Efficient biosynthesis of prunin in methanol cosolvent system by an organic solvent-tolerant α-L-rhamnosidase from Spirochaeta thermophila.

Prunin of desirable bioactivity and bioavailability can be transformed from plant-derived naringin by the key enzyme α-L-rhamnosidase. However, the production was limited by unsatisfactory properties of α-L-rhamnosidase such as thermostability and organic solvent tolerance. In this study, biochemical characteristics, and hydrolysis capacity of a novel α-L-rhamnosidase from Spirochaeta thermophila (St-Rha) were investigated, which was the first characterized α-L-rhamnosidase for Spirochaeta genus. St-Rha showed a higher substrate specificity towards naringin and exhibited excellent thermostability and methanol tolerance. The Km of St-Rha in the methanol cosolvent system was decreased 7.2-fold comparing that in the aqueous phase system, while kcat/Km value of St-Rha was enhanced 9.3-fold. Meanwhile, a preliminary conformational study was implemented through comparative molecular dynamics simulation analysis to explore the mechanism underlying the methanol tolerance of St-Rha for the first time. Furthermore, the catalytic ability of St-Rha for prunin preparation in the 20% methanol cosolvent system was explored, and 200 g/L naringin was transformed into 125.5 g/L prunin for 24 h reaction with a corresponding space-time yield of 5.2 g/L/h. These results indicated that St-Rha was a novel α-L-rhamnosidase suitable for hydrolyzing naringin in the methanol cosolvent system and provided a better alternative for improving the efficient production yield of prunin.

Extraction, Modification and Biomedical Application of Agarose Hydrogels: A Review.

Numerous compounds present in the ocean are contributing to the development of the biomedical field. Agarose, a polysaccharide derived from marine red algae, plays a vital role in biomedical applications because of its reversible temperature-sensitive gelling behavior, excellent mechanical properties, and high biological activity. Natural agarose hydrogel has a single structural composition that prevents it from adapting to complex biological environments. Therefore, agarose can be developed into different forms through physical, biological, and chemical modifications, enabling it to perform optimally in different environments. Agarose biomaterials are being increasingly used for isolation, purification, drug delivery, and tissue engineering, but most are still far from clinical approval. This review classifies and discusses the preparation, modification, and biomedical applications of agarose, focusing on its applications in isolation and purification, wound dressings, drug delivery, tissue engineering, and 3D printing. In addition, it attempts to address the opportunities and challenges associated with the future development of agarose-based biomaterials in the biomedical field. It should help to rationalize the selection of the most suitable functionalized agarose hydrogels for specific applications in the biomedical industry.

Effect of ß-glucosidase on the aroma of liquid-fermented black tea juice as an ingredient for tea-based beverages.

The effects of ß-glucosidase on the volatile profiles and aroma stability of black tea juice were evaluated using gas-chromatography-mass spectrometry coupled with sensory analysis. During liquid fermentation of tea leaves, the addition of ß-glucosidase increased the concentration of aldehydes, strengthening the undesirable "green grassy" odour. However, the "green grassy" odour was counteracted by adding green tea extract during fermentation. At the same time, "flowery" flavour notes were enhanced, improving the overall aroma quality and strengthening the characteristic "sweet" aroma of black tea. Increased addition of ß-glucosidase released more free aroma alcohols from their glucosides. Two "fruity" and "floral" aroma components, benzyl alcohol and phenylethyl alcohol, were not significantly affected by heat treatment (95 °C water bath) and the overall aroma stability was not significantly affected by ß-glucosidase treatment. ß-Glucosidase treatment improved the aroma, colour and overall suitability of fermented black tea juice as an ingredient for tea-based beverages.

A Novel κ-Carrageenase from Marine Bacterium Rhodopirellula sallentina SM41: Heterologous Expression, Biochemical Characterization and Salt-Tolerance Mechanism Investigation.

κ-carrageenases are members of the glycoside hydrolase family 16 (GH16) that hydrolyze sulfated galactans in red algae, known as κ-carrageenans. In this study, a novel κ-carrageenase gene from the marine bacterium Rhodopirellula sallentina SM41 (RsCgk) was discovered via the genome mining approach. There are currently no reports on κ-carrageenase from the Rhodopirellula genus, and RsCgk shares a low identity (less than 65%) with κ- carrageenase from other genera. The RsCgk was heterologously overexpressed in Escherichia coli BL21 and characterized for its enzymatic properties. RsCgk exhibited maximum activity at pH 7.0 and 40 °C, and 50% of its initial activity was retained after incubating at 30 °C for 2 h. More than 70% of its activity was maintained after incubation at pH 6.0-8.0 and 4 °C for 24 h. As a marine derived enzyme, RsCgk showed excellent salt tolerance, retaining full activity in 1.2 M NaCl, and the addition of NaCl greatly enhanced its thermal stability. Mass spectrometry analysis of the RsCgk hydrolysis products revealed that the enzyme had high degradation specificity and mainly produced κ-carrageenan disaccharide. Comparative molecular dynamics simulations revealed that the conformational changes of tunnel-forming loops under salt environments may cause the deactivation or stabilization of RsCgk. Our results demonstrated that RsCgk could be utilized as a potential tool enzyme for efficient production of κ-carrageenan oligosaccharides under high salt conditions.

Corrigendum: Key Enzymes in Fatty Acid Synthesis Pathway for Bioactive Lipids Biosynthesis.

[This corrects the article DOI: 10.3389/fnut.2022.851402.].

Key Enzymes in Fatty Acid Synthesis Pathway for Bioactive Lipids Biosynthesis.

Dietary bioactive lipids, one of the three primary nutrients, is not only essential for growth and provides nutrients and energy for life's activities but can also help to guard against disease, such as Alzheimer's and cardiovascular diseases, which further strengthen the immune system and maintain many body functions. Many microorganisms, such as yeast, algae, and marine fungi, have been widely developed for dietary bioactive lipids production. These biosynthetic processes were not limited by the climate and ground, which are also responsible for superiority of shorter periods and high conversion rate. However, the production process was also exposed to the challenges of low stability, concentration, and productivity, which was derived from the limited knowledge about the critical enzyme in the metabolic pathway. Fortunately, the development of enzymatic research methods provides powerful tools to understand the catalytic process, including site-specific mutagenesis, protein dynamic simulation, and metabolic engineering technology. Thus, we review the characteristics of critical desaturase and elongase involved in the fatty acids' synthesis metabolic pathway, which aims to not only provide extensive data for enzyme rational design and modification but also provides a more profound and comprehensive understanding of the dietary bioactive lipids' synthetic process.

Biochemical Characterization and Cold-Adaption Mechanism of a PL-17 Family Alginate Lyase Aly23 from Marine Bacterium Pseudoalteromonas sp. ASY5 and Its Application for Oligosaccharides Production.

As an important enzyme involved in the marine carbon cycle, alginate lyase has received extensive attention because of its excellent degradation ability on brown algae, which is widely utilized for alginate oligosaccharide preparation or bioethanol production. In comparison with endo-type alginate lyases (PL-5, PL-7, and PL-18 families), limited studies have focused on PL-17 family alginate lyases, especially for those with special characteristics. In this study, a novel PL-17 family alginate lyase, Aly23, was identified and cloned from the marine bacterium Pseudoalteromonas carrageenovora ASY5. Aly23 exhibited maximum activity at 35 °C and retained 48.93% of its highest activity at 4 °C, representing an excellent cold-adaptation property. Comparative molecular dynamics analysis was implemented to explore the structural basis for the cold-adaptation property of Aly23. Aly23 had a high substrate preference for poly ß-D-mannuronate and exhibited both endolytic and exolytic activities; its hydrolysis reaction mainly produced monosaccharides, disaccharides, and trisaccharides. Furthermore, the enzymatic hydrolyzed oligosaccharides displayed good antioxidant activities to reduce ferric and scavenge radicals, such as hydroxyl, ABTS+, and DPPH. Our work demonstrated that Aly23 is a promising cold-adapted biocatalyst for the preparation of natural antioxidants from brown algae.

Applications of Synthetic Biotechnology on Carbon Neutrality Research: A Review on Electrically Driven Microbial and Enzyme Engineering.

With the advancement of science, technology, and productivity, the rapid development of industrial production, transportation, and the exploitation of fossil fuels has gradually led to the accumulation of greenhouse gases and deterioration of global warming. Carbon neutrality is a balance between absorption and emissions achieved by minimizing carbon dioxide (CO2) emissions from human social productive activity through a series of initiatives, including energy substitution and energy efficiency improvement. Then CO2 was offset through forest carbon sequestration and captured at last. Therefore, efficiently reducing CO2 emissions and enhancing CO2 capture are a matter of great urgency. Because many species have the natural CO2 capture properties, more and more scientists focus their attention on developing the biological carbon sequestration technique and further combine with synthetic biotechnology and electricity. In this article, the advances of the synthetic biotechnology method for the most promising organisms were reviewed, such as cyanobacteria, Escherichia coli, and yeast, in which the metabolic pathways were reconstructed to enhance the efficiency of CO2 capture and product synthesis. Furthermore, the electrically driven microbial and enzyme engineering processes are also summarized, in which the critical role and principle of electricity in the process of CO2 capture are canvassed. This review provides detailed summary and analysis of CO2 capture through synthetic biotechnology, which also pave the way for implementing electrically driven combined strategies.

Preparation and Characterization of κ-Carrageenan Modified with Maleic Anhydride and Its Application in Films.

In this work, the physicochemical properties of maleic anhydride (MAH)-modified κ-carrageenan (κCar) (MC) were characterized and compared with those of native κ-carrageenan (NC). The Fourier transform infrared spectrum of MC exhibited that κCar was successfully modified. Thermogravimetric analysis indicated that the thermal stability of MC was decreased. When the degree of substitution was 0.032, MC exhibited a low gel strength (759 g/cm2), gelling temperature (33.3 °C), and dehydration rate (60.3%). Given the excellent film-forming ability of κCar, MC films were then prepared and were found to have better mechanical and barrier properties (UV and water) than NC films. With regard to optical properties, MC films could completely absorb UV light in the range of 200-236 nm. The water contact angle of MC films was higher than that of NC films. Moreover, the elongation at break increased from 26.9% to 163%. These physicochemical property changes imply that MC can be employed in polysaccharide-based films.

Agarose Stearate-Carbomer940 as Stabilizer and Rheology Modifier for Surfactant-Free Cosmetic Formulations.

Some commonly used surfactants in cosmetic products raise concerns due to their skin-irritating effects and environmental contamination. Multifunctional, high-performance polymers are good alternatives to overcome these problems. In this study, agarose stearate (AS) with emulsifying, thickening, and gel properties was synthesized. Surfactant-free cosmetic formulations were successfully prepared from AS and carbomer940 (CBM940) mixed systems. The correlation of rheological parameter with skin feeling was determined to study the usability of the mixed systems in cosmetics. Based on rheological analysis, the surfactant-free cosmetic cream (SFC) stabilized by AS-carbomer940 showed shear-thinning behavior and strongly synergistic action. The SFC exhibited a gel-like behavior and had rheological properties similar to commercial cosmetic creams. Scanning electron microscope images proved that the AS-CBM940 network played an important role in SFC's stability. Oil content could reinforce the elastic characteristics of the AS-CBM940 matrix. The SFCs showed a good appearance and sensation during and after rubbing into skin. The knowledge gained from this study may be useful for designing surfactant-free cosmetic cream with rheological properties that can be tailored for particular commercial cosmetic applications. They may also be useful for producing medicine products with highly viscous or gel-like textures, such as some ointments and wound dressings.

Convenient Agarose Preparation with Hydrogen Peroxide and Desulfation Process Analysis.

Agarose is a natural seaweed polysaccharide and widely used in the medicine, food, and biological fields because of its high gel strength, non-toxicity, and electrical neutrality. The sulfate group is one of the main charged groups that affect the performance of agarose. In the present study, a simple, eco-friendly, and efficient method was explored for agarose preparation. After desulfation with hydrogen peroxide (H2O2), the sulfate content of agar reached 0.21%. Together with gel strength, electroendosmosis, gelling and melting temperature, the indicators of desulfated agar met the standards of commercially available agarose. Notably, the desulfated agar can be used as an agarose gel electrophoresis medium to separate DNA molecules, and the separation effect is as good as that of commercially available agarose. Further, the H2O2 desulfation process was analyzed. The addition of a hydroxyl radical (HO•) scavenger remarkably decreased the H2O2 desulfation rate, indicating that HO• has a certain role in agar desulfation. Sulfate content detection indicated that sulfur was removed from agar molecules in the form of sulfate ions (SO42-) and metal sulfate. The band absence at 850 cm-1 indicated that the sulfate groups at C-4 of D-galactose in sulfated galactan were eliminated.

Chitosan Activated with Genipin: A Nontoxic Natural Carrier for Tannase Immobilization and Its Application in Enhancing Biological Activities of Tea Extract.

In this work, a non-toxic chitosan-based carrier was constructed via genipin activation and applied for the immobilization of tannase. The immobilization carriers and immobilized tannase were characterized using Fourier transform infrared spectroscopy and thermogravimetric analysis. Activation conditions (genipin concentration, activation temperature, activation pH and activation time) and immobilizations conditions (enzyme amount, immobilization time, immobilization temperature, immobilization pH, and shaking speed) were optimized. The activity and activity recovery rate of the immobilized tannase prepared using optimal activation and immobilization conditions reached 29.2 U/g and 53.6%, respectively. The immobilized tannase exhibited better environmental adaptability and stability. The immobilized tannase retained 20.1% of the initial activity after 12 cycles and retained 81.12% of residual activity after 30 days storage. The catechins composition analysis of tea extract indicated that the concentration of non-ester-type catechins, EGC and EC, were increased by 1758% and 807% after enzymatic treatment. Biological activity studies of tea extract revealed that tea extract treated with the immobilized tannase possessed higher antioxidant activity, higher inhibitory effect on α-amylase, and lower inhibitory effect on α-glucosidase. Our results demonstrate that chitosan activated with genipin could be an effective non-toxic carrier for tannase immobilization and enhancing biological activities of tea extract.

Characterization and Application of an Alginate Lyase, Aly1281 from Marine Bacterium Pseudoalteromonas carrageenovora ASY5.

Alginate extracted from widely cultured brown seaweed can be hydrolyzed by alginate lyase to produce alginate oligosaccharides (AOS) with intriguing biological activities. Herein, a novel alginate lyase Aly1281 was cloned from marine bacterium Pseudoalteromonas carrageenovora ASY5 isolated from mangrove soil and found to belong to polysaccharide lyase family 7. Aly1281 exhibited maximum activity at pH 8.0 and 50 °C and have broad substrate specificity for polyguluronate and polymannuronate. Compared with other alginate lyases, Aly1281 exhibited high degradation specificity and mainly produced di-alginate oligosaccharides which displayed good antioxidant function to reduce ferric and scavenge radicals such as hydroxyl, ABTS+ and DPPH. Moreover, the catalytic activity and kinetic performance of Aly1281 were highly improved with the addition of salt, demonstrating a salt-activation property. A putative conformational structural feature of Aly1281 was found by MD simulation analysis for understanding the salt-activation effect.

Thermostable Tannase from Aspergillus Niger and Its Application in the Enzymatic Extraction of Green Tea.

Tannase is widely used in tea beverage processing because of its ability to catalyze the hydrolysis of hydrolysable tannins or gallic acid esters and effectively improve the quality of tea extracts through enzymatic extraction. A new thermophilic tannase was cloned from Aspergillus niger FJ0118 and characterized. The tannase exhibited an optimal reaction temperature of 80 °C and retained 89.6% of the initial activity after incubation at 60 °C for 2 h. The enzymatic extraction of green tea at high temperature (70 °C) for a short time (40 min) was devised on the basis of the superior thermal stability of tannase. The enzymatic reaction significantly increased the total polyphenol content of green tea extract from 137 g·kg-1 to 291 g·kg-1. The enzymatic reaction effectively degraded the ester catechins into non-ester catechins compared with the water extraction method. Results suggested that the thermally stable tannase exhibited potential applications in the enzymatic extraction of green tea beverage.

Antioxidant capacity and prebiotic effects of Gracilaria neoagaro oligosaccharides prepared by agarase hydrolysis.

Gracilaria is a genus of red algae widely cultivated in Asia and is notable for its economic importance as food ingredients. Neoagaro oligosaccharides (NAOSs) are products of Gracilaria that have excellent water solubility, antioxidant activity and prebiotic effect. In this study, Gracilaria crude polysaccharide was treated with agarase and hydrolyzed into NAOSs with different degrees of polymerization (DP). The compositions of the hydrolyzed NAOSs were analyzed by electrospray ionization-time of flight-mass spectrometry and thin layer chromatography. The antioxidant capacity and prebiotic effects of NAOSs with different DPs were investigated and the results showed that DP could affect the antioxidant capacity of NAOSs. The prebiotic effects of NAOSs with different DP were evaluated based on the influence on the growth of four intestinal bacteria. NAOSs promoted the growth of Lactobacillus delbrueckii subsp. bulgaricus and Sterptococcus thermophilus. The protective effect of Gracilaria NAOSs in simulated gastrointestinal juice was also studied. Finally, NAOSs with best prebiotic effects were used in Procambarus feeding experiment and exhibited promotion effect on Procambarus growth. The present study showed that Gracilaria NAOSs can be utilized as antioxidant and prebiotic additive, which had a considerable potential in food and feed industry.

Eucheuma cottonii Sulfated Oligosaccharides Decrease Food Allergic Responses in Animal Models by Up-regulating Regulatory T (Treg) Cells.

In the present study, the anti-food allergy activity of Eucheuma cottonii sulfated oligosaccharide (ESO) was investigated. ESO was obtained by enzymatic degradation and purified by column chromatography. RBL-2H3 cells and BALB/c mouse model were used to test the anti-food allergy activity of ESO. The effects of ESO on the regulatory T (Treg) cells and bone marrow-derived mast cells (BMMCs) were investigated by flow cytometry. The results of in vivo assay showed that ESO decreased the levels of mast cell protease-1 and histamine and inhibited the levels of specific IgE by 77.7%. In addition, the production of interleukin (IL)-4 and IL-13 was diminished in the ESO groups compared to the non-ESO-treated group. Furthermore, ESO could up-regulate Treg cells by 22.2-97.1%. In conclusion, ESO decreased the allergy response in mice by reducing basophil degranulation, up-regulating Treg cells via Forkhead box protein 3 (Foxp3), and releasing IL-10. ESO may have preventive and therapeutic potential in allergic disease.

Investigation of sunlight-induced deterioration of aroma of pummelo (Citrus maxima) essential oil.

Deterioration of aromas of pummelo essential oil (EO) induced by sunlight was compared to those induced by heat and oxygen exposure using the techniques of sensory evaluation and GC-MS analysis. The sunlight-exposed EO was found to possess an oily off-flavor odor, which was significantly different from its counterparts induced by oxygen and heat. The strong oily note of the sunlight-exposed EO was attributed to the existence of linalool oxides and limonene oxides, as well as the lack of neral and geranial, for which UV sunlight was revealed to be the critical contributor causing the chemical reactions for the aroma changes. The results demonstrated that UV sunlight could significantly affect the aroma of the pummelo EO, providing valuable information that will benefit the production and storage of EO-based aromatic products.

Development and evaluation of an HPLC method for accurate determinations of enzyme activities of naringinase complex.

An HPLC method that can separate naringin, prunin, and naringenin was used to help accurately measure the activities of naringinase and its subunits (α-L-rhamnosidase and ß-D-glucosidase). The activities of the naringinase and ß-d-glucosidase were determined through an indirect calculation of the naringenin concentration to avoid interference from its poor solubility. The measured enzymatic activities of the naringinase complex, α-L-rhamnosidase, and ß-D-glucosidase were the as same as their theoretical activities when the substrates' (i.e., naringin or prunin) concentrations were 200 µg/mL, and the enzyme concentrations were within the range of 0.06-0.43, 0.067-0.53, and 0.15-1.13 U/mL, respectively. The ß-D-glucosidase had a much higher Vmax than either naringinase or α-L-rhamnosidase, implying the hydrolysis of naringin to prunin was the limiting step of the enzyme reaction. The reliability of the method was finally validated through the repeatability test, indicating its feasibility for the determinations of the naringinase complex.

[Detection of intracellular reactive oxygen species by flow cytometry in Pichia pastoris fermentation].

In Pichia pastoris fermentation, methanol was oxidized into carbon oxide and produced a byproduct H2 O2, one of the partially reduced forms of molecular oxygen known as reactive oxygen species (ROS) . ROS are highly damaging towards cellular constituents. Flow cytometry (FCM) is an excellent method that permits the rapid, optical analysis of individual cells and has many advantages over conventional cytometry. However, its use in detecting intracellular ROS levels during Pichia fermentation was rarely reported. In our work, by means of flow cytometry, two fluorescent dye 2', 7'-dichlorofluorescin diacetate (DCFH-DA) and propidium iodide (PI) were used to detect ROS. The effect of intracellular ROS on Pichia pastoris cells during fermentation was studied through the comparison between DCFH-DA/PI double-stained cells and PI single-stained cells. In this study, the loss of cell viability during fermentation was correlated with the accumulation of ROS. At the glycerol batch and fed-batch phase, little ROS was accumulated intracellularly and cell viability reached almost 100%. At the early methanol fed-batch phase, intracellular ROS accumulation took place but 98.5% cells still kept viable. At the later methanol fed-batch phase, 94.0% cells accumulated high ROS. As a result, some cells lost their viability because of the damage of ROS. 25.4% dead cells accumulated high ROS in the total 29.1% dead cells.