5-aminolevulinic acid (ALA) is a non-protein amino acid that has been widely used in the fields of medicine and agriculture. This study aims to engineer the C5 pathway of the ALA biosynthesis in BL21 to enhance ALA production. The ALA synthase genes , and were overexpressed in BL21 to lead to the increase in the production of ALA. The sRNA RyhB was also overexpressed to downregulate the expression of ALA dehydratase to reduce the downstream bioconversion of ALA to porphobilinogen. Next, the gene was knocked out by CRISPR-Cas9 technology to open the TCA cycle to promote the respiratory metabolism of the strain to reduce the feedback inhibition of heme to ALA. The fermentation conditions of the engineered strain were optimized by response surface experiments. The time-course analysis of the ALA production was carried out in a 1 L shake flask. Through these efforts, the production of ALA in engineered strain reached 2953 mg/L in a 1 L shake flask. This study contributes to the industrial production of ALA by the engineered in the future.

Nanocarrier-based immobilization has created new avenues for enhancing the biophysical properties of enzymes. Nanomatrices such as magnetite nanoparticles (MNPs), chitin, and chitosan with large surface areas and tunable morphology have been developed to circumvent the bottlenecks of free enzymes. The present study used MNPs to immobilize the enzyme arginine deiminase (ADI) for improved morphological control, recovery, operational stability, and easy recyclability. Hybrid magnetic arginine deiminase cross-linked enzyme aggregate (mADI-CLEA) was developed for the first time by co-aggregating ADI with magnetite nanocomposites, followed by its cross-linkage with glutaraldehyde. Structural analysis by DLS/ZETA, SEM, and FT-IR revealed their highly stable and robust nature. The resulting mADI-CLEA exhibited higher pH resistivity and thermostability than ADI-CLEA. Reusability and storage stability assay indicated that mADI-CLEA maintained more than 60% residual activity even after seven batch cycles and was stable for more than 70 days. These hybrid magnetic aggregates of ADI offer an economical and stable alternative for biomedical applications of ADI.

(PS) are the dried mature seeds of L. or Willd. in the Plantago family. Its polysaccharides are important components of . Response surface methodology was used to optimize the ultrasonic-assisted deep eutectic solvent (DES) extraction process of PS polysaccharides (PSP). The results showed that the optimal extraction parameters were a solid-liquid ratio of 1:35 g/mL, an extraction time of 73 min, and a molar ratio of 2:1. The yield of PSP was 0.64% and 1.20% by water immersion and ultrasonic water extraction, respectively, indicating that the DES extraction method (2.21 ± 0.06%) is superior to these two methods, and the optimization effect is good. Through the α-glucosidase and α-amylase inhibition activities experiment, it was found that the IC values of PSPs-1 were 1122 and 220.5 μg/mL. DPPH·and ABTS scavenging activity experiments showed that the IC values of PSPs-1 were 19.2 and 4.3 μg/mL, respectively. Its molar ratio of monosaccharide composition is rhamnose: galactose: galacturonic acid: glucose: glucuronic acid: arabinose: mannose: xylose = 33.6:13.3:6.5:3:2.6:2:1.4:1. Therefore, this study can provide an experimental basis for the establishment of an industrialized production process of polysaccharides and the study of their biological activities.

The present study aimed to optimize the microwave-assisted extraction process for both the peel and pulp of (white dragon fruit) cultivated in Tunisia, using response surface methodology. Total phenolic content, total flavonoid content, FRAP (ferric reducing antioxidant power), and DPPH (1,1-diphenyl-2-picrylhydrazyl) antioxidant activities were optimized. A central composite design (CCD) was applied, considering three key variables: extraction time, extraction temperature, and liquid-to-solid ratio. The optimized extraction parameters for peel and pulp were determined respectively (9.57 min, 42.20 °C and 27.79 mL/g) and (10.08 min, 40.84 °C and 31.52 mL/g). The main phenolic compounds identified in peel and pulp extracts using HPLC-DAD were chlorogenic and caffeic acids and rutin, quercetin, luteolin-7--glucoside as flavonoids. Therefore, this research has revealed the potential of a sustainable and eco-friendly process hold promise a directional option and encouraging a circular economy approach for industrial production of antioxidant-rich extracts.

Selenium (Se) plays a crucial role in human health, influencing conditions such as cancer, diabetes, and neurological disorders. With global population growth and unequal nutrient distribution threatening food security, new approaches are needed to meet the nutritional needs of the world. Se is essential for immune function, metabolism, and antioxidant defense, and in regions suffering from food insecurity and malnutrition, selenium-enriched food could offer an affordable solution. , microalgae, can bioaccumulate Se from its environment, enhancing its nutritional value. This study explores how different light spectra (red, white, yellow, and blue LEDs) affect Se bioaccumulation in when NaSeO is added to the culture medium in photobioreactors. The results show that red light made the highest Se bioaccumulation (0.118 mg.L), followed by white, yellow, and blue light. Se addition also increased cell dry weight by 46%, 33%, 22%, and 60%, respectively, compared to photobioreactors without Se, with biomass productivity highest under red light. Furthermore, Se boosted maximum Chl α concentration, improving photosynthetic efficiency. These findings suggest that optimizing light conditions can significantly enhance the nutritional value of , offering a potential solution to global hunger by providing a sustainable, selenium-enriched food source.

In the current work, cellulase from was successfully immobilized on a novel epoxy-affixed chromium metal-organic framework/chitosan (Cr@-MIL-101/CS) support via covalent method using glutaraldehyde as a crosslinker. The bare and cellulase-bound support was characterized by using various microscopic and spectroscopic techniques. Immobilized cellulase exhibited a high immobilization yield of 0.7 ± 0.01 mg/cm, retaining 87.5 ± 0.04% of its specific activity and displaying enhanced catalytic performance. The immobilized enzyme was maximally active at pH 5.0, temperature 65 °C and 0.9 × 10-2 mg/ml saturating substrate concentration and the half-lives of free and immobilized cellulases were approximately 9 and 19 days, respectively. The decrease in activation energy, enthalpy change, and Gibbs free energy change, coupled with an increase in entropy change upon immobilization, indicated that the enzyme's efficiency, stability, and spontaneity in catalyzing the reaction were enhanced by immobilization. Additionally, the immobilized cellulase efficiently converted rice husk cellulose to glucose, with a quantification limit of 0.05%, linear measurement ranging from 0.1 to 0.9%, and 8.5% conversion efficiency. The present method exhibited a strong correlation (R = 0.998) with the DNS method, validating its reliability. Notably, the epoxy/Cr@-MIL-101/CS-bound cellulase demonstrated impressive thermal and pH stabilities, retaining 50% of its activity at 75 °C and over 96% at pH levels of 4.5 and 5.0 after 12 h. Furthermore, it showed excellent reusability, preserving 80% of its activity after 15 cycles and maintaining 50% of its activity even after 20 days of storage. These results suggest that epoxy/Cr@-MIL-101/CS/cellulase composites could be very effective for large-scale cellulose hydrolysis applications.

S100A8 serves as a biomarker for periodontitis and is involved in inflammatory processes, making its detection highly important. In this study, we produced recombinant 5A11 (r5A11) through mammalian cell culture. By employing a three-step process of transfection, suspension cell culture, and purification, we conveniently produced r5A11 with high yield and purity. The limit of detection for the r5A11-based immunoassay was 1.7 ± 0.2 × 10ng/mL, which was higher than that of the commercially available anti-S100A8 antibody. These findings suggest the potential use of this novel antibody in various research applications and practical approaches for simple and sensitive S100A8 detection.

, an airborne plant pathogen, holds the potential to synthesize sesquiterpenes, which have been used for the industrial production of abscisic acid. Previously, through our genetic technology, we obtained strain ZX2, whose main product 1´,4´--ABA-diol is physiologically active in plants. In this study, 50 L of fed fermentation was carried out with ZX2 strain to study the stability of expression of , , , , , and genes. Four kinds of software (GeNorm, NormFinder, BestKeeper and Delta Ct) were used to analyze the expression stability of candidate genes, and finally the best reference gene was screened by RefFinder. Based on the results, the was the most stable gene. It was used to normalize the expression levels of two genes related to 1´,4´--ABA-diol production ( and ) when fed-batch fermentation. Guide the selection of appropriate internal reference genes during the fermentation process to accurately quantify the relative transcription levels of target genes in ZX2.

Investigating the biotechnological potential of wild microorganisms is paramount for optimizing bioprocesses. Given this premise, we looked for yeasts in Brazilian native stingless bees, considering the recognized potential of pollinating insect-associated microorganisms for the production of volatile organic compounds (VOCs). Two yeast strains of the species were isolated from bees and evaluated for their fermentative capacity. Both yeasts were capable of fermenting sucrose (the main sugar used in the Brazilian ethanol industry) with over 90% efficiency and yields of up to 0.504 g/g. Through an experimental design analysis (CCD), it was verified that the ethanol productivity of these yeasts can also benefit from high concentrations of sucrose and low pH values, desirable traits for microorganisms in this biofuel production. At the same time, CCD analyses also showed the great capacity of these strains to produce another alcohol of broad biotechnological interest, 2-phenylethanol. Interestingly, the statistical analyses demonstrated that greater production of this compound can occur at high sugar concentrations and low availability of nitrogen sources, which can be easily achieved using residual low-cost feedstocks. Thus, our results suggest that these strains may be efficiently used in both ethanol and 2-phenylethanol production.

In recent years, driven by increasing consumer demand for natural and healthy convenient foods, the food industry has been shifting from synthetic to natural products. This shift is also reflected in the growing popularity of non-conventional extraction methods for pigments, which are favored for sustainability and environment-friendliness compared to conventional processes. This review aims to investigate the extraction of carotenoids from a variety of natural sources, including marine sources like fungus, microalgae, and crustaceans, as well as widely studied plants like tomatoes and carrots. Additionally, it delves into the recovery of valuable carotenoids from waste products like pomace and peels, highlighting the nutritional and environmental benefits. The review also emphasizes the role of green solvents such limonene, vegetable oils, ionic liquids, supercritical fluids, and natural deep eutectic solvents in effective and ecologically friendly carotenoid extraction. These technologies support the ideas of a circular and sustainable economy in addition to having a smaller negative impact on the environment. Overall, the present study highlights the crucial importance of green extraction technologies in achieving the dual goals of sustainability and public safety.

The present study reports the green synthesis of cellulose nanocrystals from the shells of (SFS) cellulose. Three different methods, alkali, acid and organic acid, were screened for the maximum cellulose extraction. A maximum cellulose yield, 30.6 ± 0.84 , was obtained using 90% formic acid at 110 °C in 120 min. The extracted cellulose was characterized and identified by instrumental analyses. SEM analysis showed skeletal rod-like microfibril structures and similar intra-fibrillar widths. CP/MAS C NMR and FTIR spectrum revealed the purity of cellulose and the absence of other components like hemicellulose and lignin. XRD study revealed a cellulose crystallinity index of 88.07%. BET analysis showed a good surface area (3.3213 m/g) and a micro-pore area of 1.871 m/g. The cellulose nanocrystals were synthesized from the extracted cellulose using deep eutectic solvents (DES), choline chloride and lactic acid (1:2 ratio). The cellulose nanocrystals (CNC) synthesized from DES-based exhibited zeta potential and particle size of -16.7 mV and 576.3 d.nm. DES-synthesized cellulose nanocrystals were spherical-like shapes, as observed from TEM images. The present results exposed that formic acid is an effective and green catalyst for the extraction of cellulose and DES for the sustainable synthesis of CNC.

polysaccharide (BSP) is one of the main active ingredients of the traditional Chinese medicine (Thunb) Richb.f and the extraction method of BSP has a significant impact on its properties. This study investigated the effects of four extraction methods, namely hot water extraction, ultrasonic extraction, enzyme extraction, and microwave extraction, on the structure and antioxidant properties of BSP. Characterization results from FTIR and NMR showed that all four BSP consisted mainly of glucose and mannose, forming α-glycosidic and β-glycosidic linkages to form glucomannan. Hot water extraction had the lowest extraction rate of BSP at 21.78% ± 0.73%. The polysaccharide BSP-H obtained from hot water extraction had the smallest absolute Zeta potential and Grain size, but the largest molecular weight at 204 kDa. It exhibited the best thermal stability and superior antioxidant activity compared to polysaccharides extracted using the other three methods, as evaluated by three different antioxidant assays. Although the antioxidant activity of BSP-V was slightly weaker, it showed a significant improvement compared to the remaining two polysaccharides. These results suggest that hot water extraction is the most suitable method for large-scale application of BSP, preserving its activity effectively, thus facilitating practical production and product development.

In this study, biocatalytic transesterification reaction using Novozyme 435 (N435) lipase was employed to enhance the hydrophobicity of esculin, aiming to improve its solubility for commercial applications and enhance its bioactivity and oral viability. The acylation reaction of esculin with vinyl acetate was conducted at 60 °C and 200 rpm for 24 h. After chromatographic and spectroscopic analysis, two products were identified: the first one was monoacylated at the 6'-OH position of the glucosyl moiety of esculin (T: 10.3 min and 382.93 [M + H]), and the second one was diacylated at the 6'-OH and 3'-OH positions (T: 13.0 min and 424.93 [M + H]). The latter was the major product, with a conversion rate of 53.550 ± 0.368%, while the monoacetylated one showed 8.715 ± 0.064%. Both products were isolated by high-speed counter-current chromatography (HSCCC) using a two-phase system HEMWat 1:9:1:9 and characterized by NMR. In this way, these results improve the practical application of esculin, through the obtention of esculin mono and diacetates by fast and efficient biocatalysis reaction.

This study investigated the ultrasound-assisted extraction (UAE) techniques used to enhance the polyphenols and antioxidants of mango peel extract (MPE). Additionally, it explored the bacteriostatic activity of MPE against various microorganisms. The UAE method was optimized using response surface methodology (RSM) at different times, temperatures, and ratios, with optimal conditions found to be 35 minutes, 45 °C, and a 1:35 ratio. The optimized yield results for total polyphenol content (TPC) were 17.33 ± 1.57 mg GAE/g, total flavonoid content (TFC) was 12.14 ± 0.29 mg QE/g, and radical scavenging activity (RSA) was 72.11 ± 2.19%. These response models were extremely significant with p-values less than 0.05. MPE showed selective effectiveness against The results highlight the potential of mango peel as a sustainable source of bioactive compounds, contributing to waste reduction in the food industry and the development of natural antimicrobial agents. This study contributes to further research on the application of MPE in processed foods.

is a widespread medicinal plant with a sufficiently well-studied chemical composition. Secondary metabolites synthesized by plants have pharmacological value for treating numerous diseases, and various types of aseptic in vitro cultures can be used as a source of these compounds. From this perspective, hairy roots attract considerable attention for the production of bioactive chemicals, including flavonoids with antioxidant activity. This paper shows the possibility of hairy roots obtaining with 100% frequency by genetic transformation. Hairy root lines differed in growth rate and flavonoid content. In particular, flavonoids were accumulated in the amount of up to 6.68 ± 0.28 mg/g of wet weight. Methyl jasmonate in the concentration of 10 µM inhibited root growth to a small extent but stimulated the synthesis of flavonoids. The antioxidant activity and the reducing power increased in the roots grown in the medium with methyl jasmonate. The strong correlation of antioxidant activity and reducing power with flavonoid content was detected. The influence of extraction conditions on the content of flavonoids in the extracts and their bioactivity was determined. The potent reducing activity of extracts from hairy roots allowed the production of silver nanoparticles, which was confirmed by transmission electron microscopy.

Proteases are enzymes that hydrolyze peptide bonds present in proteins and peptides. They are widely used for various industrial applications, such as in the detergent, food, and dairy industries. Cheese is one of the most important products of the dairy industry, and the coagulation stage is crucial during the cheese-making process. Enzymatic coagulation is the most common technique utilized for this purpose. Microbial enzymes are frequently used for coagulation due to their advantages in terms of availability, sustainability, quality control, product variety, and compliance with dietary and cultural/religious requirements. In the present study, we identified and subsequently characterized milk coagulant activity from the fungus PLO13, obtained during a solid-state fermentation process, using the agro-industrial residue, wheat bran, as the fermentation medium. Maximum enzyme production and caseinolytic activity occurred 120 h after cultivation. When the enzyme activity against various protease-specific synthetic substrates and inhibitors was analyzed, the enzyme was found to be a serine protease, similar to elastase 2. This elastase-2-like serine protease was able to coagulate pasteurized whole and reconstituted skim milk highly efficiently in the presence and absence of calcium, even at room temperature. The coagulation process was influenced by factors such as temperature, time, and calcium concentration. We demonstrate here, for the first time, an elastase-2-like enzyme in a microorganism and its potential application in the food industry for cheese production.

This study reports the design and development of a disposable amperometric biosensor for the determination of L-glutamate. Glutamate oxidase (GlOx) was immobilized onto a screen-printed carbon electrode (SPE) modified with poly-L-Aspartic acid (PAsp), carbon quantum dots (CQD), and platinum nanoparticles (PtNP) for the construction of the biosensor. The surface composition of the modified SPE was optimized using the one variable at a time method. The morphological properties of the biosensor were characterized by scanning electron microscopy and energy-dispersive X-ray spectroscopy. The electrochemical behavior of the modified electrodes was studied by cyclic voltammetry. Under the optimized experimental conditions the linear working range, detection limit and sensitivity of the GlOx/PtNP/CQD/PAsp/SPE were found to be 1.0 - 140 µM, 0.3 µM and 0.002 µA µM, respectively. The GlOx/PtNP/CQD/PAsp/SPE biosensor also exhibited good measurement repeatability. The as-developed biosensor was applied for the determination of L-glutamate in spiked serum samples and the average analytical recovery of added glutamate was 98.9 ± 3.9%.

This study explores a novel enzymatic pretreatment approach in anaerobic reactors for dairy wastewater, using lipase AY Amano to enhance methane production and modify microbial and archaeal community composition. Batch and semi-batch reactors with a total volume of 2000 mL were used to treat dairy wastewater with initial COD of 2000 and 15,000 mg L, respectively. In a new novel approach, the semi-batch reactors underwent a three-phase operation: 30 days of acclimation, 30 days of rest, and 30 days of active operation. Adding lipase (0.05% wv) as a pretreatment significantly increased methane yield over the 90 days by 135-138% compared with the control (without enzyme addition). The organic loading rate reached 0.22 g COD day L. Furthermore, 30 days after the end of the semi-batch reactor approach (120 days from the start), reusing sludge in batch reactors increased methane yield by 114-122% compared to the control. This increase was linked to the emergence and shift of new methanogenic communities within the sludge. Integrating hydrolytic enzymes into the anaerobic treatment enhances performance and sustainability by fostering methanogen-enriched microbial communities. This is crucial for maximizing methane production but may increase costs, requiring further economic feasibility research.

2,3-Butanediol (2,3-BD) is a highly valued building block, and optimizing its production by fermentation, particularly with crude glycerol, is crucial. is a key microorganism for this process; however, there are limited studies addressing the inhibition effects of products and by-products on 2,3-BD production. This study investigates these inhibition effects to maximize 2,3-BD production. Final concentrations of 2,3-BD plus acetoin reached 89.3, 92.7, and 71.1 g.L with productivities of 1.22, 1.69, and 0.99 g.L.h in pure glycerol, glucose, and crude glycerol media, respectively. Acetic acid was the main by-product, with concentrations ranging from 10 to 15 g.L. The reinoculation of cells highlighted the strong effect of 2,3-BD and acetic acid on microbial growth and metabolism, with the cultivation environment exerting selective pressure. Notably, cells reuse enhanced performance in crude glycerol media, achieving a specific productivity in relation to biomass (Y) of 9.18 g.g; about 25% higher than in fed-batch without cells reuse. By combining results from two fed-batch cycles, the total final concentration of 2,3-BD plus acetoin reached 99.4 g.L, alongside a 33% reduction in total acetic acid production with reused cells.

The Exopolysaccharide (EPS) producing novel strains of previously isolated from the vaginal source of pregnant women were selected based on ropy structure formation. The two selected strains, and were found to be producing 2.87 g/l and 3.14 g/l EPS, respectively, in the minimal media (M17 media) after 24-hour fermentation under anaerobic condition. Both the strains have probiotic properties and have the potential to be used for industrial applications. The production media and fermentation conditions were optimized to enhance the EPS production using the one-factor method, Placket-Burman factorial designing and Central composite design (CCD) of Response surface methodology (RSM). The most relevant factors affecting the EPS yield were sucrose, yeast extract and pH for E.villorum SB2 and sucrose, yeast extract and magnesium sulfate for the as determined by Placket-Burman design, whose concentrations were further optimized using CCD. The optimized fermentation conditions gave the total EPS of 9.76 g/l (4 times the initial production) from and 7.74 g/l (2.5 times the initial production) from , respectively, after 36-hour incubation at 37 °C. These optimization studies might be helpful during scale-up process for the industrial scale production of these exopolysaccharide.

Given the escalating demand for renewable biofuels amidst the continual consumption of fossil energy, the exploration and identification of microalgal strains for biodiesel production have become crucial. In this study, a microalgal strain named HDMA-12 was isolated from Lake Chenjiadayuan in China to evaluate its biodiesel potential. Phylogenetic analysis of its internal transcribed spacer sequences revealed HDMA-12 as a new molecular record in the genus . When cultivated in BG11 basal medium, HDMA-12 achieved a biomass of 635.7 mg L and a lipid content of 26.4%. Furthermore, the fatty acid methyl ester profile of HDMA-12 exhibited favorable combustion characteristics. Subjected to 200 mM NaCl stress, HDMA-12 reached its maximum biomass of 751.5 mg L and a lipid content of 28.9%. These findings indicate the promising prospects of HDMA-12 as a promising microalgal strain for further advancements in biodiesel production.