Current status and future development of plastics: Solutions for a circular economy and limitations of environmental degradation.

Plastics are the most advanced materials, regarding their application and property range, with an annual worldwide demand of over 400 million tonnes, but have been facing strong public opposition lately. In the debate on sustainable plastics, plastic waste and plastic littering, bio-based and especially biodegradable plastics have gained attention as potential solution to these problems. This has been accompanied by misunderstanding, false and inadequate labeling of plastics as well as insufficient communication with the public and authorities. The plastic waste and littering problems are addressed by already implemented and planned bans on single-use plastics, improved regulations for labeling and communication of biodegradable plastics, and enhancement of plastic recycling. The future will show how successful these strategies are. If plastics are produced with renewable carbon from biomass, direct CO2 utilization and recycling, the production can be completely independent from fossil resources in a long-term future perspective which supports the circular renewable carbon economy.

Lipase-Catalyzed Transamidation of Urethane-Bond-Containing Ester.

Significant improvement in mechanical properties and shape recovery in polyurethanes can be obtained by cross-linking, usually performed in a traditional chemical fashion. Here, we report model studies of enzymatic transamidations of urethane-bond-containing esters to study the principles of an enzymatic build-up of covalent cross-linked polyurethane networks via amide bond formation. The Lipase-catalyzed transamidation reaction of a urethane-bond-containing model ester ethyl 2-(hexylcarbamoyloxy)propanoate with various amines is discussed. A side product was formed, that could be successfully identified, and its synthesis reduced to a minimum (<1%). Furthermore, a noncatalyzed transamidation that is performed without CalB as the catalyst could be observed. Both observations are due to the known high reactivity of amines with urethane bonds.

Contribution of single amino acid and codon substitutions to the production and secretion of a lipase by Bacillus subtilis.

BACKGROUND: Bacillus subtilis produces and secretes proteins in amounts of up to 20 g/l under optimal conditions. However, protein production can be challenging if transcription and cotranslational secretion are negatively affected, or the target protein is degraded by extracellular proteases. This study aims at elucidating the influence of a target protein on its own production by a systematic mutational analysis of the homologous B. subtilis model protein lipase A (LipA). We have covered the full natural diversity of single amino acid substitutions at 155 positions of LipA by site saturation mutagenesis excluding only highly conserved residues and qualitatively and quantitatively screened about 30,000 clones for extracellular LipA production. Identified variants with beneficial effects on production were sequenced and analyzed regarding B. subtilis growth behavior, extracellular lipase activity and amount as well as changes in lipase transcript levels. RESULTS: In total, 26 LipA variants were identified showing an up to twofold increase in either amount or activity of extracellular lipase. These variants harbor single amino acid or codon substitutions that did not substantially affect B. subtilis growth. Subsequent exemplary combination of beneficial single amino acid substitutions revealed an additive effect solely at the level of extracellular lipase amount; however, lipase amount and activity could not be increased simultaneously. CONCLUSIONS: Single amino acid and codon substitutions can affect LipA secretion and production by B. subtilis. Several codon-related effects were observed that either enhance lipA transcription or promote a more efficient folding of LipA. Single amino acid substitutions could improve LipA production by increasing its secretion or stability in the culture supernatant. Our findings indicate that optimization of the expression system is not sufficient for efficient protein production in B. subtilis. The sequence of the target protein should also be considered as an optimization target for successful protein production. Our results further suggest that variants with improved properties might be identified much faster and easier if mutagenesis is prioritized towards elements that contribute to enzymatic activity or structural integrity.

Activity-independent screening of secreted proteins using split GFP.

The large-scale industrial production of proteins requires efficient secretion, as provided, for instance, by the Sec system of Gram-positive bacteria. Protein engineering approaches to optimize secretion often involve the screening of large libraries, e.g. comprising a target protein fused to many different signal peptides. Respective high-throughput screening methods are usually based on photometric or fluorimetric assays enabling fast and simple determination of enzymatic activities. Here, we report on an alternative method for quantification of secreted proteins based on the split GFP assay. We analyzed the secretion by Bacillus subtilis of a homologous lipase and a heterologous cutinase by determination of GFP fluorescence and enzyme activity assays. Furthermore, we identified from a signal peptide library a variant of the biotechnologically relevant B. subtilis protein swollenin EXLX1 with up to 5-fold increased secretion. Our results demonstrate that the split GFP assay can be used to monitor secretion of enzymatic and non-enzymatic proteins in B. subtilis in a high-throughput manner.

Exploring the protein stability landscape: Bacillus subtilis lipase A as a model for detergent tolerance.

A systematic study was conducted with Bacillus subtilis lipase A (BSLA) to determine the effect of every single amino acid substitution on detergent tolerance. BSLA is a minimal α/ß-hydrolase of 181 amino acids with a known crystal structure. It can be expressed in Escherichia coli and is biochemically well characterized. Site saturation mutagenesis resulted in a library of 3439 variants, each with a single amino acid exchange as confirmed by DNA sequencing. The library was tested against four detergents, namely SDS, CTAB, Tween 80, and sulfobetaine. Surface remodeling emerged as an effective engineering strategy to increase tolerance towards detergents. Amino acid residues that significantly affect the tolerance for each of the four detergents were identified. In summary, this systematic analysis provides an experimental dataset to help derive novel protein engineering strategies as well as to direct modeling efforts.