. We used bioinformatics to identify a biuret hydrolase, an enzyme that has previously resisted efforts to stabilize, purify and characterize. This newly discovered enzyme is a member of the cysteine hydrolase superfamily, a family of enzymes previously not found to be involved in s-triazine metabolism. The gene from Rhizobium leguminosarum bv. viciae strain 3841 encoding biuret hydrolase was synthesized, transformed into Escherichia coli, and expressed. The enzyme was purified and found to be stable. Biuret hydrolase catalyzed the hydrolysis of biuret to allophanate and ammonia. The k(cat)/K(M) of 1.7 × 10(5) M(-1)s(-1) and the relatively low K(M) of 23 ± 4 μM together suggested that this enzyme acts uniquely on biuret physiologically. This is supported by the fact that of the 34 substrate analogs of biuret tested, only two demonstrated reactivity, both at less than 5% of the rate determined for biuret. Biuret hydrolase does not react with carboxybiuret, the product of the enzyme immediately preceding biuret hydrolase in the metabolic pathway for cyanuric acid. This suggests an unusual metabolic strategy of an enzymatically-produced intermediate undergoing non-enzymatic decarboxylation to produce the substrate for the next enzyme in the pathway.     

Synthesis of styrene–butadiene rubber latex via miniemulsion copolymerization

High-butadiene-level styrene–butadiene rubber latexes up to high solid-contents are synthesized using the miniemulsion process. It is shown that the miniemulsion polymerization approach offers an efficient heterophase route synthesizing styrene–butadiene copolymer latexes with flexible copolymer composition and narrow size distribution of the resulting latex particles. Secondary nucleation was successfully prevented by using a hydrophobic initiator. Due to the nanoreactor situation, even at high conversions, a low crosslinking degree and, therefore, low gel contents are obtained. The microstructure of the polymers obtained in miniemulsion is independent of the synthesis parameters, especially the temperature. The molecular weight can be easily adjusted by the application of transfer agents while the insoluble gel content is substantially reduced. An up-scaling of the procedure is easily possible.     

Regioselective Ring‐Opening Polymerization of a Polyhydroxycarboxylic Acid for the Synthesis of a Nanoscale Carrier Material with pH‐Dependent Stability and Sustained Drug Release

Synthetic polyesters are usually composed of monohydroxycarboxylic acids to avoid the problem of regioselectivity during ring‐opening polymerization. In contrast, the linear polyester BICpoly contains four secondary OH groups and is nevertheless esterified regioselectively at only one of these positions. Neither the synthesis of the tricyclic monomers nor the ring‐opening polymerization requires protecting groups, making BICpoly an attractive novel and biocompatible polymer. BICpoly nanoparticles can be loaded with low‐molecular weight drugs or coated onto surfaces as thin films. The release of loaded compounds makes BICpoly an attractive depot for drug release, as shown herein by loading BICpoly with dyes or the cytostatic drug doxorubicin. BICpoly is distinguishable from other polymers by its characteristic pH‐dependent degradation.     

Covalent Protein Labeling by Enzymatic Phosphocholination

We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP‐choline derivatives to N‐termini, C‐termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG‐phosphocholine) is introduced to attach the conjugated cargo.