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Dilute acid (DA) pretreatment of biomass generates multiple inhibitory compounds within the pretreated hydrolysates. These compounds subsequently contribute to the formation of pseudo-lignin on the surface of the substrate, consequently impeding the efficiency of enzymatic digestibility. To detoxify the DA pretreated hydrolysates, post-incubation with non-catalytic proteins (amaranth protein, AP; soy protein, SP; bovine serum albumin, BSA) was performed in the present study. The enzymatic digestibility of DA-pretreated substrates was increased from 40.0% (without non-catalytic proteins) to 64.9, 53.8, and 56.4%, respectively, in the presence of AP (50 mg g−1), SP (65 mg g−1), and BSA (50 mg g−1). The post-incubation of pretreated substrates with non-catalytic proteins led to high hydrophobicity, contact angle, and accessibility, likely due to less formation of pseudo-lignin. Furthermore, gas chromatography/mass spectrometry analysis revealed that AP, SP, and BSA could lower the inhibitor concentrations in the pretreated hydrolysates by 39–100%, 5–100%, and 3–100%, respectively. The detoxification of the pretreated hydrolysates by AP demonstrated superior effectiveness compared to SP and BSA. To assess the affinity between inhibitors and non-catalytic proteins, surface plasmon resonance analysis was conducted, revealing the following affinity rates: AP (18.65 nM) > SP (17.04 nM) > BSA (16.87 nM). Additionally, molecular docking analysis revealed numerous molecular binding sites (i.e., hydrogen, polar, acidic, basic, and greasy contacts) with strong binding affinity ranging from −36.17 to −76.98 kcal mol−1 between the inhibitors and the amino acids of AP. Thus, this study highlights the potential application of AP as a cost-effective strategy for achieving a viable biorefinery. Also, the findings provide valuable insights that can be utilized to advance the development of (hemi)cellulases that exhibit enhanced resistance to lignin and inhibitors. [ABSTRACT FROM AUTHOR]

Bio-mitigation of carbon dioxide has recently attracted more research focus because it is a sustainable and ecofriendly way of carbon conversion to a variety of indispensable chemicals. However, studies regarding the recombinant strain development toward simultaneous chemical production and CO2 assimilation in E. coli are limited and the reported studies are challenged with critically low efficiency. In this context, we first expressed ribulose-1,5-bisphosphate carboxylase/oxygenases (RuBisCOs) and phosphoribulokinases (PRKs) from the Calvin cycle to optimize the CO2 assimilation capability in E. coli with a value of −4.9 g-CO2 per g-DCW. To enhance the CO2 assimilation capability and broaden the applicability of RuBisCO-equipped E. coli, we conducted stepwise integration of the recombinant genes (RuBisCO and PRK) as well as introduced the CRISPRi system for redirecting the carbon flux to the RuBisCO pathway. The final strain, SSCI with CRISPRi targeted on zwf and pfkAB, enhanced the CO2 assimilation capability to −1.58 g-CO2 per g-DCW. Finally, RuBisCO-equipped E. coli was applied for simultaneous CO2 recycling and production of high-value molecules from various proteins, including 5-aminoleuvinic acid (ALA) by ALA synthetase, carbon dioxide sequestration via carbonic anhydrase (CA), and 1,5-diaminopentane (DAP) through lysine decarboxylase. This genomic RuBisCO-equipped E. coli neutralizer is denoted as a GREEN workhorse, with the great opportunity to be a low-carbon footprint microbial cell factory. [ABSTRACT FROM AUTHOR]

Milk is a natural suspension of essential nutrients and can be exploited not only as a beverage consumed directly or transformed into a range of traditional dairy foods, but also as a source of ingredients for food product innovation. In addition, by-products from the dairy industry, which used to constitute an economical and environmental problem, are still rich in proteins, lactose and minerals. Therefore, from a green point of view, they can be valorized and are being exploited for multiple food and non-food applications. This paper aims to give an overview of the different applications of milk ingredients and by-products from the dairy industry. Additionally, enzymatic modification and production of bioactive peptides and bioemulsifiers broaden their functionality and applicability. Finally, a critical review of the most promising aspects as well as some features that need further development is presented. The unmet needs, the cross fertilization in between protein domains, the carbon footprint requirements, the environmental necessities, the health and wellness new demands, etc. are dominant factors in the search for an innovation approach outlining the potential that these “apparent” constraints oblige science and technology to account for. The adjacent technology analysis opportunity that such a review generates is, in essence, an open innovation step. [ABSTRACT FROM AUTHOR]

Microalgae-based bulk production, especially for biofuel, is not yet economically feasible. One way to improve financial performance of such systems is to maximise value through a biorefinery approach (e.g. co-production streams, waste minimisation). This can be implemented via extraction of valuable intracellular compounds upon cell disruption; however both low-cost, energy efficient, and scalable cell disruption techniques, and green chemicals are required. Waste heat is a readily available resource in most production plants, and can be used to power a hydrothermal pre-treatment (HTP) step to disrupt the cells. In this study, we use HTP (120–180 °C, 5–30 min) to disrupt Chlorella sp. cell walls. We perform a subsequent water-miscible solvent and/or urea extraction (UX) to solubilise and recover proteins. The maximum extraction efficiency of 62% was achieved after HTP at 180 °C for 10 min. Regardless of the reaction time, 120 °C was found to be ineffective for protein extraction. The efficiency can be further increased to 84% if a filter-washing step with acetone and urea is implemented. Most of the recovered proteins are between 8 to 14 kDa across all temperature ranges, suggesting that extraction efficiency depended both on partial protein hydrolysis and on accessibility to solvent (i.e. cell wall degradation, solid dispersion, membrane solubilisation). The most significant amino acid losses on a per mole basis were lysine (22%) and arginine (14%). The production of flexible and high-value protein-based products via HTP-UX may enable a circular and sustainable production step for microalgal biorefineries, while improving the downstream fuel processes through nitrogen heteroatom reduction. [ABSTRACT FROM AUTHOR]

A downstream process combining PEGylation reaction and the use of enzyme conjugates acting as phase-forming components of aqueous biphasic systems (ABS) is proposed here. In this approach, citrate buffer (pH = 7.0) was used simultaneously to stop the reaction (avoiding the use of hydroxylamine) and as a phase forming agent inducing the phase separation of the PEGylated proteins. The partition of the bioconjugates was assessed using two model enzymes of small size [cytochrome c (Cyt-c) and lysozyme (LYS)], and two of large size [ L -asparaginase (ASNase) and catalase (CAT)] as well as reactive PEG of 5, 10, 20 and 40 kDa. The effect of the reaction time on the PEGylation and recovery steps was also evaluated. All reactive PEGs allowed high selectivity in the separation of PEGylated proteins from native proteins (S > 100). A positive effect in terms of selectivity was found for longer reaction times. It allowed greater amounts of PEGylated proteins, with an increase of the PEG-protein rich-phase volume (top phase), allowing 100% of recovery of PEGylated proteins. More selective systems were obtained for Cyt-c and LYS (S > 100) compared to those for ASNase and CAT (40 < S < 60); nevertheless for all, the native and PEGylated proteins had their biological activity preserved. Envisioning the industrial potential evaluation, an integrated process diagram was defined combining the PEGylation reaction with the purification of the protein conjugates. Two different scenarios were investigated considering the PEGylation reaction performance. For both approaches (complete and incomplete PEGylation reaction), high recovery yields and purities were achieved for the PEGylated conjugates (92.1 ± 0.4% < %RecTCyt-c-PEG < 98.1 ± 0.1%; 84.6% < purity < 100%) and for the unreacted enzyme (%RecBCyt-c = 81 ± 1%; purity = 97.7%), while maintaining their structural integrity. [ABSTRACT FROM AUTHOR]

In the last few decades there has been increasing interest in the spinning of regenerated silk fibers. Although this research started with the use of harsh chemicals to dissolve and then coagulate silk proteins, it has shifted to biomimetic approaches that allow the use of environmentally friendly chemistries. This shift is not surprising since silk fibers in Nature are spun under extremely mild conditions. In this work we use a biomimetic spinning process called straining flow spinning to spin regenerated silkworm silk fibers. Our results show that straining flow spinning is a versatile technique that allows the spinning of silk fibers using different coagulating systems and provides the possibility of developing an industrially scalable and environmentally friendly spinning process. [ABSTRACT FROM AUTHOR]

A waste-to-protein system that integrates a range of waste-to-protein upgrading technologies has the potential to converge innovations on zero-waste and protein security to ensure a sustainable protein future. We present a global overview of food-safe and feed-safe waste resource potential and technologies to sort and transform such waste streams with compositional quality characteristics into food-grade or feed-grade protein. The identified streams are rich in carbon and nutrients and absent of pathogens and hazardous contaminants, including food waste streams, lignocellulosic waste from agricultural residues and forestry, and contaminant-free waste from the food and drink industry. A wide range of chemical, physical, and biological treatments can be applied to extract nutrients and convert waste-carbon to fermentable sugars or other platform chemicals for subsequent conversion to protein. Our quantitative analyses suggest that the waste-to-protein system has the potential to maximise recovery of various low-value resources and catalyse the transformative solutions toward a sustainable protein future. However, novel protein regulation processes remain expensive and resource intensive in many countries, with protracted timelines for approval. This poses a significant barrier to market expansion, despite accelerated research and development in waste-to-protein technologies and novel protein sources. Thus, the waste-to-protein system is an important initiative to promote metabolic health across lifespans and tackle the global hunger crisis. [ABSTRACT FROM AUTHOR]

Ionic liquids (ILs) have numerous applications in industrial processes as a benign alternative to conventional volatile organic solvents. However, many of them are toxic to organisms and are poorly biodegradable. In this work, a series of environmentally friendly cholinium ILs have been designed and synthesized. It was found that these ILs could form aqueous two-phase systems (ATPSs) with polypropylene glycol 400 (PPG400) which is thermo-sensitive, non-toxic and biodegradable. In order to understand the phase formation processes and possible application of these ATPSs for extraction/separation of proteins, the binodal curves and tie lines of these ATPSs were measured at 25 °C, and the effects of anionic structure of the ionic liquids, nature of the proteins and difference in the concentration of top- and bottom-phases on the partitioning behavior of some typical proteins were investigated systematically. It was shown that bovine serum albumin (BSA), trypsin, papain and lysozyme could be enriched effectively into the ionic liquid-rich phase of the ATPSs, and single-step extraction efficiency could be as high as 86.4–99.9% under the optimized conditions. Furthermore, enzyme activity of the native trypsin in water and in aqueous ionic liquid solutions was determined by using N-a-benzoyl-l-arginine ethyl ester as a substrate, and activity increases to about 127% was observed after 13 months storage. In addition, PPG400 has been recovered simply by heating and reused in the next extraction processes. This avoids the non-sustainable issue of highly salty water produced in the application of the polyethylene glycol (PEG) + salt and ionic liquid + salt ATPSs. [ABSTRACT FROM AUTHOR]