Marieke E. Bruins

Thermozymes and their applications

Enzymes from thermophilic microorganisms, thermozymes, have unique characteristics such as temperature, chemical, and pH stability. They can be used in several industrial processes, in which they replace mesophilic enzymes or chemicals. Thermozymes are often used when the enzymatic process is compatible with existing (high-temperature) process conditions. The main advantages of performing processes at higher temperatures are reduced risk of microbial contamination, lower viscosity, improved transfer rates, and improved solubility of substrates. However, cofactors, substrates, or products might be unstable or other side reactions may occur. Recent developments show that thermophiles are a good source of novel catalysts that are of great industrial interest. Thermostable polymer-degrading enzymes such as amylases, pullulanases, xylanases, proteases, and cellulases are expected to play an important role in food, chemical, pharmaceutical, paper, pulp, and waste-treatment industries. Considerable research efforts have been made to better understand the stability of thermozymes. There are no major conformational differences with mesophilic enzymes, and a small number of extra salt bridges, hydrophobic interactions, or hydrogen bounds seem to confer the extra degree of stabilization. Currently, overexpression of thermozymes in standard Escherichia coli allows the production of much larger quantities of enzymes, which are easy to purify by heat treatment. With wider availability and lower cost, thermophilic enzymes will see more application in industry.

Buffer selection for HP treatment of biomaterials and its consequences for enzyme inactivation studies

Biochemical systems are best studied under buffered conditions and it is, therefore, necessary to have good knowledge on the pressure-induced changes in buffer pH. For experiments conducted at a set temperature, it is possible to use a relatively stable pressure buffer. However, when experiments have to be performed in a relatively large window of both pressure and temperature, it is not possible to find a buffer that is both pressure and temperature independent. An example of such an experiment is the measurement of pressure and temperature stability of enzymes. Here, we present the pressure dependence of pH of several buffers. We show that the pH of MES buffer pH 6.0 in a P–T plane from 0.1 to 1000 MPa and 10–90 °C has a pH range from 5.5 to 6.5. The effect of pH changes on enzyme inactivation is illustrated by an inactivation experiment with the β-glycosidase from the hyperthermophile Pyrococcus furiosus. Within the measured range, a decrease in pH of 0.5 units causes the enzyme inactivation constant to increase by an additional factor 2–3.

Towards plant protein refinery: Review on protein extraction using alkali and potential enzymatic assistance

The globally increasing protein demands require additional resources to those currently available. Furthermore, the optimal usage of protein fractions from both traditional and new protein resources, such as algae and leaves, is essential. Here, we present an overview on alkaline plant protein extraction including the potentials of enzyme addition in the form of proteases and/or carbohydrolases. Strategic biomass selection, combined with the appropriate process conditions can increase protein yields after extraction. Enzyme addition, especially of proteases, can be useful when alkaline protein extraction yields are low. These additions can also be used to enable processing at a pH closer to 7 to avoid the otherwise severe conditions that denature proteins. Finally, a protein biorefinery concept is presented that aims to upcycle residual biomass by separating essential amino acids to be used for food and feed, and non‐essential amino acids for production of bulk chemicals.

Enzyme inactivation due to Maillard reactions during oligosaccharide synthesis by a hyperthermophilic glycosidase: influence of enzyme immobilisation

The extremely thermostable beta-glycosidase from Pyrococcus furiosus was used for the production of oligosaccharides with lactose as a substrate. Using a thermozyme made it possible to operate at higher reaction temperatures, and thus to increase the substrate concentration. This increased the substrate concentration and the subsequent lower water concentration suppressed hydrolysis and therefore improved the oligosaccharide yield. During the reaction, brown pigments were formed, caused by Maillard reactions. This changes the structure of the enzyme and causes faster inactivation of the enzyme, compared to normal inactivation by temperature. This faster inactivation is the main design criterion for the reaction system. Reduction of Maillard reactions can be done by altering the process conditions or through modification of the enzyme, either chemically or by altering the enzyme structure through genetic modifications. In this work, chemical modification of the enzyme was chosen by covalent immobilisation on Eupergit. Unfortunately, the immobilisation did not reduce Maillard reactivity

Increased susceptibility of beta-glucosidase from the hyperthermophile Pyrococcus furiosus to thermal inactivation at higher pressures.

The stability of β‐glucosidase from the hyperthermophile Pyrococcus furiosus was studied as a function of pressure, temperature and pH. The conformational stability was monitored using FTIR spectroscopy, and the functional enzyme stability was monitored by inactivation studies. The enzyme proved to be highly piezostable and thermostable, with an unfolding pressure of 800 MPa at 85 °C. The tentative pressure–temperature stability diagram indicates that this enzyme is stabilized against thermal unfolding at low pressures. The activity measurements showed a two‐step inactivation mechanism due to pressure that was most pronounced at lower temperatures. The first part of this inactivation took place at pressures below 300 MPa and was not visible as a conformational transition. The second transition in activity was concomitant with the conformational transition. An increase in pH from 5.5 to 6.5 was found to have a stabilizing effect.

Pressure-Aided Proteolysis of β-Casein

Beta-casein, which is present in the form of micelles at atmospheric pressure, has been hydrolyzed during pressure treatment to improve the accessibility of the protein. Two proteolytic enzymes with different specificities were used. Trypsin was aimed at mainly hydrolyzing hydrophilic segments of beta-casein and chymotrypsin at hydrolyzing hydrophobic segments of beta-casein. Measurements on aggregation during hydrolysis at atmospheric pressure showed that probably not micelle disruption, but disruption of much larger aggregates, occurs in the process. Peptide profiles were measured via reversed-phase chromatography. Measurements on enzyme activity after pressure treatments showed that trypsin was inactivated by pressure, which could explain all differences in peptide profiles compared to atmospheric experiments. Pressure did not influence the reaction mechanism, probably because the hydrophilic part of beta-casein is sufficiently accessible. However, chymotryptic proteolysis under pressure yielded new peptides that could not be explained by a change in enzyme activity. Here, pressure altered the mechanism of hydrolysis, by changing either enzyme specificity or substrate accessibility, which led to different peptides that can have different properties.

Synergy between bio-based industry and the feed industry through biorefinery

Processing biomass into multi-functional components can contribute to the increasing demand for raw materials for feed and bio-based non-food products. This contribution aims to demonstrate synergy between the bio-based industry and the feed industry through biorefinery of currently used feed ingredients. Illustrating the biorefinery concept, rapeseed was selected as a low priced feed ingredient based on market prices versus crude protein, crude fat and apparent ileal digestible lysine content. In addition it is already used as an alternative protein source in diets and can be cultivated in European climate zones. Furthermore, inclusion level of rapeseed meal in pig diet is limited because of its nutritionally active factors. A conceptual process was developed to improve rapeseeds nutritional value and producing other bio-based building blocks simultaneously. Based on the correlation between market prices of feed ingredients and its protein and fat content, the value of refined products was estimated. Finally, a sensitivity analysis, under two profit scenario, shows that the process is economically feasible. This study demonstrates that using biorefinery processes on feed ingredients can improve feed quality. In conjunction, it produces building blocks for a bio-based industry and creates synergy between bio-based and feed industry for more efficient use of biomass.

Controlling ethanol use in chain elongation by CO2 loading rate

Chain elongation is an open-culture biotechnological process which converts volatile fatty acids (VFAs) into medium chain fatty acids (MCFAs) using ethanol and other reduced substrates. The objective of this study was to investigate the quantitative effect of CO2 loading rate on ethanol usages in a chain elongation process. We supplied different rates of CO2 to a continuously stirred anaerobic reactor, fed with ethanol and propionate. Ethanol was used to upgrade ethanol itself into caproate and to upgrade the supplied VFA (propionate) into heptanoate. A high CO2 loading rate (2.5 LCO2·L–1·d–1) stimulated excessive ethanol oxidation (EEO; up to 29%) which resulted in a high caproate production (10.8 g·L–1·d–1). A low CO2 loading rate (0.5 LCO2·L–1·d–1) reduced EEO (16%) and caproate production (2.9 g·L–1·d–1). Heptanoate production by VFA upgrading remained constant (∼1.8 g·L–1·d–1) at CO2 loading rates higher than or equal to 1 LCO2·L–1·d–1. CO2 was likely essential for growth of chain elongating microorganisms while it also stimulated syntrophic ethanol oxidation. A high CO2 loading rate must be selected to upgrade ethanol (e.g., from lignocellulosic bioethanol) into MCFAs whereas lower CO2 loading rates must be selected to upgrade VFAs (e.g., from acidified organic residues) into MCFAs while minimizing use of costly ethanol.

Methylation in methanol-water mixtures: the effect of solvent composition and high pressure.

The effect of pressure (0.1 to 400 MPa) and solvent composition (methanol concentration of 5 to 30%) on the synthesis of beta-methylgalactoside was studied. Galactose was used as a reactant and the reaction was catalyzed by beta-galactosidase from Aspergillus oryzae. Under the applied conditions the enzyme was sufficiently stable and the reaction equilibrium was reached. Higher methanol concentrations obviously influenced the product yield positively due to an increase in reactant concentration but also due to a solvent effect. This solvent effect can be explained by measurement of the activities of galactose and methylgalactoside. These results may be generalized to other methylations in methanol-water systems, where methanol positively affects synthesis yields. Pressure had a small, negative effect on synthesis yields.

Solubility of the Proteinogenic α-Amino Acids in Water, Ethanol, and Ethanol–Water Mixtures

The addition of organic solvents to α-amino acids in aqueous solution could be an effective method in crystallization. We reviewed the available data on the solubility of α-amino acids in water, water–ethanol mixtures, and ethanol at 298.15 K and 0.1 MPa. The solubility of l-alanine, l-proline, l-arginine, l-cysteine, and l-lysine in water and ethanol mixtures and the solubility of l-alanine, l-proline, l-arginine, l-cysteine, l-lysine, l-asparagine, l-glutamine, l-histidine, and l-leucine in pure ethanol systems were measured and are published here for the first time. The impact on the solubility of amino acids that can convert in solution, l-glutamic acid and l-cysteine, was studied. At lower concentrations, only the ninhydrin method and the ultraperfomance liquid chromatography (UPLC) method yield reliable results. In the case of α-amino acids that convert in solution, only the UPLC method was able to discern between the different α-amino acids and yields reliable results. Our results demonstrate that α-amino acids with similar physical structures have similar changes in solubility in mixed water/ethanol mixtures. The solubility of l-tryptophan increased at moderate ethanol concentrations.