Immobilization-Stabilization of β-Glucosidase for Implementation of Intensified Hydrolysis of Cellobiose in Continuous Flow Reactors

Cellulose saccharification to glucose is an operation of paramount importance in the bioenergy sector and the chemical and food industries, while glucose is a critical platform chemical in the integrated biorefinery. Among the cellulose degrading enzymes, β-glucosidases are responsible for cellobiose hydrolysis, the final step in cellulose saccharification, which is usually the critical bottleneck for the whole cellulose saccharification process. The design of very active and stable β-glucosidase-based biocatalysts is a key strategy to implement an efficient saccharification process. Enzyme immobilization and reaction engineering are two fundamental tools for its understanding and implementation. Here, we have designed an immobilized-stabilized solid-supported β−glucosidase based on the glyoxyl immobilization chemistry applied in porous solid particles. The biocatalyst was stable at operational temperature and highly active, which allowed us to implement 25 °C as working temperature with a catalyst productivity of 109 mmol/min/gsupport. Cellobiose degradation was implemented in discontinuous stirred tank reactors, following which a simplified kinetic model was applied to assess the process limitations due to substrate and product inhibition. Finally, the reactive process was driven in a continuous flow fixed-bed reactor, achieving reaction intensification under mild operation conditions, reaching full cellobiose conversion of 34 g/L in a reaction time span of 20 min.

Simultaneous Enzymatic Cellulose Hydrolysis and Product Separation in a Radial-Flow Membrane Bioreactor

Hydrolysis is the heart of the lignocellulose-to-bioethanol conversion process. Using enzymes to catalyze the hydrolysis represents a more environmentally friendly pathway compared to other techniques. However, for the process to be economically feasible, solving the product inhibition problem and enhancing enzyme reusability are essential. Prior research demonstrated that a flat-sheet membrane bioreactor (MBR), using an inverted dead-end filtration system, could achieve 86.7% glucose yield from purified cellulose in 6 h. In this study, the effectiveness of flat-sheet versus radial-flow MBR designs was assessed using real, complex lignocellulose biomass, namely date seeds (DSs). The tubular radial-flow MBR used here had more than a 10-fold higher membrane surface area than the flat-sheet MBR design. With simultaneous product separation using the flat-sheet inverted dead-end filtration MBR, a glucose yield of 10.8% from pretreated DSs was achieved within 8 h of reaction, which was three times higher than the yield without product separation, which was only 3.5% within the same time and under the same conditions. The superiority of the tubular radial-flow MBR to hydrolyze pretreated DSs was confirmed with a glucose yield of 60% within 8 h. The promising results obtained by the novel tubular MBR could pave the way for an economic lignocellulose-to-bioethanol process.

Kinetic Analysis Misinterpretations Due to the Occurrence of Enzyme Inhibition by Reaction Product: Comparison between Initial Velocities and Reaction Time Course Methodologies

The Michaelis–Menten equation (MME) has been extensively used in biochemical reactions, but it is not appropriate when the reaction product inhibits the enzyme. Under these circumstances, each determined initial velocity, v0, is one experimental point that actually belongs to a different MME because enzymatic product inhibition occurs as the reaction starts. Furthermore, the inhibition effect is not constant, since the concentration of the product inhibitor rises as time increases. To unveil the hidden enzyme inhibition and to simultaneously demonstrate the superiority of an integrated Michaelis–Menten equation (IMME), the same range of data points, assuming product inhibition and the presence of a second different inhibitor, was used for kinetic analysis with both methodologies. This study highlights the superiority of the IMME methodology for when the enzyme is inhibited by the reaction product, giving a more coherent inhibition model and more accurate kinetic constants than the classical MME methodology.

Syngas Fermentation for the Production of Bio-Based Polymers: A Review

Increasing environmental awareness among the general public and legislators has driven this modern era to seek alternatives to fossil-derived products such as fuel and plastics. Addressing environmental issues through bio-based products driven from microbial fermentation of synthetic gas (syngas) could be a future endeavor, as this could result in both fuel and plastic in the form of bioethanol and polyhydroxyalkanoates (PHA). Abundant availability in the form of cellulosic, lignocellulosic, and other organic and inorganic wastes presents syngas catalysis as an interesting topic for commercialization. Fascination with syngas fermentation is trending, as it addresses the limitations of conventional technologies like direct biochemical conversion and Fischer–Tropsch’s method for the utilization of lignocellulosic biomass. A plethora of microbial strains is available for syngas fermentation and PHA production, which could be exploited either in an axenic form or in a mixed culture. These microbes constitute diverse biochemical pathways supported by the activity of hydrogenase and carbon monoxide dehydrogenase (CODH), thus resulting in product diversity. There are always possibilities of enzymatic regulation and/or gene tailoring to enhance the process’s effectiveness. PHA productivity drags the techno-economical perspective of syngas fermentation, and this is further influenced by syngas impurities, gas–liquid mass transfer (GLMT), substrate or product inhibition, downstream processing, etc. Product variation and valorization could improve the economical perspective and positively impact commercial sustainability. Moreover, choices of single-stage or multi-stage fermentation processes upon product specification followed by microbial selection could be perceptively optimized.

Production of Phenylpyruvic Acid by Engineered L-Amino Acid Deaminase from Proteus Mirabilis

Objectives This study aimed to develop an efficient enzymatic strategy for the industrial production of phenylpyruvate (PPA) from L-phenylpyruvic acid (L-Phe). Results L-amino acid deaminase from Proteus mirabilis (L-pmAAD) was expressed in E. coli BL21(DE3) and modified to release product inhibition by employing conformational dynamics engineering. Based on structural analysis, two residues (E145A/L341A) were identified for reducing interactions between the product and enzyme and increasing flexibility of the protein, thereby facilitating the product release. The mutant M2E145A/E341A exhibited a 3.84-fold reduction in product inhibition and a 1.35-fold increase in catalytic efficiency in comparison to the wild type. Finally, 81.2 g/L PPA production with a conversion of 99.6% was obtained in a 5-L bioreactor. Conclusion The engineered catalyst can significantly reduce product inhibition and facilitate the effective industrial synthesis of PPA.

Two Fungal Flavonoid-specific Glucosidases/rutinosidases for Rutin Hydrolysis and Rutinoside Synthesis Under Homogeneous and Heterogeneous Reaction Conditions

The glycosidases within GH5-23 cleave the glycosidic bond of β-glucosylated or rutinosylated flavonoids. Moreover, by virtue of their transglycosylation activity, glycoconjugates with glucosyl and rutinosyl moieties are accessible. Here we report the biochemical characterization and biotechnological assessment of two heterologously expressed members of GH5-23 – McGlc from Mucor circinelloides and PcGlc from Penicillium chrysogenum. Both enzymes exhibited the highest hydrolytic activities with quercetin-3-β-O-glucopyranoside, whereas lower specificity constants were determined with the rutinosides narcissin, rutin and hesperidin. High stabilities against thermal, ethanol and dimethyl sulfoxide-induced inactivation, a very limited secondary hydrolysis of the formed transglycosylation products, and no detectable product inhibition were additional features appropriate for biotechnological applications. The enzymes were compared in their efficiencies to hydrolyze rutin and to synthesize 2‑phenylethyl rutinoside under homogeneous and heterogeneous reaction conditions using high rutin concentrations of 100 and 300 mM. Highest transglycosylation efficiencies were achieved with fully dissolved rutin in reaction mixtures containing 25 % dimethyl sulfoxide. Molecular docking and multiple sequence alignments suggest that the hydrophobic environment of aromatic residues within the +1 subsite of GH5-23 glycosidases is very important for the binding of flavonoid glucosides and rutinosides.

Two fungal flavonoid-specific glucosidases/rutinosidases for rutin hydrolysis and rutinoside synthesis under homogeneous and heterogeneous reaction conditions

The glycosidases within GH5-23 cleave the glycosidic bond of β-glucosylated or rutinosylated flavonoids. Moreover, by virtue of their transglycosylation activity, glycoconjugates with glucosyl and rutinosyl moieties are accessible. Here we report the biochemical characterization and biotechnological assessment of two heterologously expressed members of GH5-23—McGlc from Mucor circinelloides and PcGlc from Penicillium chrysogenum. Both enzymes exhibited the highest hydrolytic activities with quercetin-3-β-O-glucopyranoside, whereas lower specificity constants were determined with the rutinosides narcissin, rutin and hesperidin. High stabilities against thermal, ethanol and dimethyl sulfoxide-induced inactivation, a very limited secondary hydrolysis of the formed transglycosylation products, and no detectable product inhibition were additional features appropriate for biotechnological applications. The enzymes were compared in their efficiencies to hydrolyze rutin and to synthesize 2-phenylethyl rutinoside under homogeneous and heterogeneous reaction conditions using high rutin concentrations of 100 and 300 mM. Highest transglycosylation efficiencies were achieved with fully dissolved rutin in reaction mixtures containing 25% dimethyl sulfoxide. Molecular docking and multiple sequence alignments suggest that the hydrophobic environment of aromatic residues within the + 1 subsite of GH5-23 glycosidases is very important for the binding of flavonoid glucosides and rutinosides.