Three in One: Temperature, Solvent and Catalytic Stability by Engineering the Cofactor-Binding Element of Amine Transaminase.

Amine transaminase (ATA) catalyse enantioselectively the direct amination of ketones, but insufficient stability during catalysis limits their industrial applicability. Recently, we revealed that ATAs suffer from substrate-induced inactivation mechanism involving dissociation of the enzyme-cofactor intermediate. Here, we report on engineering the cofactor-ring-binding element, which also shapes the active-site entrance. Only two point mutations in this motif improved temperature and catalytic stability in both biphasic media and organic solvent. Thermodynamic analysis revealed a higher melting point for the enzyme-cofactor intermediate. The high cofactor affinity eliminates the need for pyridoxal 5'-phosphate supply, thus making large-scale reactions more cost effective. This is the first report on stabilising a tetrameric ATA by mutating a single structural element. As this structural "hotspot" is a common feature of other transaminases it could serve as a general engineering target.

Development of in situ product removal strategies in biocatalysis applying scaled-down unit operations.

An experimental platform based on scaled-down unit operations combined in a plug-and-play manner enables easy and highly flexible testing of advanced biocatalytic process options such as in situ product removal (ISPR) process strategies. In such a platform, it is possible to compartmentalize different process steps while operating it as a combined system, giving the possibility to test and characterize the performance of novel process concepts and biocatalysts with minimal influence of inhibitory products. Here the capabilities of performing process development by applying scaled-down unit operations are highlighted through a case study investigating the asymmetric synthesis of 1-methyl-3-phenylpropylamine (MPPA) using ω-transaminase, an enzyme in the sub-family of amino transferases (ATAs). An on-line HPLC system was applied to avoid manual sample handling and to semi-automatically characterize ω-transaminases in a scaled-down packed-bed reactor (PBR) module, showing MPPA as a strong inhibitor. To overcome the inhibition, a two-step liquid-liquid extraction (LLE) ISPR concept was tested using scaled-down unit operations combined in a plug-and-play manner. Through the tested ISPR concept, it was possible to continuously feed the main substrate benzylacetone (BA) and extract the main product MPPA throughout the reaction, thereby overcoming the challenges of low substrate solubility and product inhibition. The tested ISPR concept achieved a product concentration of 26.5 gMPPA · L-1 , a purity up to 70% gMPPA · gtot-1 and a recovery in the range of 80% mol · mol-1 of MPPA in 20 h, with the possibility to increase the concentration, purity, and recovery further. Biotechnol. Bioeng. 2017;114: 600-609. © 2016 Wiley Periodicals, Inc.

Genome-scale metabolic model of the versatile bacterium Paracoccus denitrificans Pd1222.

A genome scale metabolic model of the bacterium Paracoccus denitrificans has been constructed. The model containing 972 metabolic genes, 1,371 reactions, and 1,388 unique metabolites has been reconstructed. The model was used to carry out quantitative predictions of biomass yields on 10 different carbon sources under aerobic conditions. Yields on C1 compounds suggest that formate is oxidized by a formate dehydrogenase O, which uses ubiquinone as redox co-factor. The model also predicted the threshold methanol/mannitol uptake ratio, above which ribulose biphosphate carboxylase has to be expressed in order to optimize biomass yields. Biomass yields on acetate, formate, and succinate, when NO3- is used as electron acceptor, were also predicted correctly. The model reconstruction revealed the capability of P. denitrificans to grow on several non-conventional substrates such as adipic acid, 1,4-butanediol, 1,3-butanediol, and ethylene glycol. The capacity to grow on these substrates was tested experimentally, and the experimental biomass yields on these substrates were accurately predicted by the model.IMPORTANCEParacoccus denitrificans has been broadly used as a model denitrifying organism. It grows on a large portfolio of carbon sources, under aerobic and anoxic conditions. These characteristics, together with its amenability to genetic manipulations, make P. denitrificans a promising cell factory for industrial biotechnology. This paper presents and validates the first functional genome-scale metabolic model for P. denitrificans, which is a key tool to enable P. denitrificans as a platform for metabolic engineering and industrial biotechnology. Optimization of the biomass yield led to accurate predictions in a broad scope of substrates.

Enhancement of biogas production rate from bioplastics by alkaline pretreatment.

The effect of alkali-based pretreatment on the methanization of bioplastics was investigated. The tested bioplastics included PHB [poly(3-hydroxybutyrate)], PHBH [poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)], PHBV [poly(3-hydroxybutyrate-co-3-hydroxyvalerate], PLA (polylactic acid), and a PLA/PCL [poly(caprolactone)] 80/20 blend. Prior to methanization tests, the powdered polymers (500-1000 µm) at a concentration of 50 g/L were subjected to alkaline pretreatment using NaOH 1 M for PLA and PLA/PCL, and NaOH 2 M for PHB-based materials. Following 7 days of pretreatment, the amount of solubilized carbon for PLA and its blend accounted for 92-98% of the total initial carbon, while lower carbon recoveries were recorded for most PHB-based materials (80-93%), as revealed by dissolved total organic carbon analysis. The pretreated bioplastics were then tested for biogas production by means of mesophilic biochemical methane potential tests. Compared to unpretreated PHBs, methanization rates of pretreated PHBs were accelerated by a factor of 2.7 to 9.1 with comparable (430 NmL CH4/g material feed) or slightly lower (15% in the case of PHBH) methane yields, despite featuring a 1.4-2.3 times longer lag phases. Both materials, PLA and the PLA/PCL blend, were only extensively digested when pretreated, yielding about 360-380 NmL CH4 per gram of material fed. Unpretreated PLA-based materials showed nearly zero methanization under the timeframe and experimental conditions tested. Overall, the results suggested that alkaline pretreatment can help to enhance the methanization kinetics of bioplastics.

Screening Enzymes That Can Depolymerize Commercial Biodegradable Polymers: Heterologous Expression of Fusarium solani Cutinase in Escherichia coli.

In recent years, a number of microbial enzymes capable of degrading plastics have been identified. Biocatalytic depolymerization mediated by enzymes has emerged as a potentially more efficient and environmentally friendly alternative to the currently employed methods for plastic treatment and recycling. However, the functional and systematic study of depolymerase enzymes with respect to the degradation of a series of plastic polymers in a single work has not been widely addressed at present. In this study, the ability of a set of enzymes (esterase, arylesterase and cutinase) to degrade commercial biodegradable polymers (PBS, PBAT, PHB, PHBH, PHBV, PCL, PLA and PLA/PCL) and the effect of pre-treatment methods on their degradation rate was assessed. The degradation products were identified and quantified by HPLC and LC-HRMS analysis. Out of the three enzymes, Fusarium solani cutinase (FsCut) showed the highest activity on grinded PBAT, PBS and PCL after 7 days of incubation. FsCut was engineered and heterologous expressed in Escherichia coli, which conferred the bacterium the capability of degrading solid discs of PBAT and to grow in PBS as the sole carbon source of the medium.

Production of volatile fatty acids (VFAs) from five commercial bioplastics via acidogenic fermentation.

The feasibility of producing volatile fatty acids (VFAs) from five commercial bioplastics via acidogenic fermentation by a non-pretreated anaerobic sludge was investigated. Mesophilic, anaerobic, acidogenic batch assays at 1, 10 and 20 g/L feed concentrations revealed the feasibility of producing VFAs from polyhydroxyalkanoates (PHA), i.e., PHB and PHBV, but not from PBS, PCL and PLA under the test conditions and time. However, only high PHA substrate concentrations (10-20 g/L) resulted in organic overloading and decreasing the pH of the culture broth down to 4-5, which in turn induced the accumulation of VFAs via kinetic imbalance between acidogenesis and methanogenesis. Gaseous carbon (C-CO2 and C-CH4) accounted for 8-35% of the total initial carbon, while C-VFAs represented 10-18%, mainly as acetate and butyrate. This study represents the first systematically assessed proof-of-concept to produce VFAs from PHA, which is key for the design of bioplastic-to-bioplastic recycling (bio)technologies.

Directed Discovery of Tetrapeptide Emulsifiers.

Oil in water emulsions are an important class of soft material that are used in the food, cosmetic, and biomedical industries. These materials are formed through the use of emulsifiers that are able to stabilize oil droplets in water. Historically emulsifiers have been developed from lipids or from large biomolecules such as proteins. However, the ability to use short peptides, which have favorable degradability and toxicity profiles is seen as an attractive alternative. In this work, we demonstrate that it is possible to design emulsifiers from short (tetra) peptides that have tunability (i.e., the surface activity of the emulsion can be tuned according to the peptide primary sequence). This design process is achieved by applying coarse grain molecular dynamics simulation to consecutively reduce the molecular search space from the 83,521 candidates initially considered in the screen to four top ranking candidates that were then studied experimentally. The results of the experimental study correspond well to the predicted results from the computational screening verifying the potential of this screening methodology to be applied to a range of different molecular systems.

Biodegradation of bioplastics under aerobic and anaerobic aqueous conditions: Kinetics, carbon fate and particle size effect.

The biodegradation of PHB, PHBV, PBS, PBAT, PCL, PLA, and a PLA-PCL blend was compared under aerobic and anaerobic aqueous conditions assessing biodegradation kinetics, extent, carbon fate and particle size influence (in the range of 100-1000 µm). Under standard test conditions, PHB and PBHV were biodegraded anaerobically (83.9 ± 1.3% and 81.2 ± 1.7%, respectively) in 77 days or aerobically (83.0 ± 1.6% and 87.4 ± 7.5%) in 117 days, while PCL was only biodegraded (77.6 ± 2.4%) aerobically in 177 days. Apparent biomass growth accounted for 10 to 30.5% of the total initial carbon depending on the bioplastic and condition. Maximum aerobic and anaerobic biodegradation rates were improved up to 331 and 405%, respectively, at the lowest particle size tested (100-250 µm). This study highlights the usefulness of analysing biodegradation kinetics and carbon fate to improve both the development and testing of biodegradable materials, and waste treatments in the context of a circular bioeconomy.

Insights into Broad-Specificity Starch Modification from the Crystal Structure of Limosilactobacillus Reuteri NCC 2613 4,6-α-Glucanotransferase GtfB.

GtfB-type α-glucanotransferase enzymes from glycoside hydrolase family 70 (GH70) convert starch substrates into α-glucans that are of interest as food ingredients with a low glycemic index. Characterization of several GtfBs showed that they differ in product- and substrate specificity, especially with regard to branching, but structural information is limited to a single GtfB, preferring mostly linear starches and featuring a tunneled binding groove. Here, we present the second crystal structure of a 4,6-α-glucanotransferase (Limosilactobacillus reuteri NCC 2613) and an improved homology model of a 4,3-α-glucanotransferase GtfB (L. fermentum NCC 2970) and show that they are able to convert both linear and branched starch substrates. Compared to the previously described GtfB structure, these two enzymes feature a much more open binding groove, reminiscent of and evolutionary closer to starch-converting GH13 α-amylases. Sequence analysis of 287 putative GtfBs suggests that only 20% of them are similarly "open" and thus suitable as broad-specificity starch-converting enzymes.

Inspired by nature: Microbial production, degradation and valorization of biodegradable bioplastics for life-cycle-engineered products.

The global environmental pollution by micro- and macro-plastics reveals the consequences of an extensive use of recalcitrant plastic products together with inappropriate waste management practices that fail to sufficiently recycle the broad types of conventional plastic waste. Biobased and biodegradable plastics are experiencing an uprising as their properties offer alternative waste management solutions for a more circular material economy. However, although the production of such bioplastics has advanced on scale, the end-of-life (EOL) (bio)technologies to promote circularity are lacking behind. While composting and biogas plants are the only managed EOL options today, advanced biotechnological recycling technologies for biodegradable bioplastics are still in an embryonic stage. Thus, developing efficient biotechnologies capable of transforming bioplastic waste into high-value chemical building blocks or into the constituents of the original polymer offers promising routes towards life-cycle-engineered products. This review aims at providing a comprehensive state-of-the-art overview of microbial-based processes involved in the complete lifecycle of bioplastics. The current trends in the bioplastic market, the beginning and EOL scenarios of bioplastics, and a critical discussion on the key factors and mechanisms governing microbial degradation are systematically presented. Also, a critical evaluation of terminology and international standards to quantify polymer biodegradability is provided together with the latest biotechnological recycling strategies, including the use of different pre-treatments for (bio)plastic waste. Finally, the challenges and future perspectives for the development of life-cycle-engineered biobased and biodegradable plastic products are discussed.

Application of iterative robust model-based optimal experimental design for the calibration of biocatalytic models.

The aim of model calibration is to estimate unique parameter values from available experimental data, here applied to a biocatalytic process. The traditional approach of first gathering data followed by performing a model calibration is inefficient, since the information gathered during experimentation is not actively used to optimize the experimental design. By applying an iterative robust model-based optimal experimental design, the limited amount of data collected is used to design additional informative experiments. The algorithm is used here to calibrate the initial reaction rate of an ω-transaminase catalyzed reaction in a more accurate way. The parameter confidence region estimated from the Fisher Information Matrix is compared with the likelihood confidence region, which is not only more accurate but also a computationally more expensive method. As a result, an important deviation between both approaches is found, confirming that linearization methods should be applied with care for nonlinear models. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1278-1293, 2017.

Generic HPLC platform for automated enzyme reaction monitoring: Advancing the assay toolbox for transaminases and other PLP-dependent enzymes.

Methods for rapid and direct quantification of enzyme kinetics independent of the substrate stand in high demand for both fundamental research and bioprocess development. This study addresses the need for a generic method by developing an automated, standardizable HPLC platform monitoring reaction progress in near real-time. The method was applied to amine transaminase (ATA) catalyzed reactions intensifying process development for chiral amine synthesis. Autosampler-assisted pipetting facilitates integrated mixing and sampling under controlled temperature. Crude enzyme formulations in high and low substrate concentrations can be employed. Sequential, small (1 µL) sample injections and immediate detection after separation permits fast reaction monitoring with excellent sensitivity, accuracy and reproducibility. Due to its modular design, different chromatographic techniques, e.g. reverse phase and size exclusion chromatography (SEC) can be employed. A novel assay for pyridoxal 5'-phosphate-dependent enzymes is presented using SEC for direct monitoring of enzyme-bound and free reaction intermediates. Time-resolved changes of the different cofactor states, e.g. pyridoxal 5'-phosphate, pyridoxamine 5'-phosphate and the internal aldimine were traced in both half reactions. The combination of the automated HPLC platform with SEC offers a method for substrate-independent screening, which renders a missing piece in the assay and screening toolbox for ATAs and other PLP-dependent enzymes.