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The aim of the present study was to generate a consistent data set for biorefinery intermediates from novel energy crop Silphium perfoliatum compared with Fagus sylvatica, including process streams from liquid hot water pretreatment, enzymatic hydrolysis, and fermentation with Bacillus licheniformis. The consistency of the data set was further supported by the application of a consistent analytical method based on high performance anion exchange chromatography with pulsed amperometric detection and validated for process intermediates, which renders the technique a versatile analytical tool also for alcoholic compounds. For the first time, Silphium perfoliatum was used for liquid hot water pretreatment which resulted in a maximal absolute glucose recovery after enzymatic hydrolysis for feedstock pretreated with 200 °C, 20 min, 20% solid loading. Under these conditions, 68% glucose were recovered for Silphium perfoliatum and 62% for Fagus sylvatica. Further, enzymatic hydrolyzates of both feedstocks were successfully used as single carbon sources for 2,3-butanediol fermentation with Bacillus licheniformis resulting in a 2,3-butanediol yield of 39% and 31% of the theoretical yield for Silphium perfoliatum and Fagus sylvatica, respectively. Thus, the technical suitability of Silphium perfoliatum as feedstock for a liquid hot water–based biorefinery process was comprehensively demonstrated and successfully bore the comparison with Fagus sylvatica.

A variety of enzymes can be easily incorporated and overexpressed within Escherichia coli cells by plasmids, making it an ideal chassis for bioelectrosynthesis. It has recently been demonstrated that microbial electrosynthesis (MES) of chiral alcohols is possible by using genetically modified E. coli with plasmid‐incorporated and overexpressed enzymes and methyl viologen as mediator for electron transfer. This model system, using NADPH‐dependent alcohol dehydrogenase from Lactobacillus brevis to convert acetophenone into (R)‐1‐phenylethanol, is assessed by using a design of experiment (DoE) approach. Process optimization is achieved with a 2.4‐fold increased yield of 94±7 %, a 3.9‐fold increased reaction rate of 324±67 μm h−1, and a coulombic efficiency of up to 68±7 %, while maintaining an excellent enantioselectivity of >99 %. Subsequent scale‐up to 1 L by using electrobioreactors under batch and fed‐batch conditions increases the titer of (R)‐1‐phenylethanol to 12.8±2.0 mm and paves the way to further develop E. coli into a universal chassis for MES in a standard biotechnological process environment., Powered up to implementation: Design of experiments and response surface methodology boosts the yields and efficiency of the enantioselective microbial electrosynthesis of (R)‐1‐phenylethanol by using resting Escherichia coli. Subsequent scale‐up to a 1 L electrobioreactor system, as well as fed‐batch operation, is a further important step towards commercial application.

Cellulose was successfully extracted from Ulva lactuca, an abundant and unexploited green macroalga that causes several environmental disorders by its excessive eutrophication. The extracted cellulose purity was checked by acid hydrolysis and high performance anion exchange chromatography coupled to pulsed amperometric detection, showing mainly glucose, and by Fourier transform infrared spectroscopy analysis, revealing the same functional groups as the commercial cellulose AVICEL and the absence of uronic esters from hemicellulosic contaminants. The crystallinity index was 59% compared to 82% for the AVICEL cellulose, showing the semi crystalline character of the extracted cellulose. The thermal behavior of both celluloses was the same as they showed a degradation peak at 360 °C as demonstrated by the thermogravimetric analysis. Beside the physicochemical properties, the alga cellulose presented several interesting rheological properties such as a high specific surface area (5.74 m2/g of dry matter) and consequently a higher water retention (43.07 g/100g of dry matter). These results show that alga can be an alternative source of cellulose to the conventional ones such as wood.

Cello-oligomers have a wide application range and are gained from three different cello-oligomer production paths, which show different drawbacks, namely acid or alkaline hydrolysis of cellulose, chemical or enzymatic synthesis, and enzymatic hydrolysis of cellulose using cellulase mixtures. Therefore, this study aims at a novel route towards cello-oligomers using purified endoglucanases. From the renewable raw material cellulose, pretreated by an ionic liquid, cello-oligomers with a weight average degree of polymerization (DPW) of 65 were directly obtained by enzymatic hydrolysis. During hydrolysis, between 14.1 and 24.5 mass percent of monomeric and dimeric sugars were formed as byproduct. A second ionic liquid pretreatment of the cellulose, remaining after the first hydrolysis, and a second enzymatic hydrolysis resulted in cello-oligomers with a DP of 35. XRD deconvolution and Raman analysis confirmed that crystallinity remained unchanged during enzymatic cellulose hydrolysis.

Chiral alcohols constitute important building blocks that can be produced enantioselectively by using nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-dependent oxidoreductases. For NAD(P)H regeneration, electricity delivers the cheapest reduction equivalents. Enzymatic electrosynthesis suffers from cofactor and enzyme instability, whereas microbial electrosynthesis (MES) exploits whole cells. Here, we demonstrate MES by using resting Escherichia coli as biocatalytic chassis for a production platform towards fine chemicals through electric power. This chassis was exemplified for the synthesis of chiral alcohols by using a NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis for synthesis of (R)-1-phenylethanol from acetophenone. The E. coli strain and growth conditions affected the performance. Maximum yields of (39.4±5.7) % at a coulombic efficiency of (50.5±6.0) % with enantiomeric excess >99 % was demonstrated at a rate of (83.5±13.9) μm h-1 , confirming the potential of MES for synthesis of high-value compounds.

In this paper, we present a systematic robust dynamic optimization framework applied to the benzaldehyde lyase-catalyzed carboligation of propanal and benzaldehyde to produce (R)-2-hydroxy-1-phenylbutan-1-one (BA). First, the elementary process functions approach was used to screen between different dosing concepts, and it was found that simultaneously dosing propanal and benzaldehyde leads to the highest final concentration of BA. Next, we applied global sensitivity analysis and found that 10 out of 13 kinetic parameters are relevant. Time-varying back-offs were then used to handle parametric uncertainties due to these 10 parameters. A major contribution in our work is the use of the point estimate method instead of Monte Carlo simulations to calculate the back-offs in an efficient and reproducible manner. We show that this new approach is at least 10 times faster than the conventional Monte Carlo approach while achieving low approximation errors.

Unspecific peroxygenases have attracted interest due to their ability to catalyze the oxygenation of various types of C-H bonds using only hydrogen peroxide as a cosubstrate. Due to the instability of these enzymes at even low hydrogen peroxide concentrations, careful fed-batch addition of the cosubstrate or ideally in situ production is required. While various approaches for hydrogen peroxide addition have been qualitatively assessed, only limited kinetic data concerning enzyme inactivation and peroxide accumulation has been reported so far. To obtain quantitative insights into the kinetics of such a process, a detailed data set for a peroxygenase-catalyzed benzylic hydroxylation coupled with electrochemical hydrogen peroxide production is presented. Based on this data set, we set out to model such an electroenzymatic process. For this, initial velocity data for the benzylic hydroxylation is collected and an extended Ping-Pong-Bi-Bi type rate equation is established, which sufficiently describes the enzyme kinetic. Moreover, we propose an empirical inactivation term based on the collected data set. Finally, we show that the full model does not only describe the process with sufficient accuracy, but can also be used predictively to control hydrogen peroxide feeding rates To limit the concentration of this critical cosubstrate in the system.

High-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) was used for developing a method for identifying and quantifying aldehydes in biomass hydrolyzates. This method was optimized to the requirements of HPAEC-PAD in order to allow for a simultaneous determination of aldehydes by respective Cannizzaro alcohols. To this end, sodium hydroxide concentration (0.1 to 5.0 mol/L), temperature (30 to 40 °C), and reaction time (0 to 24 h) were investigated for sufficient and reproducible disproportionation of the biomass-derived aldehydes. The optimized method for aldehyde disproportionation and subsequent measurement are 1 mol/L sodium hydroxide, 40 °C, and 1 h reaction time. The detection limits resulting from this method are lower than 68.55 mg/L and the sensitivity above 0.024 (nC min)/(mg/L) for 3,4-dimethoxybenzaldehyde. Linearity for aldehyde calibration always exceeded 0.98. Thus, HPAEC-PAD analysis allows for the quantification of biomass-derived compounds from all natural polymers and, therefore, it has exemplarily been used to quantify aldehyde concentration of beech wood, orange peel, and algae biomass hydrolyzates. Graphical abstract.

We present a workflow for kinetic modeling of biocatalytic reactions which combines methods from Bayesian learning and uncertainty quantification for model calibration, model selection, evaluation, and model reduction in a consistent statistical frame-work. Our workflow is particularly tailored to sparse data settings in which a considerable variability of the parameters remains after the models have been adapted to available data, a ubiquitous problem in many real-world applications. Our workflow is exemplified on an enzyme-catalyzed two-substrate reaction mechanism describing the symmetric carboligation of 3,5-dimethoxy-benzaldehyde to (R)-3,3',5,5'-tetramethoxybenzoin catalyzed by benzaldehyde lyase from Pseudomonas fluorescens. Results indicate a substrate-dependent inactivation of enzyme, which is in accordance with other recent studies.

BACKGROUND: Extraction is the method of choice for separating sensitive products. For example, it has been applied in aromatics and rare‐earth metal separation. Recently, biotechnological processes entered the field of bulk chemicals and might benefit from process intensification by in situ extraction of inhibiting products, however in the presence of cells and/or aeration. This study applies reactive extraction of the polyamide monomer hexane‐1,6‐diamine from cell‐containing medium. RESULTS: To prove technical feasibility of this reactive extraction, simulation of the extraction column based on single‐drop measurements was validated at pilot‐scale. Simulated results show good accordance with experimental data with an error below 20%. The extraction column was subsequently used as a bioreactor with integrated aeration and product extraction applying a four‐phase system investigating the effect on drop distributions and holdup of the dispersed phases. Interestingly, aeration increased the holdup up to five‐fold and decreased the mean drop size of the organic extractive phase by 30%, thereby potentially improving extraction efficiency. CONCLUSION: The ReDrop simulation tool is capable of predicting challenging separation processes like the reactive extraction of hexane‐1,6‐diamine with D2EHPA diluted in oleyl alcohol from a biotransformation medium. The process design enabled four‐phase operation of the column with aeration, extractive organic phase and cell‐containing continuous aqueous phase. © 2018 Society of Chemical Industry

Thiamine diphosphate (ThDP)-dependent enzymes catalyze a broad range of reactions with excellent enantioselectivity. Among these reactions, carboligations of aldehydes are of particular interest since the products, chiral hydroxy ketones, are valuable building blocks in the pharmaceutical industry. However, the substrates, for example, benzaldehyde, inactivate the biocatalysts, for example the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens (PfBAL). Because only few mechanistic kinetic models for carboligation and simultaneous inactivation are available today, we quantitatively determined the reaction kinetics and inactivation of the self-carboligation of benzaldehyde yielding the product (R)-benzoin catalyzed by PfBAL directly from progress curves using model-based experimental analysis. Discrimination of several inactivation models identified the substrate-dependent inactivation by benzaldehyde to be significant. Sensitivity analysis and optimal experimental design improved parameter precision significantly, to between 4 and 26% relative standard deviation while maintaining the necessary number of 13 experiments moderate. The developed mechanistic kinetic model will enable to perform a model-based process optimization to circumvent the substrate-dependent enzyme inactivation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1081-1092, 2018.

Aims The purpose of the study was to demonstrate feasibility of the Conserved Domains Database (CDD) for identification of novel biocatalysts with desirable properties from a class of well-characterized biocatalysts. Methods and results The thermostable ADH from Sulfolobus solfataricus with a broad substrate range was applied as a template for the search for novel thermostable ADHs via CDD. From the resulting hits, a putative ADH gene from the thermophilic organism Chloroflexus aurantiacus was cloned and expressed in Escherichia coli. The resulting enzyme was purified and characterized. With a temperature activity optimum of 70°C and a broad substrate spectrum especially for diketones, a versatile new biocatalyst was obtained. Conclusions Database-based mining in CDD is a suitable approach to obtain novel biocatalysts with desirable properties. Thereby, the available diversity of similar but not equal enzymes within this class can be increased. Significance and impact of the study For industrial applications, there is a demand for larger diversity of similar well-characterized enzymes in order to test them for a given process (biodiversity screening). For fundamental science, the comparison of enzymes with similar function but different sequence can provide insight into structure function relationships or the evolution of enzymes. This study gives a good example on how this demand can be efficiently met.

While the demand for paper and packaging material is increasing, industry and consumers are searching for more sustainable raw materials. This study evaluates three non-wood perennials to find alternative raw materials for the paper and pulp industry. Meadow hay, cup plant (Silphium perfoliatum L.) and Virginia mallow (Sida hermaphrodita (L.) Rusby) are attracting attention as potential raw materials for the bioeconomy due to biodiversity benefits, low fertilizer requirements as well as high yields. For the first time a detailed view on their fibre morphology and use as paper feedstock is given. After three different mechanical grinding methods, the plant material was screened, pulped in NaOH and beaten in a PFI mill. Birch fibre has been chosen as short-fibre control and blend base. Hand-sheets with different pulp blends of birch and one of the three raw materials were made, and paper properties were measured. For meadow hay, Virginia mallow and cup plant fibre lengths of 0.5, 1.3, and 0.9 mm were measured. Therefore, all perennial plant materials have comparable fibre lengths to hardwoods. Meadow hay blends with birch pulp percentages of 50 % and 75 % generated higher paper strength compared to the pure birch paper at a beating intensity of 5000 revolutions (PFI). The paper strength of cup plant and Virginia mallow blends is comparable to the strength of the birch control. Due to these promising results, all analyzed raw materials could find their application, especially in the growing area of sustainable packaging materials.

In a biorefinery, only a complete raw material cellulose saccharification, at high consistencies, leads to high sugar concentrations and thereby to high yields in the subsequent fermentation. However, enzymatic hydrolysis is significantly hampered by a strong slowdown of the reaction after its start, which may be explained, e.g., by enzyme inactivation, inhibition, or compositional and structural changes of the biomass. Effective pulp enzymatic hydrolysis strongly depends on the biomass pretreatment. We studied enzyme adsorption to three cellulosic materials and its effect on the reaction rate during hydrolysis. The maximal cellulase adsorption decreased from 137.7 mg protein/g biomass via 106.1 mg protein/g biomass to 88.8 mg/g biomass for α-cellulose, organocat, and organosolv material, respectively. The initial reaction rates directly correlated to the amount of adsorbed enzyme as predicted by the adsorption isotherms. However, the adsorbed cellulase loading increased dramatically during the reaction a...

Extraction is a downstream-process option in bio-based processes. Since the knowledge of phase-separation behavior is essential for designing efficient separation processes, this study investigates the settling and coalescence behavior of bio-based extraction systems using a standard lab-scale settling cell. The influence of different buffer media as well as of E. coli cells on coalescence was determined for the reactive extraction of hexane-1.6-diamine with isostearic acid and D2EHPA using kerosene and oleyl alcohol as diluents. As a result, an increasing pH value of the buffer significantly increases settling time. The presence of E. coli cells hinders the phase separation of the investigated systems, in particular with dispersed organic phases.

Progress curve experiments combined with optimal experimental design (OED) are an efficient approach to determine enzyme kinetics. However, it is hardly possible to verify why specific experiments are suggested for nonlinear enzyme kinetic model identification. Therefore, we systematically investigated the surface and contour plots of the sensitivities and of the OED criteria which are based on sensitivities. The model reaction was an enzyme catalyzed self-ligation of aldehydes to chiral 2-hydroxyketones. The visualization improved the understanding of OED and allowed for deducing and confirming five suggestions for kinetic identification: 1. Avoid experiments vicinal to the reaction equilibrium, 2. Choose the design space as large as possible, 3. Prefer D(eterminant)- and E(igenvalue)-criteria over the A(verage)-criterion, 4. Apply enzyme concentrations such that the reaction does not complete too fast, and 5. Few optimal experiments result in significantly improved parameter estimations. The graphical analysis also provides information about selecting appropriate optimization algorithms. This article is protected by copyright. All rights reserved.

To study the interaction of laccases, mediators, and substrates in laccase-mediator systems (LMS), an on-line measurement was developed using high performance anion exchange chromatography equipped with a CarboPac™ PA 100 column coupled to pulsed amperometric detection (HPAEC-PAD). The developed method was optimized for overall chromatographic run time (45 to 120 min) and automated sample drawing. As an example, the Trametes versicolor laccase induced oxidation of 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (adlerol) using 1-hydroxybenzotriazole (HBT) as mediator was measured and analyzed on-line. Since the Au electrode of the PAD detects only hydroxyl group containing substances with a limit of detection being in the milligram/liter range, not all products are measureable. Therefore, this method was applied for the quantification of adlerol, and-based on adlerol conversion-for the quantification of the LMS activity at a specific T. versicolor laccase/HBT ratio. The automated chromatographic activity assay allowed for a defined reaction start of all laccase-mediator-system reactions mixtures, and the LMS reaction progress was automatically monitored for 48 h. The automatization enabled an integrated monitoring overnight and over-weekend and minimized all manual errors such as pipetting of solutions accordingly. The activity of the LMS based on adlerol consumption was determined to 0.47 U/mg protein for a laccase/mediator ratio of 1.75 U laccase/g HBT. In the future, the automated method will allow for a fast screening of combinations of laccases, mediators, and substrates which are efficient for lignin modification. In particular, it allows for a fast and easy quantification of the oxidizing activity of an LMS on a lignin-related substrate which is not covered by typical colorimetric laccase assays. ᅟ.

BACKGROUND: The production of polymers constitutes the major mass flow in the chemical industry. The foreseeable future change from fossil to renewable resources leads to the demand for more production routes based on biotechnological conversions. These routes preferably require biocompatible components and possess certain limitations of temperature and pH value for the corresponding downstream processing, which can be met by liquid–liquid extraction. This study investigates the extraction of the monomers hexane‐1,6‐diamine and butane‐1,4‐diamine from fermentation media using bis(2‐ethylhexyl) hydrogen phosphate and isostearic acid as reactive extractants and kerosene as well as oleyl alcohol as diluents. RESULTS: As expected, the degree of extraction strongly depends on the pH‐value but only weakly on the diluents. A higher diluent polarity leads to a slightly higher degree of extraction at a given pH value. The fraction of reactive extractant can be used to adjust the minimal pH value to reach the desired degree of extraction. No significant influence of fermentation medium, buffer, presence of an E. coli microorganism, or temperature was detected. CONCLUSION: Reactive extraction of bio‐based diamines with organic acids is feasible. The degree of extraction can be adjusted by the fraction of reactive extractant. © 2016 Society of Chemical Industry

The kinetic description of enzyme-catalyzed reactions is a core task in biotechnology and biochemical engineering. In particular, mechanistic kinetic models help from the discovery of the biocatalyst throughout its application. Chemo- or enantioselective enzyme reactions often undergo two alternative pathways for the release of two different products from a central intermediate. For these types of reaction, no explicit reaction equations have been derived so far. To this end, we extend the commonly used Cleland's notation for branched reaction pathways and explicitly derive the rate expressions for two-coupled ordered bi-uni reactions. This mechanism also leads to a ping-pong bi-bi mechanism for a transfer reaction between the two products via the same central intermediate of the reaction system. Using the cross-ligation of benzaldehyde and propanal catalyzed by the thiamine diphosphate-dependent enzyme benzaldehyde lyase from Pseudomonas fluorescens yielding (R)-2-hydroxy-1-phenylbutan-1-one as a case study, we performed model-based experimental analysis to show that such a reaction mechanism can be modeled mechanistically and leads to reasonable kinetic parameters.

Screw press processing of biomass can be considered as a suitable mechanically based pretreatment for biofuel production since it disrupts the structure of lignocellulosic biomass with high shear and pressure forces. The combination with chemical treatment has been suggested to increase the conversion of lignocellulosic biomass to fermentable sugars. Within the study, the synergetic effect of alkaline (sodium hydroxide, NaOH) soaking and screw press pretreatment on wheat straw was evaluated based on, e.g., sugar recovery and energy efficiency. After alkaline soaking (at 0.1 M for 30 min) and sequential screw press pretreatment with various screw press configurations and modified screw barrel, the lignin content of pretreated wheat straw was quantified. In addition, the structure of pretreated wheat straw was investigated by scanning electron microscopy and measurement of specific surface area. It could be shown that removal of lignin is more important than increase of surface area of the biomass to reach a high sugar recovery. The rate constant of the enzymatic hydrolysis increased from 1.1 × 10−3 1/h for the non-treated material over 2.3 × 10−3 1/h for the alkaline-soaked material to 26.9 × 10−3 1/h for alkaline-assisted screw press pretreated material, indicating a nearly 25-fold improvement of the digestibility by the combined chemo-mechanical pretreatment. Finally, the screw configuration was found to be an important factor for improving the sugar recovery and for reducing the specific energy consumption of the screw press pretreatment.

A novel control concept was developed for a two phase biocatalytic oxidoreduction system. The hydrophobic substrate acetophenone dissolved in n -heptane is reduced to ( S )-1-phenylethanol by Candida parapsilosis carbonyl reductase 2, immobilized in a polyvinyl alcohol hydrogel. The cofactor NADH is regenerated via formic acid oxidation using likewise immobilized Candida boidinii formate dehydrogenase, increasing the pH-value of the aqueous phase. Therefore, the measured amount of CO 2 leaving the reactor is used to calculate the amount of formic acid to be replaced. Experiments lead to unexpectedly poor conversions motivating the development of a holistic process model, which was exclusively based on literature data and did not require parameter fitting. Simulation studies identified the CO 2 -solubility in n -heptane as the root cause for the time-lag in co-substrate feed and the resulting pH-shifts leading to poor conversions. They also indicated enzyme activity and stability as improvement targets, and the choice of an organic phase with low CO 2 -solubility. Exemplarily, increased buffer concentration to stabilize the pH within the hydrogel resulted in a predicted 13% productivity improvement, which could be validated experimentally, thus highlighting the potential of process models for complex biocatalytic process evaluation.

The development of novel enzymes for biocatalytic processes requires knowledge on substrate profile and selectivity; this can be derived from databases and from publications. Often, these sources lack time-course data for the substrate or product, and an unambiguous link between experiment and enzyme sequence. The lack of integrated, original data hampers the comprehensive analysis of enzyme kinetics and the evaluation of sequence-function relationships. In order to accelerate enzyme engineering, BioCatNet integrates protein sequence, protein structure, and experimental data for a given enzyme family. BioCatNet explicitly assigns the enzyme sequence to the experimental data, which consists of information on reaction conditions and time-course data. BioCatNet facilitates the consistent documentation of reaction conditions, the archiving of time-course data, and the efficient exchange of experimental data among collaborators. Data integration is demonstrated for three case studies by using the TEED (Thiamine diphosphate-dependent Enzymes Engineering Database).

Here, we report on how to stabilize and break emulsions that are compatible with enzymatic reaction conditions. Many substrates of enzymatic reactions are soluble in unpolar organic solvents whereas the enzymes themselves often need an aqueous environment. We use a buffer solution (triethanolamine hydrochloride) as aqueous and MtBE (tert-butyl methyl ether) as organic phase which provide good enzyme compatibility. We are able to break emulsions in a desired temperature range by using NiPAM–NiPMAM microgels with different monomer compositions and architecture, respectively. Our microgels need to deswell to about 55% of its swollen size at room temperature to let the emulsion break. Emulsions can be broken such that the microgels are either colloidally stable in the aqueous phase or flocculated. The temperature interval in which the microgels stay colloidally stable while the emulsion is broken is broader for the core–shell microgel than for the copolymer microgel. The behavior of the microgels in aqueous solution allows predicting: (i) the temperature at which the emulsion breaks and (ii) whether microgels flocculate or not during breaking the emulsion. However, the partial miscibility of the organic phase with the aqueous phase has to be taken into account. Thus, we are able to stabilize and break emulsions by employing microgels as responsive emulsifiers and to adapt the microgels to the requirements of biocatalytic processes.

Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument-caused deviations in enzyme kinetics between two MTP-readers, we comprehensively quantified temperature distribution in 96-well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96-well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature-induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development.

Biotechnology for biofuels 11(1), 279-289 (2018). doi:10.1186/s13068-018-1273-y, Published by BioMed Central, London

Green macroalgae are an abundant and undervalued biomass with a specific cell wall structure. In this context, different pretreatments, namely ethanol organosolv (Org), alkaline, liquid hot water (LHW), and ionic liquid (IL) pretreatments, were applied to the green macroalgae Ulva lactuca biomass and then evaluated. Their effects on chemical composition, biomass crystallinity, enzymatic digestibility, and theoretical ethanol potential were studied. The chemical composition analysis showed that the Org and LHW pretreatments allowed the highest glucan recovery (80.8 ± 3.6 and 62.9 ± 4.4 g/100 g DM, respectively) with ulvan (80.0 and 99.1%) and hemicellulose (55.0 and 42.3%) removal. These findings were in agreement with both thermogravimetric analysis and scanning electron microscopy results that confirm significant structural changes of the pretreated biomasses. It was found that the employed pretreatments did not significantly affect the cellulose crystallinity; however, they both increased the whole crystallinity and the enzymatic digestibility. This later reached 97.5% in the case of LHW pretreatment. Our results showed high efficiency saccharification of Ulva lactuca biomass that will constitute the key step of the implementation of a biorefinery process.

Modern biorefinery concepts focus on lignocellulosic biomass as a feedstock for the production of next generation biofuels and platform chemicals. Lignocellulose is a recalcitrant composite consisting of several tightly packed components which are stuck together by the phenolic polymer lignin hampering the access to the carbohydrate compounds of biomass. Certain saprophytic organisms are able to degrade lignin by the use of an enzymatic cocktail. Laccases have been found to play a major role during lignin degradation and have therefore been intensively researched with regard to potential applications for biomass processing. Within this review, we go along the process chain of a third generation biorefinery and highlight the process steps which could benefit from laccase applications. Laccases can assist the pretreatment of biomass and promote the subsequent enzymatic hydrolysis of cellulose by the oxidative modification of residual lignin on the biomass surface. In combination with mediator molecules laccases are often reported being able to catalyze the depolymerization of lignin. Studies with lignin model compounds confirm the chemical possibility of a laccase-catalyzed cleavage of lignin bonds, but the strong polymerization activity of laccase counters the decomposition of lignin by repolymerizing the degradation products. Therefore, it is a key challenge to shift the catalytic performance of laccase towards lignin cleavage by optimizing the process conditions. Another field of application for laccases is the detoxification of biomass hydrolyzates by the oxidative elimination of lignin-derived phenolics which inhibit hydrolytic enzymes and are toxic for fermentation organisms. This review critically discusses the potential applications for laccases in biorefinery processes and emphasizes the challenges and perspectives which go along with the use of this enzyme for the technical utilization of lignocellulose.

pH-shifts are a serious challenge in cofactor dependent biocatalytic oxidoreductions. Therefore, a pH control strategy was developed for reaction systems, where the pH value is not directly measurable. Such a reaction system is the biphasic aqueous-organic reaction system, where the oxidoreduction of hydrophobic substrates in organic solvents is catalysed by hydrogel-immobilized enzymes, and enzyme-coupled cofactor regeneration is accomplished via formate dehydrogenase, leading to a pH-shift. Dual lifetime referencing (DLR), a fluorescence spectroscopic method, was applied for online-monitoring of the pH-value within the immobilizates during the reaction, allowing for a controlled dosage of formic acid. It could be shown that by applying trisodium 8-hydroxypyrene-1, 3, 6-trisulfonate as pH indicator and Ru(II) tris(4, 7-diphenyl-1, 10-phenantroline) (Ru[dpp]) as a reference luminophore the control of the pH-value in a macroscopic gel-bead-stabilized aqueous/organic two phase system in a range of pH 6.5 to 8.0 is possible. An experimental proof of concept could maintain a stable pH of 7.5 ± 0.15 during the reaction for at least 105 h. With these results, it could be shown that DLR is a powerful tool for pH-control within reaction systems with no direct access for conventional pH-measurement.

The homodimeric Candida parapsilosis carbonyl reductase 2 (CPCR2) is an industrially attractive biocatalyst due to its broad substrate range and high stereoselectivity. In addition, CPCR2 is reasonably stable in monophasic organic solvents (e.g. alcohols) but apparently instable in biphasic organic systems. Hence, we conducted first a thorough quantitative inactivation study of CPCR2, using both wild-type and stability improved variants, in an attempt to identify critical factors influencing the enzyme stability. Possible inactivation phenomena including oxidation, shear forces, dissociation and adsorption at interfaces were assessed on a microliter scale using quantitative kinetic assays. Our results demonstrate that interface interactions and dimer dissociation are the main reasons for inactivation of CPCR2. Shear forces seems to enhance these inactivation processes whereas oxidation plays no role in CPCR2 inactivation. Secondly, an attempt was made to find suitable stabilization strategies to utilize CPCR2 in various reaction systems. To minimize the inactivation, bovine serum albumin was used as traditional blocking and crowding agent. The residual activity of the wild-type was successfully increased up to 2.5-fold by addition of 1 μg mL−1 bovine serum albumin. To avoid dimer dissociation the cofactor concentration was successively increased. The residual activity was successfully enhanced up to 5-fold, 3-fold and 1.5-fold for the wild-type, single and double mutant, respectively. Further, recently gained data from the enzyme crystal structure were used to interpret the effects of stabilization. We propose conformational change of a flexible region in CPCR2 upon binding of the cofactor leading to internal stabilization of the enzyme. In conclusion, we propose the addition of bovine serum albumin and the cofactor NADH as a suitable stabilization strategy to utilize CPCR2 in various reaction systems.

Analytical methods 7(18), 7866-7873(2015). doi:10.1039/C5AY01371B, Published by RSC Publ., Cambridge

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Lignocellulosic biomass can potentially be transformed into a wide variety of products ranging from biofuels and bulk chemicals to high value products. Through enzymatic hydrolysis, cellulose and hemicellulose can be utilized. However, lignin remains difficult to degrade which is partially due to its insolubility in aqueous solutions. Ionic liquids (IL) such as [EMIM][EtSO4] and [EMIM][Ac] increase lignin solubility and thereby, enzymatic access to lignin. Therefore, the reaction kinetics of the laccase–mediator system for the oxidation of lignin model compounds in IL solutions was investigated. Laccase in buffer solution had a higher activity compared to laccase in 5, 15, and 30% (v/v) [EMIM][EtSO4] and [EMIM][Ac]. However, the presence of 15% (v/v) IL helped to stabilize laccase activity over time. Cyclic voltammetry was used to investigate the reaction kinetics between the mediator ABTS and the lignin model compound veratryl alcohol in different ILs and IL concentrations. The increased conductivity at low IL concentrations expectedly lead to a rising reaction rate of mediator with substrate, whereas further increasing IL concentrations lead to higher viscosities and correspondingly, lower reaction rates. As a result, the investigation of the laccase–mediator system's reaction kinetics in buffer and ionic liquids provides a basis for evaluating and optimizing the lignocellulosic biomass degradation process.

A process concept combining pretreatment of wood in ionic liquids and subsequent enzymatic hydrolysis to sugars is herein investigated to identify operating conditions which allow for (i) the processing of larger wood chips of 10. mm length, (ii) low temperature, (iii) high sugar yield, and (iv) short processing time. A careful quantitative study of the interaction of pretreatment and hydrolysis reveals that hydrolysis is most effective if beech chips are first disintegrated in [EMIM][Ac] at 115. °C for 1.5. h. The cellulose conversion varies between 70.5. wt% and 90.2. wt% for hydrolysis times between 5. h and 72. h. A complete recovery of cellulose and xylan resulting in a total saccharification of 65. wt% of the wood chips could be demonstrated. It is shown that short pretreatment times are required to enable high sugar yield as well as to limit product degradation.

For the design of bioconversion processes parallel experimentation in microtiter plates is commonly applied to reduce the experimental load, although data accuracy and reproducibility are often reduced. In an effort to quantify the impact of different microscale experimental systems on the estimation of enzyme kinetic parameters from progress curves, we comprehensively evaluated the enzymatic reduction of acetophenone in both open and closed polystyrene and quartz microtiter plates as well as quartz cuvettes. Differences in conversion of up to 50% over time were observed increasing from polystyrene MTPs to quartz MTPs to quartz cuvettes. Initial reaction velocities increased systematically from polystyrene to quartz MTPs and cuvettes. The experimental errors decreased in the same order showing highest experimental error of about 20% in polystyrene. We further evaluated reasons causing the deviations within one system as well as between the systems. The choice of reaction vessel material, temperature effects and substrate cross contaminations in MTPs were shown to be of importance in the experimental results. Although the experimental data differed between the reaction vessels, no distinct trends in estimated kinetic parameters were found. While the microkinetic parameters vary up to an order of magnitude between different systems, the corresponding macrokinetic parameters lie in the same range for all systems varying by 29-118%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:87-95, 2017.

Emulsions stabilized by stimuli-responsive microgels were used to perform enzyme catalysis. Many substrates are poorly water-soluble, while enzymes naturally require aqueous environments, thus resulting in a two-phase aqueous-organic system. Smart microgels allow an enzyme-catalyzed reaction to be performed in an emulsion that can be broken under controlled conditions to separate the reaction product and to recycle the enzyme (E) and the microgel.

The efficacy of glycerol as an additive at enzyme immobilization on the catalytic performance (activity, stability, enantioselectivity and productivity) of immobilized (R)-specific Parvibaculum lavamentivorans alcohol dehydrogenase in a continuous gas phase reactor was investigated. The enantioselective reduction of 4-methyl-2-pentanone with concomitant NADH regeneration by 2-propanol as co-substrate was used as a model reaction. Non-porous glass beads were used as immobilization carriers. The immobilized alcohol dehydrogenase performance was markedly enhanced by the addition of glycerol. Interestingly, the enantioselectivity was stabilized during the reaction progress. The optimal glycerol amount, thermodynamic activity of water in the feed gas phase, and reaction temperature were obtained at 10 mg/g-glass carriers, 0.8 and 313 K in terms of the productivity, respectively. As a function of the thermodynamic activity of substrate in the feed gas phase, it was also optimized at 0.103 for 2-propanol and 0.250 for 4-methyl-2-pentanone. Compared to the glycerol-free immobilized enzyme system, the productivity of the chiral compound was ca. 15-fold higher, on the other hand, based on the kinetic analysis the maximum reaction rate was found to be about 2-times higher, while the Michaelis constant for 2-propanol was about 2-fold lower and that for 4-methyl-2-pentanone decreased slightly. These findings indicate that the gas phase enzyme reaction exhibits an excellent catalytic performance in the presence of additive glycerol for chiral compound production, however needs to optimize the additive amount and reaction conditions for effective industrial operation.

For the efficient production of glucose for platform chemicals or biofuels, cellulosic biomass is pretreated and subsequently hydrolyzed with cellulases. Although ionic liquids (IL) are known to effectively pretreat cellulosic biomass, the hydrolysis of IL pretreated biomass has not been optimized so far. Here, we present a semi-empirical model to rationally optimize the hydrolysis of pretreated α-cellulose - regenerated from IL and containing residual IL from the pretreatment. First, the influence of the IL MMIM DMP on the individual cellulases endoglucanase I, cellobiohydrolase I and β-glucosidase was investigated. Second, an enzyme loading-dependent model was developed to describe kinetics for the individual cellulases and cellulase mixtures. Third, this model was used to optimize the cellulase mixture for the efficient hydrolysis of regenerated cellulose containing residual IL. Finally, we could significantly increase the initial hydrolysis rate in 10% (v/v) MMIM DMP by 49% and the sugar yield by 10% points.

To increase the stability of enzymes in organic solvents, they may be immobilized in a polymer matrix suspended in the solvent. It is important to know that all the immobilized enzyme particles are completely suspended in order to ensure an efficient particle-liquid mass transfer coefficient while putting as little energy into the system as necessary so that the particles are not harmed. A novel noninvasive method based on photometry and analysis of images taken with a standard digital camera is presented to estimate a mean extinction in different parts of the reactor and to finally determine the just suspending stirring speed (NJS) for complete suspension in an objective way. With this optical method, six different reactor configurations were compared based on the maximum local energy dissipation rate. Here, the 90° bladed stirrer with a flat bottom proved to be one of the best configurations for attaining complete suspension with low maximum local energy dissipation rate.

The present study investigates continuous gas phase stereoselective reduction catalyzed by commercial thermophilic alcohol dehydrogenase. The alcohol dehydrogenase and cofactor β-nicotinamide adenine dinucleotide phosphate were simultaneously immobilized on non-porous spherical glass beads. The reduction of 4-methyl-2-pentanone to (S)-4-methyl-2-pentanol with concomitant cofactor regeneration by 2-propanol was employed as a model reaction. According to the differing buffer pH dependence of the enzyme activity on both substrates, the maximal reactivity was obtained at a pH of 7.5 used for immobilized enzyme preparation. Owing to the higher stereoselective production, the water activity in the feed gas phase and reaction temperature were optimized at 0.9 and 333 K, respectively.

A new prospective cellulase assay simultaneously combining high-throughput, online analysis and insoluble cellulosic substrates is described. The hydrolysis of three different insoluble cellulosic substrates, catalysed by a commercial cellulase preparation from Trichoderma reesei (Celluclast), was monitored using the BioLector - allowing online monitoring of scattered light intensities in a continuously shaken microtiter plate. Cellulase activities could be quantitatively assayed using the BioLector. At low cellulase/cellulose ratios, the Michaelis-Menten parameters of the cellulase mixture were mainly affected by the crystallinity index of the cellulose. Here, the apparent maximum cellulase activities inversely correlated with the crystallinity index of the cellulose. At high cellulase/cellulose ratios the particle size of the cellulose, defining the external surface area accessible to the cellulases, was the key determining factor for cellulase activity. The developed technique was also successfully applied to evaluate the pH optimum of cellulases. Moreover, the non-hydrolytic deagglomeration of cellulose particles was investigated, for the first time, using high-throughput scattered light detection. In conclusion, this cellulase assay ideally links high-throughput, online analysis and realistic insoluble cellulosic substrates in one simple system. It will considerably simplify and accelerate fundamental research on cellulase screening.

The continuous enzymatic gas/solid bio-reactor serves both for the production of volatile fine chemicals and flavors on an industrial scale and for thermodynamically controlled investigation of substrate and water effects on enzyme preparations for research purposes. Here, we comparatively investigated the molecular effects on the operational stability of NADPH-dependent Lactobacillus brevis alcohol dehydrogenase and an NADH-dependent variant thereof, LbADH G37D, in the gas/solid bioreactor. The reference reaction is the reduction of acetophenone to (R)-1-phenylethanol with concomitant oxidation of 2-propanol to acetone for the purpose of regeneration of the redox cofactor. It could be clearly shown that not the thermostability of the cofactor, but the thermostability of the proteins in the solid dry state govern the order of magnitude of the operational stability of both purified enzymes in the gas/solid reactor at low thermodynamic activity of water and substrate. However, at higher thermodynamic activity the operational stability in the gas/solid reactor is overlaid by stabilizing and destabilizing effects of the substrates that require further investigation. We demonstrated first evidence that the substrate affinity of the two variants in the gas/solid reactor is similar to the affinity in aqueous medium. We could also show that partial unfolding of the proteins with subsequent aggregation are the factors governing protein thermo-in-stability both in the dissolved and in the dry state. Thus, stability investigations of enzymes in the dry state are suggested to predict their basal level of operational stability in gas/solid reactions.

The estimation of kinetic parameters provides valuable insights into the function of biocatalysts and is indispensable in optimizing process conditions. Frequently, kinetic analysis relies on the Michaelis-Menten model derived from initial reaction rates at different initial substrate concentrations. However, by analysis of complete progress curves, more complex kinetic models can be identified. This case study compares two previously published experiments on benzaldehyde lyase-catalyzed self-ligation for the substrates benzaldehyde and 3,5-dimethoxybenzaldehyde to investigate 1) the effect of using different kinetic model equations on the kinetic parameter values, and 2) the effect of using models with and without enzyme inactivation on the kinetic parameter values. These analyses first highlight possible pitfalls in the interpretation of kinetic parameter estimates and second suggest a consistent strategy for data management and validation of kinetic models: First, Michaelis-Menten parameters need to be interpreted with care, complete progress curves are necessary to describe the reaction dynamics, and all experimental conditions have to be taken into consideration when interpreting parameter estimates. Second, complete progress curves should be stored together with the respective reaction conditions, to consistently annotate experimental data and avoid misinterpretation of kinetic parameters. Such a data management strategy is provided by the BioCatNet database system.

For the analysis of enzyme kinetics, a variety of programs exists. These programs apply either algebraic or dynamic parameter estimation, requiring different approaches for data fitting. The choice of approach and computer program is usually subjective, and it is generally assumed that this choice has no influence on the obtained parameter estimates. However, this assumption has not yet been verified comprehensively. Therefore, in this study, five computer programs for progress curve analysis were compared with respect to accuracy and minimum data amount required to obtain accurate parameter estimates. While two of these five computer programs (MS-Excel, Origin) use algebraic parameter estimation, three computer programs (Encora, ModelMaker, gPROMS) are able to perform dynamic parameter estimation. For this comparison, the industrially important enzyme penicillin amidase (EC 3.5.1.11) was studied, and both experimental and in silico data were used. It was shown that significant differences in the estimated parameter values arise by using different computer programs, especially if the number of data points is low. Therefore, deviations between parameter values reported in the literature could simply be caused by the use of different computer programs.

For the rational design of processes using immobilized enzymes a mechanistic kinetic model is required, which accounts for all kinetic and thermodynamic phenomena, including the enzyme reaction, the mass transfer of the reactants between both phases, and their diffusion inside the immobilizate. For the example of enzymes immobilized in hydrogel beads suspended in an organic solvent, such a mechanistic kinetic model was obtained by a model-based experimental analysis approach. It was proven that the usually applied concentration measurements in the bulk phase are not sufficient to draw mechanistic conclusions. The most suitable measurement technique was found to be the quantification of the concentration along the radius of the hydrogel bead. These line scans, achieved by two-photon laser scanning microscopy, for the first time allowed to estimate intrinsic reaction and mass transfer parameters simultaneously. Thus, the obtained intrinsic parameter estimates for the biphasic hydrogel system could be directly compared with those obtained in individual systems. This comparison revealed for the first time that the enzyme reaction was not significantly affected by the mild hydrogel encapsulation. However, a significant impact on the transport parameters was found that underlines the need for analyzing the real reaction system using mechanistic models.

The immobilization conditions of commercial ketoreductase for continuous enantioselective reduction in the gas-phase reaction were investigated with respect to the immobilization efficiency (residual activity and protein loading) and the gas-phase reaction efficiency (initial reaction rate, half-life, and enantioselectivity). For the analyses, ketoreductase was first immobilized by physical deposition on glass supports and the reduction of 2-butanone to (S)-2-butanol with the concomitant regeneration of NADH by 2-propanol was used as a model reaction. The optimal conditions of enzyme immobilization were obtained using an absolute pressure of 100 hPa for drying, a pH between 6.5 and 7.0, and a buffer concentration of 50 mM. The buffer concentration in particular had a strong effect on both the enzyme activity and enantioselectivity. Under optimal immobilization conditions, the thermostability of ketoreductase in the gas-phase system was enhanced compared to the aqueous-phase system, while the enantioselectivity was successfully maintained at a level identical to that of the native enzyme. These results indicate that the gas-phase reaction has a great potential for industrial production of chiral compounds, but requires careful optimization of immobilization conditions for the reaction to progress effectively.