In contrast to the application of soluble enzymes in industry, immobilized enzymes often offer advantages in view of stability, volume specific biocatalyst loading, recyclability as well as ...simplified downstream processing. In this tutorial review the focus is set on the evaluation of immobilized enzymes in respect to mass transport limitations, immobilization yield and stability, to enable industrial applications.
Evaluation of immobilized enzymes in respect to mass transport limitations, immobilization yield and stability, to enable industrial applications.
‘Ideal’ solvents in biocatalysis have to fulfill a large number of requirements, such as high substrate solubility, high enzyme activity and stability, and positive effects on reaction equilibrium. ...In the past decades, many enzymatic synthesis routes in water-based and nonaqueous (organic solvents, ionic or supercritical fluids) reaction media have been developed. However, no solvent meets every demand for different reaction types at the same time, and there is still a need for novel solvents suited for different reaction types and applications. Deep eutectic solvents (DESs) have recently been evaluated as solvents in different biocatalytic reactions. They can improve substrate supply, conversion, and stability. The best results were obtained when the DES is formed by the substrates of an enzymatic reaction.
A wide range of inexpensive renewables can be used as components of deep eutectic solvents; therefore, the solvents are often biodegradable, nontoxic, nonvolatile, and nonflammable.
A broad range of enzymatic and chemo-enzymatic synthesis reactions can be performed in deep eutectic solvents. Most of the reactions, covering transesterifications, epoxidations, and C–C bond formations, are catalyzed by lipases.
Deep eutectic solvents can be used to enable new biocatalytic synthesis routes that cannot be realized in conventional reaction media (i.e., aqueous buffers).
Components of deep eutectic solvents can be used as substrate and solvent at the same time; this virtually solvent-free approach enables processes with high substrate conversion and high atom efficiency.
The occurrence of organically bound phosphorus (P) as phytate in plant-based feeding material is a challenge for livestock farming due to limited utilization during the digestion by the animal. Its ...excretion into the environment through the manure pathway, poses a challenge, due to increased eutrophication and restrictions for P. Hence, while the routine supplementation of phytase enzymes in monogastric diets is common practice, metabolically triggering endogenous plant enzymes by wet-treatment prior to feeding can also lead to a better utilization of phytate bound P and increased digestibility by the animal. Nonetheless, traditional quantification of residual phytate content in plant material is both labor- and chemical-intense. The aim of this study is, therefore, to predict the remaining phytate content during wet-treatment through a straightforward and flexible methodological approach based on real-time analysis. For this, rye bran is used as a model substrate. A partial least squares regression algorithm relates the infrared spectra to the concentrations and predict the amount of P species that are transferred from the bran matrix to the liquid phase. By applying a mass balance for P and considering the effect of water compression, the amount of residual phytate content in rye bran at different time points of wet-treatment is determined. Results are compared to wet chemical methods, resulting in a RMSEP of 0.28 g
∙100 g
. In addition, the study demonstrates the feasibility of this approach and provides insights into phytate degradation in plant residuals. The method holds the potential for further applications for the screening and investigation of feed material conditioning and also offers the possibility to employ various real-time analytical techniques for assessing phytate remnants in biological samples during wet-treatment.
The fixation of CO2 by enzymatic carboxylation for production of valuable carboxylic acids is one way to recycle carbon. Unfortunately, this type of reaction is limited by an unfavourable ...thermodynamic equilibrium. An excess of the C1 substrate is required to increase conversions. Solvents with a high CO2 solubility, such as amines, can provide the C1 substrate in excess. Here, we report on the effect of CO2 pressures up to 1100 kPa on the enzymatic carboxylation of resorcinol in aqueous triethanolamine. Equilibrium yields correlate to the bicarbonate concentration. However, inhibition is observed at elevated pressure, severely reducing the enzyme activity. The reaction yields were reduced at higher pressures, whereas at ambient pressure, higher yields were achieved. Overall, CO2 pressures above 100 kPa have been demonstrated to be counterproductive for improving the biotransformation, as productivity decreases rapidly for only a modest improvement in conversion. It is expected that CO2 carbamylation intensifies at elevated CO2 pressures, causing the inhibition of the enzyme. To further increase the reaction yield, the in situ product precipitation is tested by the addition of the quaternary ammonium salt tetrabutylammonium bromide.
Ulvan is a sulfated polysaccharide extracted from green macroalgae with unique structural and compositional properties. Due to its biocompatibility, biodegradability, and film-forming properties, as ...well as high stability, ulvan has shown promising potential as an ingredient of biopolymer films such as sustainable and readily biodegradable biomaterials that could replace petroleum-based plastics in diverse applications such as packaging. This work investigates the potential of Ulva fenestrata as a source of ulvan. Enzyme-assisted extraction with commercial cellulases (Viscozyme L and Cellulysin) and proteases (Neutrase 0.8L and Flavourzyme) was used for cell wall disruption, and the effect of the extraction time (3, 6, 17, and 20 h) on the ulvan yield and its main characteristics (molecular weight, functional groups, purity, and antioxidant capacity) were investigated. Furthermore, a combined process based on enzymatic and ultrasound extraction was performed. Results showed that higher extraction times led to higher ulvan yields, reaching a maximum of 14.1% dw with Cellulysin after 20 h. The combination of enzymatic and ultrasound-assisted extraction resulted in the highest ulvan extraction (17.9% dw). The relatively high protein content in U. fenestrata (19.8% dw) makes the residual biomass, after ulvan extraction, a potential protein source in food and feed applications.
Enzymes are able to perform reactions under mild conditions, e.g., pH and temperature, with remarkable chemo-, regio-, and stereoselectivity. Due to this feature the number of biocatalysts used in ...organic synthesis has rapidly increased during the last decades, especially for the production of chiral compounds. The present review highlights biotechnological processes for the production of chiral alcohols by reducing prochiral ketones with whole cells. Microbial transformations feature different characteristics in comparison to isolated enzymes. Enzymes that are used in whole-cell biotransformations are often more stable due to the presence of their natural environment inside the cell. Because reductase-catalyzed reactions are dependent on cofactors, one major task in process development is to provide an effective method for regeneration of the consumed cofactors. Many whole-cell biocatalysts offer their internal cofactor regeneration that can be used by adding cosubstrates, glucose or, in the case of cyanobacteria, simply light. In this paper, various processes carried out on laboratory and industrial scales are presented. Thereby, attention is turned to process parameters, e.g., conversion, yield, enantiomeric excess, and process strategies, e.g., the application of biphasic systems. The biocatalytic production of chiral alcohols utilizing isolated enzymes is presented in part I of this review (Goldberg et al., Appl Microbiol Biotechnol, 2007).
This review highlights the state of the art in the field of coupled chemo(enzymatic) reactions in continuous flow. Three different approaches to such reaction systems are presented herein and ...discussed in view of their advantages and disadvantages as well as trends for their future development.
Bacterial cell-cell communication (quorum sensing, QS) represents a fundamental process crucial for biofilm formation, pathogenicity, and virulence allowing coordinated, concerted actions of bacteria ...depending on their cell density. With the widespread appearance of antibiotic-resistance of biofilms, there is an increasing need for novel strategies to control harmful biofilms. One attractive and most likely effective approach is to target bacterial communication systems for novel drug design in biotechnological and medical applications. In this study, metagenomic large-insert libraries were constructed and screened for QS interfering activities (quorum quenching, QQ) using recently established reporter strains. Overall, 142 out of 46,400 metagenomic clones were identified to interfere with acyl-homoserine lactones (AHLs), 13 with autoinducer-2 (AI-2). Five cosmid clones with highest simultaneous interfering activities were further analyzed and the respective open reading frames conferring QQ activities identified. Those showed homologies to bacterial oxidoreductases, proteases, amidases and aminotransferases. Evaluating the ability of the respective purified QQ-proteins to prevent biofilm formation of several model systems demonstrated highest inhibitory effects of QQ-2 using the crystal violet biofilm assay. This was confirmed by heterologous expression of the respective QQ proteins in Klebsiella oxytoca M5a1 and monitoring biofilm formation in a continuous flow cell system. Moreover, QQ-2 chemically immobilized to the glass surface of the flow cell effectively inhibited biofilm formation of K. oxytoca as well as clinical K. pneumoniae isolates derived from patients with urinary tract infections. Indications were obtained by molecular and biochemical characterizations that QQ-2 represents an oxidoreductase most likely reducing the signaling molecules AHL and AI-2 to QS-inactive hydroxy-derivatives. Overall, we propose that the identified novel QQ-2 protein efficiently inhibits AI-2 modulated biofilm formation by modifying the signal molecule; and thus appears particularly attractive for medical and biotechnological applications.
One way to enhance the flow properties of packed bed reactors, including efficient mass transfer and high catalyst conversion rates, is the use of 3D printing. By creating optimized structures that ...prevent channeling and high pressure drops, it is possible to achieve the desired target. Nevertheless, additively manufactured structures most often possess a limited surface-area-to-volume-ratio, especially as porous printed structures are not standardized yet. One way to achieve surface-enhanced 3D-printed structures is surface modification to introduce surface-initiated polymers. In addition, when stimuli-sensitive polymers are chosen, autonomous process control is prospective. The current publication deals with the application of surface-induced polymerization on 3D-printed structures with the subsequent application as an enzyme carrier. Surface-induced polymerization can easily increase the number of enzymes by a factor of six compared to the non-modified 3D-printed structure. In addition, the swelling behavior of polyacrylic acid is proven, even with immobilized enzymes, enabling smart reaction control. The maximum activity of Esterase 2 (Est2) from Alicyclobacillus acidocaldarius per g carrier, determined after 2 h of polymer synthesis, is 0.61 U/gsupport. Furthermore, universal applicability is shown in aqueous and organic systems, applying an Est2 and Candida antarctica lipase B (CalB) catalyzed reaction and leaving space for improvement due to compatibility of the functionalization process and the here chosen organic solvent. Overall, no enzyme leaching is detectable, and process stability for at least five subsequent batches is ensured.
Immobilisation plays an important role in the industrial application of enzymes. The stabilisation and reusability of immobilised enzymes reduce the cost of the catalyst and facilitate their use in ...continuously operated reactors. For this purpose, an applicable type of immobilisation needs to be identified. In this study, we investigate the conversion of CDP and PolyP to CTP by NDP polyphosphate phosphotransferase 3 from Ruegeria pomeroyi (RpPPK2-3) and describe the covalent immobilisation of RpPPK2-3. In order to select a suitable carrier for the immobilisation of RpPPK2-3, a screening with different amino methacrylate (glutaraldehyde-pre-activated) and epoxy methacrylate carriers was carried out. The epoxy methacrylate carrier ECR8209M (Purolite®) was found to be the most suitable. With a half-life of 462 d when stored at 6 °C and a 50-fold reusability with a 93% residual activity, the immobilised enzyme showed a higher stability compared to the soluble enzyme with a half-life of 0.04 d. Although the half-life of the soluble enzyme could be increased to 32 d by adding PPi, it could not reach the stability of the immobilisate. Due to the resilience of the immobilisate, it is suitable for application in continuous reactor set-ups, e.g., packed-bed reactors.
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