Engineering Phi29-DNAP Variants for Customized DNA Hydrogel Materials.

DNA hydrogels, which hold potential for use in medicine, biosensors, and tissue engineering, can be produced through enzymatic rolling circle amplification (RCA) using phi29 DNA polymerase (DNAP). This paper introduces new DNAP variants designed for RCA-based DNA hydrogel production, featuring enzymes with modified DNA binding, enhanced thermostability, reduced exonuclease activity, and protein tags for fluorescence detection or specific immobilization. We evaluated these enzymes by quantifying DNA output via quantitative PCR (qPCR) and assessing hydrogel mechanical properties through micromechanical indentation. The results showed that most variants generated similar DNA amounts and hydrogels with comparable mechanical properties. Additionally, all variants successfully incorporated non-natural nucleotides, such as base-modified dGTP derivatives and 2'-fluoro-dGTP, during RCA. This study's robust analytical approach offers a strong foundation for selecting new enzymes and producing DNA hydrogels with tailored material properties.

Enhanced Bioactivity of Tailor-Made Glycolipid Enriched Manuka Honey.

Glycolipids can be synthetized in deep eutectic solvents (DESs) as they possess low water content allowing a reversed lipase activity and thus enables ester formation. Based on this principle, honey can also serve as a media for glycolipid synthesis. Indeed, this supersaturated sugar solution is comparable in terms of physicochemical properties to the sugar-based DESs. Honey-based products being commercially available for therapeutic applications, it appears interesting to enhance its bioactivity. In the current work, we investigate if enriching medical grade honey with in situ enzymatically-synthetized glycolipids can improve the antimicrobial property of the mixture. The tested mixtures are composed of Manuka honey that is enriched with octanoate, decanoate, laurate, and myristate sugar esters, respectively dubbed GOH, GDH, GLH, and GMH. To characterize the bioactivity of those mixtures, first a qualitative screening using an agar well diffusion assay has been performed with methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Candida bombicola, Escherichia coli, and Pseudomonas putida which confirmed considerably enhanced susceptibility of these micro-organisms to the different glycolipid enriched honey mixtures. Then, a designed biosensor E. coli strain that displays a stress reporter system consisting of three stress-specific inducible, red, green, and blue fluorescent proteins which respectively translate to physiological stress, genotoxicity, and cytotoxicity was used. Bioactivity was, therefore, characterized, and a six-fold enhancement of the physiological stress that was caused by GOH compared to regular Manuka honey at a 1.6% (v/v) concentration was observed. An antibacterial agar well diffusion assay with E. coli was performed as well and demonstrated an improved inhibitory potential with GOH upon 20% (v/v) concentration.

Microwave-Assisted One-Pot Lipid Extraction and Glycolipid Production from Oleaginous Yeast Saitozyma podzolica in Sugar Alcohol-Based Media.

Glycolipids are non-ionic surfactants occurring in numerous products of daily life. Due to their surface-activity, emulsifying properties, and foaming abilities, they can be applied in food, cosmetics, and pharmaceuticals. Enzymatic synthesis of glycolipids based on carbohydrates and free fatty acids or esters is often catalyzed using certain acyltransferases in reaction media of low water activity, e.g., organic solvents or notably Deep Eutectic Systems (DESs). Existing reports describing integrated processes for glycolipid production from renewables use many reaction steps, therefore this study aims at simplifying the procedure. By using microwave dielectric heating, DESs preparation was first accelerated considerably. A comparative study revealed a preparation time on average 16-fold faster than the conventional heating method in an incubator. Furthermore, lipids from robust oleaginous yeast biomass were successfully extracted up to 70% without using the pre-treatment method for cell disruption, limiting logically the energy input necessary for such process. Acidified DESs consisting of either xylitol or sorbitol and choline chloride mediated the one-pot process, allowing subsequent conversion of the lipids into mono-acylated palmitate, oleate, linoleate, and stearate sugar alcohol esters. Thus, we show strong evidence that addition of immobilized Candida antarctica Lipase B (Novozym 435®), in acidified DES mixture, enables a simplified and fast glycolipid synthesis using directly oleaginous yeast biomass.

Lipase-Catalyzed Production of Sorbitol Laurate in a "2-in-1" Deep Eutectic System: Factors Affecting the Synthesis and Scalability.

Surfactants, such as glycolipids, are specialty compounds that can be encountered daily in cleaning agents, pharmaceuticals or even in food. Due to their wide range of applications and, more notably, their presence in hygiene products, the demand is continuously increasing worldwide. The established chemical synthesis of glycolipids presents several disadvantages, such as lack of specificity and selectivity. Moreover, the solubility of polyols, such as sugars or sugar alcohols, in organic solvents is rather low. The enzymatic synthesis of these compounds is, however, possible in nearly water-free media using inexpensive and renewable building blocks. Using lipases, ester formation can be achieved under mild conditions. We propose, herein, a "2-in-1" system that overcomes solubility problems, as a Deep Eutectic System (DES) made of sorbitol and choline chloride replaces either a purely organic or aqueous medium. For the first time, 16 commercially available lipase formulations were compared, and the factors affecting the conversion were investigated to optimize this process, owing to a newly developed High-Performance Liquid Chromatography-Evaporative Light Scattering Detector (HPLC-ELSD) method for quantification. Thus, using 50 g/L of lipase formulation Novozym 435® at 50 °C, the optimized synthesis of sorbitol laurate (SL) allowed to achieve 28% molar conversion of 0.5 M of vinyl laurate to its sugar alcohol monoester when the DES contained 5 wt.% water. After 48h, the de novo synthesized glycolipid was separated from the media by liquid-liquid extraction, purified by flash-chromatography and characterized thoroughly by one- and two-dimensional Nuclear Magnetic Resonance (NMR) experiments combined to Mass Spectrometry (MS). In completion, we provide initial proof of scalability for this process. Using a 2.5 L stirred tank reactor (STR) allowed a batch production reaching 25 g/L in a highly viscous two-phase system.

ß-Phenylalanine Ester Synthesis from Stable ß-Keto Ester Substrate Using Engineered ω-Transaminases.

The successful synthesis of chiral amines from ketones using ω-transaminases has been shown in many cases in the last two decades. In contrast, the amination of ß-keto acids is a special and relatively new challenge, as they decompose easily in aqueous solution. To avoid this, transamination of the more stable ß-keto esters would be an interesting alternative. For this reason, ω-transaminases were tested in this study, which enabled the transamination of the ß-keto ester substrate ethyl benzoylacetate. Therefore, a ω-transaminase library was screened using a coloring o-xylylenediamine assay. The ω-transaminase mutants 3FCR_4M and ATA117 11Rd show great potential for further engineering experiments aiming at the synthesis of chiral (S)- and (R)-ß-phenylalanine esters. This alternative approach resulted in the conversion of 32% and 13% for the (S)- and (R)-enantiomer, respectively. Furthermore, the (S)-ß-phenylalanine ethyl ester was isolated by performing a semi-preparative synthesis.

Intensification of Enzymatic Sorbityl Laurate Production in Dissolved and Neat Systems under Conventional and Microwave Heating.

Glycolipids such as sugar alcohol esters have been demonstrated to be relevant for numerous applications across various domains of specialty. The use of organic solvents and, more recently, deep eutectic solvents (DESs) to mediate lipase-supported bioconversions is gaining potential for industrial application. However, many challenges and limitations remain such as extensive time of production and relatively low productivities among others, which must be solved to strengthen such a biocatalytic process in industry. In this context, this study focuses on the intensification of sorbityl laurate production, as a model biocatalyzed reaction using Novozym 435, investigating the relevance of temperature, heating method, and solvent system. By increasing the reaction temperature from 50 to 90 °C, the space-time yield and product yield were considerably enhanced for reactions in DES and the organic solvent 2M2B, irrespective of the heating method (conventional or microwave heating). However, positive effects in 2M2B were more pronounced with conventional heating as 98% conversion yield was reached within 90 min at 90 °C, equating thus to a nearly 4-fold increase in performance yielding 118.0 ± 3.6 g/(L·h) productivity. With DES, the overall yield and space-time yield were lower with both heating methods. However, microwave heating enabled a 2-fold increase in both performance parameters when the reaction temperature was increased from 50 to 90 °C. Compared to conventional heating, a 7-fold increase in space-time yield at 50 °C and a 16-fold increase at 90 °C were achieved in DES by microwave heating. Furthermore, microwave irradiation enabled the usage of a neat, solvent-free system, representing an initial proof of concept with productivities of up to 13.3 ± 2.3 g/(L·h).

Identification of novel bioactive proteins and their produced oligopeptides from Torreya grandis nuts using proteomic based prediction.

Torreya grandis nut is a chief functional food in China consumed for centuries. Besides its rich protein composition, increasing studies are now focusing on T. grandis functional proteins that have not yet identified. In this study, liquid chromatography coupled with mass spectrometry detection of smaller and major proteins, revealed that the major peptide was 36935.00 Da. Proteome sequencing annotated 142 proteins in total. Bioactive proteins such as defensin 4 was annotated and its anti-microbial function was verified. Finally, functional oligopeptides were predicted by searching sequences of digested peptides in databases. Ten group of oligopeptides were suggested to exhibit antioxidant, Angiotensin-converting enzyme inhibition, anti-inflammatory. The predicted antioxidant activity was experimentally validated. It is interesting that a peptide GYCVSDNN digested from defensin 4 showed antioxidant activity. This study reports novel functional peptides from T. grandis nuts that have not been isolated and/or included as functional ingredients in nutraceuticals and in food industry.

Lipase-Mediated Mechanoenzymatic Synthesis of Sugar Esters in Dissolved Unconventional and Neat Reaction Systems.

Mechanochemical and biocatalytic approaches in modern research are two major assets to develop greener processes. In the present study, these modular tools of sustainability are pointed toward the production of versatile and daily employed compounds such as surfactants. Toward this aim, glycolipids, a class of nonionic surfactants composed of ubiquitous and primary metabolites such as sugar and fatty acid moieties, represent a promising alternative to petroleum-derived surface-active agents. Therefore, the combination of biocatalysis with mechanochemistry aiming at glycolipid synthesis seemed a logical step that was taken in this study for the first time. The monoacylated model compound glucose-6-O-decanoate was synthesized with the help of a bead mill apparatus using two different unconventional dissolved reaction systems, namely, menthol-based hydrophobic deep eutectic solvents and 2-methyl-2-butanol, thus reaching up to 12% yield in the latter based on the conversion of vinyl decanoate, after only 90 min of reaction. In addition, a neat reaction system using an excess of vinylated fatty ester as an adjuvant allowed a 27 mM/h space-time yield. The overall significant increase in productivities, up to 6 times, compared to standard heating and shaking methods, shows the tremendous potential of mechanoenzymatic synthesis.

Application of deep eutectic solvents in protein extraction and purification.

Deep eutectic solvents (DESs) are a mixture of hydrogen bond donor (HBD) and hydrogen bond acceptor (HBA) molecules that can consist, respectively, of natural plant metabolites such as sugars, carboxylic acids, amino acids, and ionic molecules, which are for the vast majority ammonium salts. Media such as DESs are modular tools of sustainability that can be pointed toward the extraction of bioactive molecules due to their excellent physicochemical properties, their relatively low price, and accessibility. The present review focuses on the application of DESs for protein extraction and purification. The in-depth effects and principles that apply to DES-mediated extraction using various renewable biomasses will be discussed as well. One of the most important observations being made is that DESs have a clear ability to maintain the biological and/or functional activity of the extracted proteins, as well as increase their stability compared to traditional solvents. They demonstrate true potential for a reproducible but more importantly, scalable protein extraction and purification compared to traditional methods while enabling waste valorization in some particular cases.

Comparative study on interfacial and foaming properties of glycolipids in relation to the gas applied for foam generation.

Glycolipids are biosurfactants with a wide range of structural diversity. They are biodegradable, based on renewables, ecocompatible and exhibit high surface activity. Still, studies comparing glycolipids and conventional surfactants in terms of interfacial properties and foaming performance are lacking. Here, we compared interfacial and foaming properties of microbial and enzymatically synthesized glycolipids to those of the widely-used, conventional surfactant sodium dodecyl sulfate (SDS). The enzymatically produced sorbose monodecanoate, as well as microbially produced di-rhamno-di-lipids exhibited high foam stabilizing properties, similar to those of SDS. However, sophorolipid and mono-rhamno-di-lipids did not produce metastable foams. An appropriate selection of head and tail groups depending on the application of interest is therefore necessary. Then, glycolipids can serve as an ecofriendly and efficient alternative to petroleum-based surfactants, even at substantially lower concentrations than e.g. SDS. Moreover, the influence of three foaming gases on the foaming properties of the glycolipids was evaluated. Slightly higher foam stability and lower coarsening rates were determined for sorbose monodecanoate when using nitrogen as the foaming gas instead of air. Foams generated with carbon dioxide were not metastable, no matter which surfactant was used.

Eleostearic phospholipids as probes to evaluate antioxidants efficiency against liposomes oxidation.

Regardless of the applications: therapeutic vehicle or membrane model to mimic complex biological systems; it is of a great importance to develop simplified, reproducible and rapid model assays allowing for a relevant assessment of the liposomal membrane oxidation and therefore antioxidant activity of selected molecules. Here, we describe a new and high-throughput assay that we called "Vesicle Conjugated Autoxidizable Triene (VesiCAT)". It is based on specific UV absorbance spectral properties of a new phospholipid probe, synthesized with natural conjugated eleostearic acid extracted from Tung oil. The VesiCAT assay has been developed with two different radical generators (2,2'-azobis(2-amidinopropane) dihydrochloride; AAPH and 2,2'-azobis(2,4-dimethylvaleronitrile); AMVN), producing a constant flux of oxidant species, either in membrane or in aqueous phase. This method appears very efficient in assessing the effect of various pure antioxidant molecules in their ability to preserve liposomes from oxidative degradation. In addition, the AAPH- and AMVN-induced oxidations offer the possibility of extracting different but complementary information with respect to the antioxidants efficacy.