Polyhydroxyalkanoates (PHA) are biodegradable polymers and have the potential to substitute with fossil-fuel based polymers since they have similar properties. Many studies on the production of PHA have been conducted, but the extraction/purification processes have received less attention. Mostly, solvent extraction has been studied, and the effect of different solvent types on the separation processes have been investigated. A better extraction method for PHA makes it a feasible alternative to fossil-fuel based polymers. In this study, a new protocol for the extraction of PHA from activated sludge by supercritical carbon dioxide disruption (sCO2) and biopolymer recovery from disrupted cells were proposed. Extraction experiments were carried out with sCO2 at different pressures, temperatures, times, biomass amounts, and modifier volumes. The operation yield was expressed based on the polyhydroxybutyrate (PHB) release efficiency. The biomass for the extraction experiments was obtained from a PHA production reactor where activated sludge was fed with anaerobically pretreated yeast industry wastewater. 80 % PHB releasing efficiency was achieved by disturbing 2 g of biomass at a density of 57 g/L (biomass/volume) with sCO2 at 200 bar pressure for 15 min at 40 °C. The PHB purity and molecular weight (Mv) of biopolymers were 80 % and 0.27•106 respectively. The use of methanol as a modifier during the sCO2 disruption increased the Mv to 0.37•106. Characterization studies by Fourier transform infrared spectroscopy (FTIR) and thermal degradation analysis (TGA) demonstrated that the biopolymer recovered with this extraction protocol was comparable to commercial PHB. As a clear advantage over the other extraction protocols; operationally fast and simple extraction procedure was achieved.

In an effort to achieve sustainable development goals, a reevaluation of the materials used in wooden buildings must be done, including the preservatives used to treat the materials. Since typical wood preservatives use toxic heavy metals, their handling and use can contaminate the environment. Therefore, substances such as lignin-derived components have been investigated as bio-based preservatives. Organosolv treatment is a promising technique for separating components of lignocellulosic biomass, which enables the utilization of each component. The present report describes components of lignocellulose with antifungal effects that were recovered after organosolv treatment using water and 1-butanol solvent at 473 K for 2 h, followed by simple solvent fractionation. The organosolv lignin was divided into three fractions: n-hexane soluble, ethyl acetate soluble, and ethyl acetate insoluble, yielding 23 wt%, 52 wt% and 13 wt%, respectively. Antifungal activity was determined using an agar plate method. White rot fungi (Trametes versicolor) was dispersed on the agar plate with a cellulose disc containing each lignin-derived fraction obtained from Japanese cedar. Results showed inhibition of fungal growth over the cellulose disc containing the n-hexane soluble fraction. To examine the effect in greater detail, the chemical structure of the n-hexane-soluble fraction on the antifungal activity was investigated. The content of phenolic hydroxyl group in n-hexane-soluble fraction was the highest (4.6 mmol/g), and the results from the chemical modification suggested that the functional group was required for antifungal action. In addition, the n-hexane-soluble fraction imparted some water resistance. The procedures used for cedar as a feedstock were applied to another type of biomass-bagasse-and its fractions showed antifungal activity similar to those of Japanese cedar.

As versatile and green biocatalysts for the asymmetric amination of ketones, the insufficient thermostability of transaminases always limits its broad application in the pharmaceutical and fine chemical industries. Here, synthetic shuffling technology was used to enhance stability of (R)-selective transaminase from Aspergillus terreus. The results showed that 30 out of 5000 mutants had improved thermostability by color-based screening method, among which mutants with residual enzyme activity higher than 50% at 45 °C for 10 min were selected for further analysis. Especially, the half-inactivation temperature (T5010), half-life (t1/2), and melting temperature (Tm) of the best mutant M14 (M280C-H210N-M150C-F115L) were 13.7 °C, 165.8 min, and 13.9 °C higher than that of the wild type (WT), respectively. M14 also exhibited a significant biocatalytic efficiency toward acetophenone and 1-acetylnaphthalene, the yield of which were 265.6% and 117.5% higher than WT, respectively. Based on molecular dynamics simulation, improved catalytic efficiency of M14 could be attributed to its increased hydrogen bonds interaction around the mutation sites. Additionally, the introduction of disulfide bond combined with above mutations has a synergistic effect on the improved protein thermostability.

The laccase enzyme family belongs to the oxidoreductase enzyme class and is one of the most commercially valuable enzymes that catalyzes the oxidation of one electron of a wide range of phenolic compounds. Separation and purification of laccases are crucial for industry since they play an important role in dye decolorization, biodegradation and food processing. Therefore, developing effective, high yielding and cost-effective methods for laccase production is vital. In this study, it was aimed to prepare cryogel columns for laccase purification following the bioproduction of laccase via Aspergillus niger. 2-hydroxyethyl methacrylate based cryogels were synthesized in the presence of 1-vinylimidazole as the affinity ligand and characterized by swelling tests, Brunauer-Emmett-Teller surface area measurement and scanning electron microscopy analysis. Surface area and water uptake ratio of cryogel columns were 35 m2/g and 93 %, respectively. The effect of pH, equilibrium laccase concentration, flow rate, interaction time and temperature on laccase adsorption were examined. The purification factor was calculated as 10.53 under optimum conditions and the enzyme recovery was found to be 86.7 % from fermentation medium. Current study revealed that laccase purification using cryogels following filtration of fermentation medium could be a promising candidate for industrial applications with eliminating the need for complex chromatographic steps.

Isomaltulose is a promising functional sweetener with broad application prospects in the food industry. Currently, isomaltulose is mainly produced through bioconversion processes based on the isomerization of sucrose, the economic feasibility of which is influenced by the cost of sucrose feedstocks, the biocatalyst preparation, and product purification. Cyanobacterial photosynthetic production utilizing solar energy and carbon dioxide represents a promising route for the supply of sugar products, which can promote both carbon reduction and green production. Previously, some cyanobacteria strains have been successfully engineered for synthesis of sucrose, the main feedstock for isomaltulose production. In this work, we introduced different sucrose isomerases into Synechococcus elongatus PCC 7942 and successfully achieved the isomaltulose synthesis and accumulation in the recombinant strains. Combinatory expression of an Escherichia coli sourced sucrose permease CscB with the sucrose isomerases led to efficient secretion of isomaltulose and significantly elevated the final titer. During a 6-day cultivation, 777 mg/L of isomaltulose was produced by the engineered Synechococcus cell factory. This work demonstrated a new route for isomaltulose biosynthesis utilizing carbon dioxide as the substrate, and provided novel understandings for the plasticity of cyanobacterial photosynthetic metabolism network.

Recombinant mammalian host cell lines, in particular CHO and HEK293 cells, are used for the industrial production of therapeutic proteins. Despite their well-known genomic instability, the control mechanisms that enable cells to respond to changes in the environmental conditions are not yet fully understood, nor do we have a good understanding of the factors that lead to phenotypic shifts in long-term cultures. A contributing factor could be inherent diversity in transcriptomes within a population. In this study, we used a full-length coverage single-cell RNA sequencing (scRNA-seq) approach to investigate and compare cell-to-cell variability and the impact of standardized and homogenous culture conditions on the diversity of individual cell transcriptomes, comparing suspension CHO-K1 and adherent HEK293FT cells. Our data showed a critical batch effect from the sequencing of four 96-well plates of CHO-K1 single cells stored for different periods of time, which was and may be therefore identified as a technical variable to consider in experimental planning. Besides, in an artificial and controlled culture environment such as used in routine cell culture technology, the gene expression pattern of a given population does not reveal any marker gene capable to disclose relevant cell population substructures, both for CHO-K1 cells and for HEK293FT cells. The variation observed is primarily driven by the cell cycle.

Oilseed cakes (OC) present high potential as feedstock for the biobased industry. Biotechnological processes allow OC valorization by the production of diverse value-added products and simultaneously altering OC structure, improving their nutritional value, and boosting OC utilization in animal feed. This work explored the use of fungi consortium of two different species as a bioprocessing approach to improve the nutritional quality of OC, obtain enzymes and antioxidants by solid-state fermentation (SSF) of sunflower cake (SFC) and rapeseed cake (RSC). Rhyzopus oryzae and Aspergillus ibericus consortium led to the highest production of cellulase (135 U/g) and β-glucosidase (265 U/g) while maximum protease (228 U/g) was obtained with A. niger and R. oryzae consortium. Maximum xylanase production (886 U/g) was observed in SSF of RSC resulting in high hemicellulose reduction. The synergistic action of lignocellulosic enzymes resulted in extracts with increased antioxidant potential with possible application as food additives against oxidative stress.

D-Pantothenic acid (D-PA) is an essential vitamin with wide applications. However, the biotechnological production of D-PA is still not competitive with the chemical synthesis in terms of production cost. Ketopantoate hydroxymethyltransferase is a crucial enzyme in the D-PA synthetic pathway in Escherichia coli encoded by the panB gene. Here a hot spots study was applied to a ketopantoate hydroxymethyltransferase from Corynebacterium glutamicum (CgKPHMT) to relieve the product inhibitory effect and thus improve the D-PA production. Compared with the wild type, the double-site variant CgKPHMT-K25A/E189S showed 1.8 times higher enzyme activity and 2.1 times higher catalytic efficiency, 1.88 and 3.32 times higher inhibitory constant of α-ketoisovalerate and D-PA, respectively. The D-PA yield using E. coli W3110 adopted the double-site variant was 41.17 g·L-1 within 48 h, a 9.80 g·L-1 increase. Structural analysis of K25A/E189S revealed the expansion of the entry channel and the change of the electric charge from negative to uncharged due to the substitution from glutamic acid to serine at site 189. Our study emphasized the positive roles of ketopantoate hydroxymethyltransferase in D-PA production and paved the way by analyzing critical enzymes in the synthetic pathway of E. coli to increase the D-PA yield.

One of the key metrics in the design of biosensors is the presence of an effective capture layer. Surface-immobilized proteins (especially as a part of antibody-antigen combinations) are the most commonly used capture ligands in biosensors. The surface coverage of these proteins in flow-based biosensors are affected by both the linker chemistry used to attach them as well as the microchannel geometry. We used streptavidin as a model protein to compare glutaraldehyde, EDC-NHS, sulfo-SMCC and sulfo-NHS-biotin as linkers inside straight, serpentine and square-wave microchannel geometries. We found that straight microchannels achieve the highest degree of protein immobilization compared to serpentine and square-wave microchannels, irrespective of the linker chemistry used. We also showed that for a given microchannel geometry, sulfo-NHS-biotin leads to the highest immobilization of streptavidin among all the linkers.

The measurement of volumetric titer is an integral step in the assessment and selection of a production cell line and cell culture process. The production of monoclonal antibodies (mAbs), a major class of therapeutic proteins, in Chinese Hamster Ovary (CHO) cell lines is challenging due to the clone-to-clone variations in the intrinsic capability to secrete a biologically complex protein. The measurement of intracellular mAb concentration could be a valuable tool to determine the ratio of intracellular to secreted product and be part of the evaluation of potential mAb productive cell lines. High throughput automation is a valuable tool that is used in bioprocess development to reduce work intensive steps. When coupled with the Simple Western (Wes) platform, automated capillary electrophoresis is an efficient method to measure recombinant protein concentration. In this study, we demonstrate the utility of using the automated Wes to rapidly measure intracellular titer and then compare the intracellular titer, volumetric titer and specific productivity between high and low production CHO clones expressing a model human IgG1 mAb.

Global interest for the minor cannabinoid cannabichromene (CBC) is growing steadily, as potential pharmaceutical applications continue to emerge. Due to low-yielding and unspecific extraction processes from its plant host Cannabis sativa, a biotechnological production is desirable. The complete heterologous biosynthesis of several other cannabinoids has recently been demonstrated as an accessible platform. However, the enzyme involved in the biosynthesis of CBC precursor cannabichromenic acid (CBCA) suffers from comparatively low catalytic efficiency, has not been crystallized, and remains poorly characterized. This study contributes to overcoming these challenges in three unique aspects. A deep‑learning‑assisted prediction of the CBCA synthase crystal structure using DeepMinds AlphaFold is performed and evaluated. The predicted CBCA synthase structure scored considerably higher in various quality assessments than the alternative template‑based homology modeling approach. A robust and practical understanding of crucial structure-function relationships for CBCA synthase is provided and a new binding mode for the substrate uncovered. Rational design approaches and computational analyses to suggest CBCAS variants with facilitated activity are applied. Through subsequent screening the substrate conversion of those variants is compared to the native enzyme. The best variant presented in this study increases CBCA production from crude lysate 22-fold and is one of five positions where substitutions had a significantly favorable impact on product formation.

L-lactate oxidase (LOX) is a biotechnologically important enzyme used in biosensors and colorimetric kits to detect lactate, a key biomarker in clinical diagnostics, sports medicine and the food industry. In this work, we produced a recombinant His-tagged Aerococcus viridans LOX (rLOX) in Escherichia coli and carried out its functional characterization for industrial applications. Our rLOX was evaluated in a colorimetric kit for human diagnostics and in an amperometric biosensor to measure the lactic acid in food products. The rLOX was fully functional for both applications, with a performance comparable to commercial untagged LOXs. As the industrial use of LOX enzyme requires a large-scale production, we scaled up the rLOX production in a fed-batch bioreactor culture and obtained a yield approximately ten times higher than that of the Erlenmeyer scale. The His-tag allowed an easy and highly efficient purification process, and a high-purity rLOX was recovered after this one-step affinity purification. In this study, we described a simple, rapid and cost-competitive approach for the production of a recombinant His-tagged LOX enzyme suitable for industrial use.

Microbial production of aromatic compounds is an attractive and sustainable biotechnological approach. With this motivation, here metabolic engineering of Corynebacterium glutamicum for l-tyrosine (l-Tyr) overproduction was attempted by pushing the carbon flux more towards l-Tyr. Translational start codon exchanges of prephenate dehydratase (pheA), anthranilate synthase (trpE), and phenylalanine aminotransferase (pat) genes revealed that reduced expression of pheA was the major contributor to increased l-Tyr titer while codon exchange in trpE was effective to a lower extent. Overexpression of aroE and qsuC, encoding shikimate dehydrogenase and 3-dehydroquinate dehydratase, respectively, and of dapC (cg1253), which is predicted to encode prephenate aminotransferase, were futile to increase l-Tyr titer. Similarly, deletion of the qsuABD gene cluster had also not enhanced titer. As for increasing precursor supply, deletion of ptsG of glucose uptake and overexpression of inositol permease (iolT2) and glucokinase (glcK) were not effective, but with utilization of xylose, enabled by overexpression of xylose isomerase (xylA) and xylulokinase (xylB), titer improved. Highest l-Tyr titer using the construct was 3.1 g/L on glucose and 3.6 g/L on a 1:3 (w/v) mixture of glucose and xylose. This result displays the potential of the constructed strain to produce l-Tyr from lignocellulosic renewable carbon sources.

As an easily obtained organic waste, by-product acetic acid could be an appropriate co-substrate with blue algae wastes (increase C/N ratio of substrates) for co-fermentation of PHA production. However, there are still acrylic acid and other chemicals in by-product acetic acid, which could cause severe inhibition for fermenting microorganisms during PHA production process. The current study represented that alkali pretreatment (pH level of 12) is a more favorable method compared with thermal pretreatment (80 ℃ for 30 min) for breaking cell walls of blue algae. It seemed that there was no synergistic effect of the combination of thermal and alkali pretreatment methods (temperature of 80 ℃ and pH level of 12). Optimal parameters during electro-fenton process for removal of inhibitors in by-product acetic acid were under current of 0.5 A, pH level of 3 and reaction time of 120 min. Both the highest dry weight of PHA and PHA concentration were achieved by applying blue algae and by-product acetic acid (after pretreatment) as co-substrates (mixed ratio of 3:1, stirring speed of 200 r/min, 24 h), indicating that using by-product acetic acid (after pretreatment) as co-substrate could increase C/N ratio and promote PHA production successfully. The current study could offer new insights for improving PHA production by co-fermentation.

This work aimed to quantify growth and biochemical parameters (viable cell density, Xv; cell viability, CV; glucose, lactate, glutamine, glutamate, ammonium, and potassium concentrations) in upstream stages to obtain rabies virus-like particles (rabies VLP) from insect cell-baculovirus system using on-line and off-line Raman spectra to calibrate global models with minimal experimental data. Five cultivations in bioreactor were performed. The first one comprised the growth of uninfected Spodoptera frugiperda (Sf9) cells, the second and third runs to obtain recombinant baculovirus (rBV) bearing Rabies G glycoprotein and matrix protein, respectively. The fourth one involved the generation of rabies VLP from rBVs and the last one was a repetition of the third one with cell inoculum infected by rBV. The spectra were acquired through a Raman spectrometer with a 785-nm laser source. The fitted Partial Least Square models for nutrients and metabolites were comparable with those previously reported for mammalian cell lines (Relative error < 15 %). However, the use of this chemometrics approach for Xv and CV was not as accurate as it was for other parameters. The findings from this work established the basis for bioprocess Raman spectroscopical monitoring using insect cells for VLP manufacturing, which are gaining ground in the pharmaceutical industry.

Tyrosinase is pivotal for melanin formation. Measuring monophenolase activity is of great importance for both fundamental research and industrial applications. For the first time, a backpropagation (BP) artificial neural network with three-dimensional fluorescence spectroscopy was applied for the real-time determination of tyrosinase monophenolase activity. Principal component analysis (PCA) was utilized for the dimension reduction of three-dimensional fluorescence data. The four principal components served as inputs for the neural network. Network parameters were optimized using a genetic algorithm (GA). BP learning algorithm was applied to train the network model to determine tyrosine levels in a binary mixture containing tyrosine and L-DOPA without any chemical separation. The time course of tyrosine consumption by monophenolase was determined to calculate the initial velocity of the enzymatic reaction. The limit of detection of the monophenolase assay was 0.0615 U·mL-1. This combined strategy of PCA, GAs, and BP artificial neural networks for three-dimensional fluorescence spectroscopy was efficient for the real-time and in-situ determination of monophenolase activity in a cascade reaction.