Enzymes revolutionize the bioproduction of value-added compounds: From enzyme discovery to special applications.

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Process investigations of extreme thermophilic fermentations for hydrogen production: effect of bubble induction and reduced pressure.

Hydrogen production via thermophilic dark fermentation is considered a sustainable way to produce renewable hydrogen. For industrial scale an optimisation of hydrogen production is of highest importance. The aim of this work was to evaluate induced bubble formation and applying reduced pressure as methods of removing produced hydrogen instead of external gas stripping. Evaluation was carried out in a continuously stirred tank reactor using the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus. The addition of a bubble formation inductor was able to maintain the fermentation, but only at low hydrogen production rates and yields. Applying reduced pressure at a level of 305 mbar, nitrogen stripping could be omitted and hydrogen yields of around 72% of the theoretical maximum were achieved. It was proven, that application of reduced pressure is a promising alternative to inert gas stripping to obtain high hydrogen productivities and yields for thermophilic dark fermentations.

Purification and characterization of a new bioscouring pectate lyase from Bacillus pumilus BK2.

An alkalophilic bacterium was isolated based on the potential of extra-cellular enzymes for bioscouring. The bacterium was identified as a new strain of Bacillus pumilus BK2 producing an extra-cellular endo-pectate lyase PL (EC 4.2.2.2). PL was purified to homogeneity in three steps and has a molecular mass of 37.3+/-4.8 kDa as determined by SDS-PAGE and an isoelectric point of pH 8.5. Peptide mass mapping by nano-LC-MS of PL revealed 15% homology with a pectate lyase from Bacillus sp. The pectate lyase exhibited optimum activity at pH 8.5 and around 70 degrees C in Tris/HCl buffer. It showed a half-life at 30 degrees C of more than 75 h. Stability decreased with increasing temperature, extremely over 60 degrees C. The enzyme did not require Ca2+ ions for activity, and was strongly inhibited by EDTA and Co2+. PL was active on polygalacturonic acid and esterified pectin, but the affinity showed a maximum for intermediate esterified pectins and decreased over a value of 50% of esterification. The best substrate was 29.5% methylated pectin. PL cleaved polygalacturonic acid via a beta-elimination mechanism as shown by NMR analysis. PL released unsaturated tetragalacturonic acid from citrus pectin and polygalacturonic acid, but did not show any side activities on other hemicelluloses. On polygalacturonic acid PL showed a Km of 0.24 gl(-1) and a vmax of 0.72 gl(-1)min(-1). The applicability of pectate lyase for the bioscouring process was tested on a cotton fabric. Removal of up to 80% of pectin was proven by means of ruthenium red dyeing and HPAEC (65%). Structural contact angle measurements clearly indicated the increased hydrophilicity of enzyme treated fabrics.