Production of marine bacterial metalloprotease A69 and evaluation of its potential in preparing soybean peptides with angiotensin-converting enzyme-inhibitory activity.

BACKGROUND: Marine bacteria secrete a variety of proteases, which are a good source to explore proteases with application value. However, only a few marine bacterial proteases with a potential in bioactive peptides preparation have been reported. RESULTS: The metalloprotease A69 from the marine bacterium Anoxybacillus caldiproteolyticus 1A02591 was successfully expressed in the food safe bacterium Bacillus subtilis as a secreted enzyme. A technique to efficiently produce protease A69 in a 15-L bioreactor was established, with a production of 8988 U mL-1 . Based on optimizing the hydrolysis parameters of A69 on soybean protein, a process for soybean protein peptides (SPs) preparation was set up, in which soybean protein was hydrolyzed by A69 at 4000 U g-1 and 60 °C for 3 h. The prepared SPs had a high content (> 90%) of peptides with a molecular mass less than 3000 Da and contained 18 amino acids. The prepared SPs showed high angiotensin-converting enzyme (ACE)-inhibitory activity, with an IC50 value of 0.135 mg mL-1 . Moreover, three ACE-inhibitory peptides, RPSYT, VLIVP and LAIPVNKP, were identified from the SPs using liquid chromatography-mass spectrometry analysis. CONCLUSION: The marine bacterial metalloprotease A69 has a promising potential for preparing SPs with good nutritional and potential antihypertensive effects, laying a good foundation for its industrial production and application. © 2023 Society of Chemical Industry.

Mechanistic Insight Into the Production of Collagen Oligopeptides by the S8 Family Protease A4095.

Collagen oligopeptides have wide applications in foods, pharmaceuticals, cosmetics, and others due to their high bioactivities and bioavailability. The S8 family is the second-largest family of serine proteases. Several collagenolytic proteases from this family have been reported to have good potential in the preparation of collagen oligopeptides, however, the underlying mechanism remains unknown. A4095 was the most abundant S8 protease secreted by the protease-producing bacterium Anoxybacillus caldiproteolyticus 1A02591. Here, we characterized A4095 as an S8 collagenolytic protease and illustrated its structural basis to produce collagen oligopeptides. Protease A4095 preferentially hydrolyzed the Y-Gly peptide bonds in denatured bovine bone collagen, leading to high production (62.48% <1000 Da) of collagen oligopeptides. Structural and mutational analyses indicated that A4095 has a unique S1' substrate-binding pocket to preferentially bind Gly, which is the structural determinant for the high production of collagen oligopeptides. This study provides mechanistic insight into the advantage of the S8 collagenolytic proteases in preparing collagen oligopeptides.

Molecular Mechanism of Polysaccharides Extracted from Chinese Medicine Targeting Gut Microbiota for Promoting Health.

The accumulating evidence revealed that gut microbiota plays an important role in pathological process of disease including obesity, type 2 diabetes mellitus, heart failure, and non-alcoholic fatty liver disease. Polysaccharides extracted from Chinese medicine (CM) can not only alleviate pathological status but also promote health by anti-inflammatory, regulating immunity, lowering blood glucose and lipids, anti-cancer, and anti-oxidation. The alterations of gut microbiota composition and metabolism pathways are the potential mechanisms of CM polysaccharides treatment. In addition, they exert functions through gut-organ axis or play an indirect role by synergistic actions with other drugs or components mediated by gut microbiota. This review summarizes the molecular mechanisms of CM polysaccharides interacted with intestinal microbial inhabitants as potential prebiotics for promoting health.

Effects of Radix Astragali and Its Split Components on Gene Expression Profiles Related to Water Metabolism in Rats with the Dampness Stagnancy due to Spleen Deficiency Syndrome.

Radix Astragali (RA) with slight sweet and warm property is a significant "qi tonifying" herb; it is indicated for the syndrome of dampness stagnancy due to spleen deficiency (DSSD). The purpose of this research was to explore effects of RA and its split components on gene expression profiles related to water metabolism in rats with the DSSD syndrome for identifying components representing property and flavor of RA. The results indicated that RA and its split components, especially polysaccharides component, significantly increased the body weight and the urine volume and decreased the water load index of model rats. Our data also indicated differentially expressed genes (DEGs) related to water metabolism involved secretion, ion transport, water homeostasis, regulation of body fluid levels, and water channel activity; the expression of AQP1, AQP3, AQP4, AQP5, AQP6, and AQP8 was improved; calcium, cAMP, MAPK, PPAR, AMPK, and PI3K-Akt signaling pathway may be related to water metabolism. In general, results indicate that RA and its split components could promote water metabolism in rats with the DSSD syndrome via regulating the expression of AQPs, which reflected sweet-warm properties of RA. Effects of the polysaccharides component are better than others.

The novel mechanism of oxymatrine affecting HERG currents at different temperatures.

BACKGROUND/AIMS: Human ether-à-go-go-related gene (hERG) has an important role in the repolarization of the cardiac action potential. Our studies were to investigate the effects of oxymatrine (one of the natural constituents extracted from Chinese herb Sophora flavescens Ait) on hERG-encoded K(+) channels at different temperatures and its underlying mechanism. METHODS: The effects of oxymatrine were examined on hERG channels stably expressed in HEK293 cells using a whole-cell patch clamp technique. RESULTS: At the temperature 30°C, oxymatrine inhibited hERG current in a concentration-dependent manner and the IC(50) was ∼665 µM. However at the temperature of 20°C, low concentration oxymatrine C≤100 µM increased hERG current density. However, high concentration oxymatrine C>100 µM inhibited the hERG current density significantly. Oxymatrine only affected the activation kinetic of hERG channels at all temperatures and had a high binding affinity for open state of hERG channels except the 300 µM-20°C group which had a high binding affinity for inactive state of hERG channels. CONCLUSION: Oxymatrine is a low potency blocker of hERG K+ channels at 30°C, low concentration oxymatrine affect the hERG activation gating with accelerating hERG tail current at 20°C, oxymatrine is a potential hERG activator at low temperatures.

[Effect of sophocarpine on HERG K+ channels].

Human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier K+ current, which has an important effect on both proarrhythmia and antiarrhythmia. To investigate the effect of sophocarpine (SC) on HERG channel stably expressing in human embryonic kidney-293 (HEK293) cells, whole-cell patch-clamp technique was used to record HERG current and kinetic curves. As the result, it was found that SC inhibited HERG current in a concentration-dependent manner (10, 30, 100, and 300 micromol x L(-1)). At 0 mV, 10, 30, 100, and 300 micromol x L(-1) SC respectively inhibited IHERG by Istep ( 10.7 +/- 2.8)% , (11.3 +/- 5.5)% , (47.0 +/- 2.3)% and (53.7 +/- 2.5)% , and Itail (1.1 +/- 3.0)%, (17.1 +/- 3.3)%, (32.7 +/- 1.9)% (P < 0.05, n = 12) and (56.0 +/- 2.4)% (P < 0.05, n = 13). The time constants of inactivation, recovery from inactivation and onset of inactivation were accelerated. SC did not change other channel kinetics (activation and deactivation). It is concluded that SC inhibited the transfected HERG channels by influencing the inactivation state, which is the probable anti-arrhythmic mechanism.

[Effect of Daming capsule on expression of connexin43 isoforms in hyperlipemic rat's cardiac muscle].

OBJECTIVE: To find the molecular mechanism of decreasing blood fat effect of Darning capsule on hyperlipemic rat, we study the expression of connexin43 in the myocardium before and after using the capsule. METHOD: Forty Wistar rats were randomly divided into 5 group: control group, hyperlipemia model group, Daming capsule group of high dose, middle dose and low dose (200, 100, 50 mg kg(-1) d(-1)). Each group had 8 rats. Hyperlipemic rat model was made firstly, the blood was obtained via vena caudalis and the indexes of TC, TG, LDL, HDL and NEFA in the serum were measured. The myocardial total RNA was extracted by Trizol method. To compare the expression of connexin43 in the following groups: hyperlipemia, normal and drug, we used the technique of RT-PCR, immunostaining and microconfoul. RESULT: The concentrations of TC, TG, LDL and NEFA in hyperlipemic serum were increased (P <0. 05), while that of HDL was decreased (P <0. 05). After treating with Daming capsule, the concentration of the preceding four indexes were decreased and the concentrations of HDL was increased up to nearly normal level. No significant difference was found in the ECG of the three groups. As compared with the normal group, the mRNA expressions of connexin43 in hyperlipemia group was weakened (P <0.05), while that of the drug group was enhanced(P <0.05). The same result in immunostaining was observed. CONCLUSION: Hyperlipemic rat model was successfully established and Daming capsule has the effect of lowering blood lipid. Furthermore, the molecular mechanism of Darning capsule is related with the change of Cx43 closely.