Effects of organic solvent, water activity, and salt hydrate pair on the sn-1,3 selectivity and activity of whole-cell lipase from Aspergillus niger GZUF36.

We previously screened a whole-cell lipase EC 3.1.1.3 from the novel strain Aspergillus niger GZUF36, which exhibited 1,3-selectivity in the synthesis of 1,3-diacylglycerol via glycerolysis. However, the mechanism of lipase selectively in catalyzing the sn-1,3 position remains ambiguous. This work was performed to investigate the 1,3-selective mechanism of lipase using glycerolysis to synthesize 1,3-diacylglycerol (1,3-DG) as a model reaction by changing solvent(s) and water activity (aw), and addition of salt hydrate pair. The measured diacylglycerol yield was also used to examine lipase activity. Results indicated that not only organic solvent and aw have strong effect on the sn-1,3 selectivity, but also ions of salt hydrate pair also affected selectivity. Lipase conformation was altered by hydrophobic interactions of the solvent, aw, or ions of salt hydrate, resulting in distinct sn-1,3 selectivity of the lipase. The salt hydrate pair changed the lipase conformation and selectivity not only by aw but also by static interactions, which was rarely reported. These parameters also affected lipase activity. The lipase displayed the highest selectivity (about 88%) and activity in solvents of t-butanol and n-hexane (1:29, v/v) at aw 0.43. The results demonstrated that the sn-1,3 selectivity and activity of the lipase from A. niger GZUF36 may be improved by control of some crucial factors. This work laid a foundation for the application of lipase in the synthesis of 1,3-DG and other structural and functional lipids.

The ß-galactosidase gene AtrBGAL2 regulates Akebia trifoliata fruit cracking.

Cracking of Akebia trifoliata fruit at maturity is problematic for the cultivation of the horticultural crop, shortening shelf-life quality and compromising commercial value. However, the molecular mechanisms underlying this feature of A. trifoliata are not known. Genes involved in cell wall metabolism were identified by genome and transcriptome sequencing, which may play important roles in fruit cracking. One of the galactose metabolism related genes, ß-galactosidase (AtrBGAL2), was identified in A. trifoliata, and overexpression (OE) of AtrBGAL2 resulted in early fruit cracking, higher water-soluble pectin contents, and lower acid-soluble pectin, cellulose, and hemicellulose content compared to the wild type. Whereas silencing of AtrBGAL2 in trifoliata by virus induced gene silencing showed opposite trends. The levels of AtrBGAL2 transcripts were 24.6 and 66.0-fold higher in OE A. trifoliata and tomato fruits, respectively, and the cell wall-related genes were also gradually greater than in control plants during fruit ripening. Whereas the expression levels of AtrBGAL2 was significantly down-regulated by 54.1 % and 73.7 % in gene silenced A. trifoliata and CRISPR/Cas9 tomato mutant plants, respectively, and cell wall-related genes were also significantly reduced. These results demonstrate that AtrBGAL2 plays important roles in regulating fruit cracking during fruit ripening.

Antioxidant and anti-inflammatory properties of an aminoglycan-rich exopolysaccharide from the submerged fermentation of Bacillus thuringiensis.

Proteins from Bacillus thuringiensis are widely used as biopesticides but little is known about its exopolysaccharides. The exopolysaccharide BPS-2 was extracted from B. thuringiensis IX-01 after high-cell-density fermentation. BPS-2 is a heteropolysaccharide (molecular weight 29.36 kDa) composed of D-galactosamine, arabinose, glucosamine, glucose, and mannose in molar ratios 5.53: 1.77:4.74:3.24:1. In vitro upper gastrointestinal simulations showed that BPS-2 has strong anti-digestive capacity, with scavenging of DPPH, hydroxyl, ABTS, and superoxide anions radicals of 31.34 ± 1.67 %, 32.43 ± 3.01 %, 34.31 ± 2.12 %, and 48.53 ± 3.55 %, respectively, after BPS-2 entered the colon. It significantly inhibited production of lipopolysaccharide-induced nitric oxide and multiple pro-inflammatory cytokines and had proliferative effects on RAW 264.7 cells. BPS-2 inhibited malondialdehyde secretion and elevated activities of glutathione peroxidase, superoxide dismutase, and total antioxidants, significantly improving the antioxidant status of inflammation model cells. This first report of the in vitro anti-inflammation and antioxidant properties of BPS-2 from B. thuringiensis provides a basis for biopharmaceutical applications.

Encapsulation of polyphenols in pH-responsive micelles self-assembled from octenyl-succinylated curdlan oligosaccharide and its effect on the gut microbiota.

An amphiphilic polymer based on octenyl succinic anhydride-modified curdlan oligosaccharide (MCOS) was synthesized. The critical micelle concentration of MCOS was 3.91 µg·mL-1. MCOS could self-assemble into spherical micelles with a particle size of 230.1 nm and a zeta potential of - 37.9 mV. When used for polyphenol encapsulation, the loading capacity of curcumin and quercetin-co-encapsulated micelles was higher than that of single-polyphenol encapsulated micelles. In vitro gastrointestinal release test showed that the MCOS micelle presented a pH-dependent release, released a little polyphenol in simulated gastric fluid, but presented sustained release in the simulated intestinal fluid. The gastrointestinal-digested polyphenol-loaded micelles exhibited excellent antioxidant ability. In vitro human fecal fermentation indicated that the MCOS carrier could promote the production of short-chain fatty acids by gut microbiota and exhibited the highest relative abundance of Megamonas. In addition, the supplementation of curcumin and quercetin-co-loaded MCOS micelles increased the relative abundance of Bifidobacterium and inhibited the growth of Escherichia_Shigella. These findings indicated that the MCOS carrier can be potentially used for the colon-targeted delivery of hydrophobic polyphenols due to its pH-responsive property.

Enhanced solubility of curcumin by complexation with fermented cyclic ß-1,2-glucans.

Curcumin (CUR) is a low-solubility polyphenolic compound with many physiological functions. Cyclic ß-1,2-glucans (cyclosophoraoses [Cys]), which contain rings of different sizes with degrees of polymerization ranging from 17 to 23, were obtained from Rhizobium radiobacter ATCC 1333, a soil microorganism. The complexation ability and solubility enhancement of cyclic ß-1,2-glucans with insoluble curcumin were investigated. Phase-solubility analysis revealed that the stoichiometric ratio of the inclusion complexes was 1:1. The stability constant of Cys was 930 M-1, which was 7.68 times that of α-cyclodextrin (α-CD) and 2.09 times that of ß-cyclodextrin (ß-CD). The characteristics of the curcumin/Cys inclusion complexes were successfully determined by using Fourier transform infrared (FTIR) spectrometry, differential scanning calorimetry (DSC), nuclear magnetic resonance (1H NMR) spectroscopy, and scanning electron microscopy (SEM). Moreover, a 1:1 molecular model of the curcumin/Cys inclusion complexes was established through molecular docking analysis. These findings indicated that cyclic ß-1,2-glucans successfully formed complexes with curcumin, which suggested that they could be used as solubility-increasing agents. To the best of our knowledge, this is the first report in which curcumin has been embedded into cyclic ß-1,2-glucans resulting in an increase in its aqueous solubility.

Purification, characterization, and chemical modification of Bacillus velezensis SN-14 fibrinolytic enzyme.

Fermented bean foods are a crucial source of fibrinolytic enzymes. The presented study aimed to purify, characterize, and chemically modify Bacillus velezensis SN-14 fibrinolytic enzyme. The fibrinolytic enzyme was purified using CTAB/isooctane/hexyl alcohol/n-butyl alcohol reverse micellar system, and the purified enzyme was chemically modified to improve its enzymatic activity and stability. Enzyme activity recovery and the purification fold for this enzyme were 44.5 ± 1.9% and 4.93 ± 0.05 fold, respectively. SDS-PAGE results showed that the molecular weight of the purified fibrinolytic enzyme was around 28 kDa. Besides, the optimum temperature and pH of the purified fibrinolytic enzyme were 37 °C and 8-9, respectively. Fe2+, mPEG5000, and pepsin were used for chemical modification and for improving the activity and stability of the purified enzyme. Thermal and acid-base stability of chemically modified enzymes increased significantly, whereas enzymatic activity increased by 7.3 times. After 30 d of frozen storage, the modified enzyme's activity was remarkably lower (33.2%) than the unmodified enzyme (60.6%). The current study on B. velezensis SN-14 fibrinolytic enzyme and chemical modification method using Fe2+, mPEG5000, and pepsin provide a reference for developing fibrinolytic drugs and foods.

Isolation of Bacillus spp. with High Fibrinolytic Activity and Performance Evaluation in Fermented Douchi.

ABSTRACT: Fibrinolytic enzymes are effective and highly safe in treating cardiovascular and cerebrovascular diseases. Therefore, screening fibrinolytic enzyme-producing microbial strains with excellent fermentation performance is of great value to industrial applications. The fibrin plate method was used in screening strains with high yields of fibrinolytic enzymes from different fermented food products, and the screened strains were preliminarily identified using molecular biology. Then, the strains were used for solid-state fermentation of soybeans. Moreover, the fermentation product douchi was subjected to fibrinolytic activity measurement, sensory evaluation, and biogenic amine content determination. The fermentation performance of each strain was comprehensively evaluated through principal component analysis. Finally, the target strain was identified based on strain morphology, physiological and biochemical characteristics, 16S rDNA sequence, and phylogenetic analysis results. A total of 15 Bacillus species with high fibrinolysin activity were selected. Their fibrinolytic enzyme-producing activity levels were higher than 5,500 IU/g. Through molecular biology analysis, we found 4 strains of Bacillus subtilis, 10 strains of Bacillus amyloliquefaciens, and 1 strain of Bacillus velezensis. The principal component analysis results showed that SN-14 had the best fermentation performance and reduced the accumulation of histamine and total amine, the fibrinolytic activity of fermented douchi reached 5,920.5 ± 107.7 IU/g, and the sensory score was 4.6 ± 0.3 (out of 5 points). Finally, the combined results of physiological and biochemical analyses showed SN-14 was Bacillus velezensis. The high-yield fibrinolytic and excellent fermentation performance strain Bacillus velezensis SN-14 has potential industrial application.

Solvothermal synthesis of functional magnetic nanoparticles for biocatalyst.

A facile and simple one-step solvothermal method has been developed to synthesize polyethyleneimine (PEI)-modified magnetic nanoparticles. Characterization of morphology, surface charges, crystal structure, and magnetic property confirmed the efficiency of this facile synthesis route. Lipase immobilized on the PEI-modified magnetic nanoparticles was used to synthesize vitamin A palmitate from vitamin A acetate and palmitic acid. The reuse of immobilized lipase can be extended to eight times by removing water during esterification with a conversion rate above 80 % for 12 h.