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.

Preparation and Characterization of Carrageenase Immobilized onto Polyethyleneimine-Modified Pomelo Peel.

In this study, carrageenase immobilization was evaluated with a concise and efficient strategy. Pomelo peel cellulose (PPC) modified by polyethyleneimine (PEI) using the physical absorption method was used as a carrier to immobilize carrageenase and achieved repeated batch catalysis. In addition, various immobilization and reaction parameters were scrutinized to enhance the immobilization efficiency. Under the optimized conditions, the enzyme activity recovery rate was more than 50% and 4.1 times higher than immobilization with non-modified pomelo peels. The optimum temperature and pH of carrageenase after immobilization by PEI-modified pomelo peel, at 60°C and 7.5 respectively, were in line with the free enzyme. The temperature resistance was reduced, inconsistent with free enzyme, and pH resistance was increased. A significant loss of activity (46.8%) was observed after reusing it thrice under optimal reaction conditions. In terms of stability, the immobilized enzyme conserved 76.0% of the initial enzyme activity after 98 days of storage. Furthermore, a modest decrease in the kinetic constant (Km) value was observed, indicating the improved substrate affinity of the immobilized enzyme. Therefore, modified pomelo peel is a verified and promising enzyme immobilization system for the synthesis of inorganic solvents.

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.

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.

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.

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.

Structure and physicochemical properties of amphiphilic agar modified with octenyl succinic anhydride.

A novel amphiphilic agar with high transparency and freeze-thaw stability was prepared using octenyl succinic anhydride (OSA). Fourier transform infrared spectroscopy and nuclear magnetic resonance spectroscopy confirmed that the hydrophobic OS groups were successfully introduced in OSA-modified agar (OSAR) backbone. The OSAR showed higher emulsion stability and oil loading capacity than the native agar (NA). Compared with gel transparency (47.1 %), syneresis (42.1 %) of NA, OSAR exhibited high gel transparency (80 %) and low syneresis (3.3 %) when the degree of substitution (DS) was 0.06 and 0.12, respectively. Meanwhile, the OSAR showed a decreased interface tension and average molecular weight after modification. Thermogravimetric analysis indicated the thermal stability of OSAR was decreased, while texture profile analysis showed the springiness of the OSAR gel was enhanced. Dynamic rheology measurements revealed the OSAR with low gel strength displayed more liquid-like properties. Moreover, the OSAR exhibited lower turbidity and melting temperatures than the NA.

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.

Extraction of sulfated agar from Gracilaria lemaneiformis using hydrogen peroxide-assisted enzymatic method.

In this study, an eco-friendly extraction method was explored to obtain high sulfate content agar and repair the deficiency of enzymatic extraction by taking full advantage of H2O2. The sulfate content of EHA (H2O2-assisted enzymatic extracted agar) reached 3.56 %, which is significantly higher than that of traditional alkali-extracted agar (AA, 1.8 %). Moreover, EHA exhibited lower viscosity (9.4 cP), which improved 26.6 % and 14 % of filtration and gel dehydration rates than EA (enzymatic extracted agar), respectively. Additionally, the physicochemical properties of the agars were evaluated and compared. Among these agars, EHA showed some favorable properties, such as high yield (16.08 %) and low dissolution temperature (88.9 °C). The surface of algae became smoother after treatment with H2O2 due to effective degradation of cellulose. Besides, mass spectrometry analysis revealed that EHA preserved a great amount of sulfate, while thermogravimetric analysis suggested that the thermal stability of EA and EHA both decreased.

Cloning, expression, and characterization of a new pH- and heat-stable alginate lyase from Pseudoalteromonas carrageenovora ASY5.

Alginate lyase is important in marine alginate degradation, and its enzymatic hydrolysates are excellent antioxidants. Here, we cloned a new alginate lyase, that is, Alg823, from the Gram-negative marine bacterium Pseudoalteromonas carrageenovora ASY5. The optimal temperature and pH of Alg823 were 55°C and pH 8.0, respectively. After 30 min of incubation at 50°C, Alg823 could maintain over 75.0% of the maximum enzyme activity, suggesting its thermostability. The recombinant alginate lyase retained more than 80.0% of the maximum enzyme activity after it was treated at pH 6.0-10.0 and 4°C for 24 hr, indicating its excellent pH stability. Mg2+ , Ca2+ , Na+ , and K+ could promote enzyme activity. Alginate oligosaccharides obtained by degradation with Alg823 displayed an excellent ability to scavenge ABTS, hydroxyl, and DPPH radicals. Alg823 showed potential for novel applications in alginate oligosaccharide production because of its pH tolerance and heat adaptation. PRACTICAL APPLICATIONS: Alginate oligosaccharides produced by alginate degradation possess favorable properties, such as low molecular weight, high stability, and co-dissolution with water. These oligosaccharides also have many biological activities. As such, they have been widely explored. Alginate oligosaccharides are prepared via three methods, namely, physical, chemical, and enzymatic methods. In chemical method, operational processes are difficult to thereby possibly damaging the unique structure of polysaccharides and causing environmental pollution. Although physical methods can overcome some of the shortcomings of chemical methods, their reaction is still difficult to control, and products are complicated. Conversely, enzymatic methods can has advantages of mild conditions, single product, and less pollution. Furthermore, oligosaccharides prepared by enzymatic methods are more biologically active than those prepared by other methods. Thus, finding novel alginate lyase with high activity and stability is important for research and commercial purposes.

Effect of starch osmo-coating on carotenoids, colour and microstructure of dehydrated pumpkin slices.

Carotenoids in pumpkins are extremely unstable during industrial drying, to avoid the carotenoid degradation, starch omso-coating was subjected to the pretreatment process for dehydrated pumpkin slices. The results showed that starch omso-coating reduced the dehydration rate of pumpkin slices. When coated with corn starch, the retention rates of lutein, α-carotene and ß-carotene in dehydrated pumpkin slices significantly increased by 11.3, 9.0 and 7.7%, respectively, and the provitamin A activity increased by 9.5%. 1% (w/v) modified corn omso-coating gave highest retention rate of total carotenoids (95.5%), while provitamin A activity reached 4148 µg RAE/100 g. In addition, the colour parameters △E and a* values reduced, but L*, b* and H values increased with coated samples. Pearson correlation analysis showed that lutein, α-carotene and ß-carotene were significantly positive correlated with L*, and exhibited negative correlations with △E. The SEM indicated starch granules was attached to tissue gap and caused the film formation of oxygen barrier. It could be concluded that modified corn coating effectively improved the quality of final product.

Thermal degradation kinetics of all-trans and cis-carotenoids in a light-induced model system.

Thermal degradation kinetics of lutein, zeaxanthin, ß-cryptoxanthin, ß-carotene was studied at 25, 35, and 45°C in a model system. Qualitative and quantitative analyses of all-trans- and cis-carotenoids were conducted using HPLC-DAD-MS technologies. Kinetic and thermodynamic parameters were calculated by non-linear regression. A total of 29 geometrical isomers and four oxidation products were detected, including all-trans-, keto compounds, mono-cis- and di-cis-isomers. Degradations of all-trans-lutein, zeaxanthin, ß-cryptoxanthin, and ß-carotene were described by a first-order kinetic model, with the order of rate constants as kß-carotene>kß-cryptoxanthin>klutein>kzeaxanthin. Activation energies of zeaxanthin, lutein, ß-cryptoxanthin, and ß-carotene were 65.6, 38.9, 33.9, and 8.6kJ/moL, respectively. cis-carotenoids also followed with the first-order kinetic model, but they did not show a defined sequence of degradation rate constants and activation energies at different temperatures. A possible degradation pathway of four carotenoids was identified to better understand the mechanism of carotenoid degradation.

Preparation and characterization of tannase immobilized onto carboxyl-functionalized superparamagnetic ferroferric oxide nanoparticles.

Tannase from Aspergillus tubingensis was immobilized onto carboxyl-functionalized Fe3O4 nanoparticles (CMNPs), and conditions affecting tannase immobilization were investigated. Successful binding between CMNPs and tannase was confirmed by Fourier transform infrared spectroscopy and thermogravimetric analysis. Vibrating sample magnetometry and X-ray diffraction showed that the CMNPs and immobilized tannase exhibit distinct magnetic responses and superparamagnetic properties. Free and immobilized tannase exhibited identical optimal temperatures of 50°C and differing pH optima at 6 and 7, respectively. The thermal, pH, and storage stabilities of the immobilized tannase were superior to those of free tannase. After six cycles of catalytic hydrolysis of propyl gallate, the immobilized tannase maintained over 60% of its initial activity. The Michaelis constant (Km) of the immobilized enzyme indicated its higher affinity for substrate binding than the free enzyme.

Statistical optimization for tannase production by Aspergillus tubingensis in solid-state fermentation using tea stalks

Background A sequential statistical strategy was used to optimize tannase production from Aspergillus tubingensis using tea stalks by solid-state fermentation. Results First, using a Plackett-Burman design, inoculum size and incubation time (among seven tested variables) were identified as the most significant factors for tannase yield. The effects of significant variables were further evaluated through a single steepest ascent experiment and central composite design with response surface analysis. Under optimal conditions, the experimental value of 84.24 units per gram of dry substrate (U/gds) closely matched the predicted value of 87.26 U/gds. Conclusions The result of the statistical approach was 2.09 times higher than the basal medium (40.22 U/gds). The results were fitted onto a second-order polynomial model with a correlation coefficient (R²) of 0.9340, which implied an adequate credibility of the model.

Study on the relationship between intracellular metabolites and astaxanthin accumulation during Phaffia rhodozyma fermentation

Background To study the relationship between intracellular anabolism and astaxanthin production, the influence of intracellular protein and fatty acids on astaxanthin production by four mutant Phaffia rhodozyma strains and their variations was investigated in this research. Results First, the content of astaxanthin in cells showed a reverse fluctuation in contrast to that of protein during the whole fermentation process. Moreover, compared with the three other strains, the astaxanthin-overproducing mutant strain of the yeast P. rhodozyma, called JMU-MVP14, had the highest specific productivity of astaxanthin as 6.8 mg/g, whereas its intracellular protein and fatty acid contents were the lowest. In addition, as a kind of sugar metabolic product, ethanol was only produced by P. rhodozyma JMU-VDL668 and JMU-7B12 during fermentation. Conclusions The results indicated that the accumulation of ethanol, intracellular protein, and fatty acids had competition effects on astaxanthin synthesis. This condition may explain why the P. rhodozyma strains JMU-VDL668 and JMU-7B12 achieved relatively lower astaxanthin production (1.7 and 1.2 mg/L) than the other two strains JMU-MVP14 and JMU-17W (20.4 and 3.9 mg/L).

Quality enhancement in the Japanese sea bass (Lateolabrax japonicas) fillets stored at 4°C by chitosan coating incorporated with citric acid or licorice extract.

The preserving effects of chitosan, chitosan and citric acid, chitosan and licorice extract on fresh Japanese sea bass fillets stored at 4 °C for 12 days were studied. Results showed that citric acid or licorice extract can enhance the preserving function of chitosan significantly by retarding lipid oxidation and inhibiting microbial growth as reflected in thiobarbituric acid reactive substances and total plate count, respectively. Both total volatile basic nitrogen values and sensory scores indicated chitosan and citric acid or licorice extract can significantly reduce the quality loss and extend the shelf life of Japanese sea bass fish fillets during refrigerated storage. Citric acid or licorice extract with chitosan could thus be applied in the seafood industry to enhance quality of fish fillets as natural preservatives.