Stabilized enzymes are crucial for the industrial application of biocatalysis due to their enhanced operational stability, which leads to prolonged enzyme activity, cost-efficiency and consequently scalability of biocatalytic processes. Over the past decade, numerous studies have demonstrated that deep eutectic solvents (DES) are excellent enzyme stabilizers. However, the search for an optimal DES has primarily relied on trial-and-error methods, lacking systematic exploration of DES structure-activity relationships. Therefore, this study aims to rationally design DES to stabilize various dehydrogenases through extensive experimental screening, followed by the development of a straightforward and reliable mathematical model to predict the efficacy of DES in enzyme stabilization. A total of 28 DES were tested for their ability to stabilize three dehydrogenases at 30°C: (S)-alcohol dehydrogenase from Rhodococcus ruber (ADH-A), (R)-alcohol dehydrogenase from Lactobacillus kefir (Lk-ADH) and glucose dehydrogenase from Bacillus megaterium (GDH). The residual activity of these enzymes in the presence of DES was quantified using first-order kinetic models. The screening revealed that DES based on polyols serve as promising stabilizing environments for the three tested dehydrogenases, particularly for the enzymes Lk-ADH and GDH, which are intrinsically unstable in aqueous environments. In glycerol-based DES, increases in enzyme half-life of up to 175-fold for Lk-ADH and 60-fold for GDH were observed compared to reference buffers. Furthermore, to establish the relationship between the enzyme inactivation rate constants and DES descriptors generated by the Conductor-like Screening Model for Real Solvents, artificial neural network models were developed. The models for ADH-A and GDH showed high efficiency and reliability (R2 > 0.75) for in silico screening of the enzyme inactivation rate constants based on DES descriptors. In conclusion, these results highlight the significant potential of the integrated experimental and in silico approach for the rational design of DES tailored to stabilize enzymes.

Pulsed light (PL) pasteurization is being explored as a substitute for the conventional thermal pasteurization of juices in recent times due to better retention of nutrients and overall quality. However, the long-term stability of the PL-pasteurized juice must be investigated to promote its application by the industry. The effect of PL treatment (effective fluence of 1.15 J·cm-2) and thermal treatment (90°C for 60 s) on microbial quality, enzyme activity, bioactive compounds, sensory acceptance, and color profile of table grape juice during storage at 4 and 25°C was investigated in this study. The PL pasteurization enhanced the microbial shelf-life of the juice (<6 log10cfu·mL-1) from 5 to 35 days at 4°C. The PL and thermally-pasteurized juice demonstrated a shelf-life of only 10 days when stored at 25°C. The total soluble solids and titratable acidity did not alter significantly throughout the storage period. The peroxidase, polyphenol oxidase, and pectin methylesterase activities were below 10% for the PL and thermally-treated beverage when stored at 4°C. The sensory acceptability of the PL-pasteurized juice after 35 days of refrigerated storage (6.9 ± 0.3) was close to the untreated juice (7.2 ± 0.3) and greater than thermally-treated juice (6.2 ± 0.2). After the 35th day of storage at 4°C, PL-treated grape juice retained 55%, 12%, and 15.3% more phenolics, flavonoids, and antioxidant capacity, respectively, than the thermally-pasteurized juice. Hence, PL pasteurization can effectively prolong the shelf-life of table grape juice while achieving microbial and enzymatic stability, along with high sensory and nutritional appeal. PRACTICAL APPLICATION: Exploring non-thermal methods like pulsed light (PL) pasteurization as a substitute for conventional thermal methods is gaining recognition for its ability to retain nutrients and improve overall juice quality. However, the industry\'s adoption depends on understanding the shelf-stability of PL-pasteurized juice. This study specifically investigates the practical applications of PL treatment in comparison with conventional thermal treatment in enhancing microbial safety and enzymatic stability in table grape juice. The findings contribute insights into optimizing the shelf life of table grape juice and preserving its quality, supported by microbial, enzymatic, and sensory evaluations.

This work explored the impact of ultrasound (US) on the activity, stability, and macrostructural conformation of cyclodextrin glycosyltransferase (CGTase) and how these changes could maximize the production of β-cyclodextrins (β-CDs). The results showed that ultrasonic pretreatment (20 kHz and 38 W/L) at pH 6.0 promoted increased enzymatic activity. Specifically, after sonication at 25 °C/30 min, there was a maximum activity increase of 93 % and 68 % when biocatalysis was carried out at 25 and 55 °C, respectively. For activity measured at 80 °C, maximum increase (31 %) was observed after sonication at 25 °C/60 min. Comparatively, US pretreatment at low pH (pH = 4.0) resulted in a lower activity increase (max. 28 %). These activation levels were maintained after 24 h of storage at 8 °C, suggesting that changes on CGTase after ultrasonic pretreatment were not transitory. These pretreatments altered the conformational structure of CGTase, revealed by an up to 11 % increase in intrinsic fluorescence intensity, and resulted in macrostructural modifications, such as a decrease in particle size and polydispersion index (up to 85 % and 45.8 %, respectively). Therefore, the sonication of CGTase under specific conditions of pH, time, and temperature (especially at pH 6.0/ 30 min/ 25 °C) promotes macrostructural changes in CGTase that induce enzyme activation and, consequently, higher production of β-CDs.

This study aimed to fabricate gum Arabic (GA)-coated Fe3O4 nanoparticles bearing numerous active aldehyde groups on their surface, followed by an assessment of their capability as a magnetic support for the covalent immobilization of the trypsin enzyme for the first time. FT-IR, XRD, TGA, and SEM results demonstrated the successful synthesis of GA-coated Fe3O4 nanoparticles, along with the covalent immobilization of the enzyme onto the support. Immobilization enhanced the relative enzymatic activity across a range of aqueous solution pH levels (ranging from 4 to 11) and temperatures (ranging from 20 to 80 °C) without altering the optimum pH and temperature for trypsin activity. Kinetic studies using Michaelis-Menten plots revealed changes in kinetic parameters, including a lower Vmax and higher Km for immobilized trypsin compared to the free enzyme. The immobilization onto magnetic gum Arabic nanoparticles resulted in an improved stability of trypsin in the presence of various solvents, maintaining a stability order comparable to that of the free enzyme due to the stabilizing effect of the support. The reusability results showed that the immobilized enzyme can retain over 93% of its activity for up to 15 cycles.

In this study, horseradish peroxidase (HRP) was immobilized for the first time on Ca alginate-starch hybrid beads and employed for the biodegradation of phenol red dye. The optimal protein loading was 50 mg/g of support. Immobilized HRP demonstrated improved thermal stability and maximum catalytic activity at 50 °C and pH 6.0, with an increase in half-life (t1/2) and enzymatic deactivation energy (Ed) compared to free HRP. After 30 days of storage at 4 °C, immobilized HRP retained 109% of its initial activity. Compared to free HRP, the immobilized enzyme exhibited higher potential for phenol red dye degradation, as evidenced by the removal of 55.87% of initial phenol red after 90 min, which was 11.5 times greater than free HRP. In sequential batch reactions, the immobilized HRP demonstrated good potential efficiency for the biodegradation of phenol red dye. The immobilized HRP was used for a total of 15 cycles, degrading 18.99% after 10 cycles and 11.69% after 15 cycles, with a residual enzymatic activity of 19.40% and 12.34%, respectively. Overall, the results suggest that HRP immobilized on Ca alginate-starch hybrid supports shows promise as a biocatalyst for industrial and biotechnological applications, particularly for the biodegradation of recalcitrant compounds such as phenol red dye.

Here we designed and synthesized analogs of two antimicrobial peptides, namely C10:0-A2, a lipopeptide, and TA4, a cationic α-helical amphipathic peptide, and used non-proteinogenic amino acids to improve their therapeutic properties. The physicochemical properties of these analogs were analyzed, including their retention time, hydrophobicity, and critical micelle concentration, as well as their antimicrobial activity against gram-positive and gram-negative bacteria and yeast. Our results showed that substitution with D- and N-methyl amino acids could be a useful strategy to modulate the therapeutic properties of antimicrobial peptides and lipopeptides, including enhancing stability against enzymatic degradation. The study provides insights into the design and optimization of antimicrobial peptides to achieve improved stability and therapeutic efficacy. TA4(dK), C10:0-A2(6-NMeLys), and C10:0-A2(9-NMeLys) were identified as the most promising molecules for further studies.

Dipeptides are minimalistic peptide building blocks that form well ordered structures through molecular self-assembly. The driving forces involved are cooperative noncovalent interactions such as π-π stacking, hydrogen bonding, and ionic as well as hydrophobic interactions. One of the most intriguing self-assembled motifs that has been extensively explored as a low molecular weight hydrogel for drug delivery, tissue engineering, imaging and techtonics, etc. is Phe-Phe (FF). The backbone of the dipeptide is very crucial for extending secondary structures in self-assembly, and any subtle change in the backbone drastically affect the molecular recognitions. The squaramide (SQ) motif has the unique advantage of hydrogen bonding which can promote the self-assembly process. In this work we have integrated the SQ unit into the dipeptide FF backbone to achieve molecular self-assembly. The resulting carbamate protected backbone modified dipeptide (BocFSAF-OH, 10) has exhibited molecular self-assembly with a fibrilar network. It formed a stable hydrogel (with CAC of 0.024 ± 0.0098 wt %) via the solvent switch method and was found to possess excellent enzymatic stability. The dipeptide and the resulting hydrogel were found to be cytocompatible. When integrated with a polysaccharide based biopolymer, e.g. sodium alginate, the resulting matrix exhibited strong hydrogel character. Therefore, the dipeptide hydrogel of 10 may find its applications in a variety of fields including drug delivery and tissue engineering.

Chemically modified nucleic acids are essential for the therapeutic application of oligonucleotides. In this study, 6\'-C-spiro-thymidine exhibiting a fixed torsion angle γ was designed, synthesized, and incorporated into oligonucleotides. The conformational analysis of the 6\'-C-spiro-thymidine monomer revealed that its torsion angle γ was in the +synclinal range (approx. 60°), which is similar to that in a natural RNA duplex, as expected. On the other hand, the sugar conformation of the RNA duplex is known to be predominantly an N-type, whereas that of the synthesized monomer was an S-type. The results of the UV melting analysis demonstrated that the duplex-forming ability of 6\'-C-spiro-thymidine was inferior to that of natural DNA. Contrarily, 6\'-C-spiro-thymidine could enhance the stability of oligonucleotides toward nucleases. Particularly, the incorporation of 6\'-C-spiro-thymidine on the 3\'-ends of the oligonucleotides significantly increased the nuclease resistance of the oligonucleotides.

Pineapple juice is preferred by consumers for its unique aroma and flavor that come from a set of amino acids, amines, phenolic compounds, and furanone. The juice is susceptible to spoilage, and a common practice is to pasteurize it at 70-95°C for 0.5-5 min. However, the characteristic flavors and phytochemicals are negatively influenced by the intense time-temperature treatment. To retain the thermosensitive compounds in the juice, some nonthermal technologies such as high-pressure processing, pulsed electric field, pulsed light, ultrasound, and ultraviolet treatments have been explored. These techniques ensured microbial safety (5-log reduction in E. coli, S. Typhimurium, or S. cerevisiae) while preserving a maximum ascorbic acid (84-99%) in the juice. The shelf life of these nonthermally treated juice varied between 14 days (UV treated at 7.5 mJ/cm2 ) and 6 months (clarified through microfiltration). Moreover, the inactivation of spoilage enzyme in the juice required a higher intensity. The present review discusses the potential of several nonthermal techniques employed for the pasteurization of pineapple juice. The pasteurization ability of the combined hurdle between mild thermal and nonthermal processing is also presented. The review also summarizes the target for pasteurization, the plan to design a nonthermal processing intensity, and the consumer perspective toward nonthermally treated pineapple juice. The techniques are compared on the common ground like safety, stability, and quality of the juice. This will help readers to select an appropriate nonthermal technology for pineapple juice production and design the intensity required to satisfy the manufacturers, retailers, and consumers.

Improving the cell selectivity of anticancer peptides (ACPs) is a major hurdle in their clinical utilisation. In this study, a new acid-activated ACP was designed by conjugating a cationic ACP LK to its anionic binding partner peptide (LEH) via a disulphide linker to trigger antitumor activity at acidic pH while masking its killing activity at normal pH. Three anionic binding peptides containing different numbers of glutamic acid (Glu) and histidine were engineered to obtain an efficient acid-activated ACP. The conjugates LK-LEH2 and LK-LEH3 exhibited 6.1- and 8.0-fold higher killing activity at pH 6.0 relative to at pH 7.4, respectively, suggesting their excellent pH-dependent antitumor activity; and their cytotoxicity was 10-fold lower than that of LK. However, LK-LEH4 had no pH-responsive killing effect. Interestingly, increasing the number of Glu from 2 to 4 increased the pH-response of the physical mixture of LK and LEH; conversely, they weakly decreased the cytotoxicity of LK, suggesting that the conjugate connection is required to achieve excellent pH dependence while maintaining minimum toxicity. LK-LEH2 and LK-LEH3 were more enzymatically stable than LK, indicating their potential for in vivo application. Our work provided a basis for designing promising ACPs with good selectivity and low toxicity.