Thrombosis has threatened human health in past decades. Bacillus nattokinase is a potential low-cost thrombolytic drug without side-effects and has been introduced into the consumer market as a functional food or dietary supplement. This review firstly summarizes the biodiversity of sources and the fermentation process of nattokinase, and systematically elucidates the structure, catalytic mechanism and enzymatic properties of nattokinase. In view of the problems of low fermentation yield, insufficient activity and stability of nattokinase, this review discusses the heterologous expression of nattokinase in different microbial hosts and summarizes the protein and genetic engineering progress of nattokinase-producing strains. Finally, this review summarizes the clinical applications of nattokinase.

Pulcherriminic acid is a cyclic dipeptide found mainly in Bacillus and yeast. Due to the ability of pulcherriminic acid to chelate Fe³⁺ to produce reddish brown pulcherrimin, microorganisms capable of synthesizing pulcherriminic acid compete with other microorganisms for environmental iron ions to achieve bacteriostatic effects. Therefore, studying the biosynthetic pathway and their enzymatic catalysis, gene regulation in the process of synthesis of pulcherriminic acid in Bacillus can facilitate the industrial production, and promote the wide application in food, agriculture and medicine industries. After initially discussing, this review summarizes current research on the synthesis of pulcherriminic acid by Bacillus, which includes the crystallization of key enzymes, molecular catalytic mechanisms, regulation of synthetic pathways, and methods to improve efficiency in synthesizing pulcherriminic acid and its precursors. Finally, possible applications of pulcherriminic acid in the fermented food, such as Chinese Baijiu, applying combinatorial biosynthesis will be summarized.

To investigate the structure–antioxidant activity relationship, Pleurotus ferulae polysaccharides were extracted using ultrasonic (U-PFPS) and microwave/ultrasonic-assisted methods (MU-PFPS). Compared to U-PFPS with a molecular weight of 1.566 × 103 kDa, MU-PFPS exhibited a lower molecular weight of 89.26 kDa. In addition, unlike U-PFPS, which is primarily composed of glucose (Glu:Man:Gal = 91.1:3.5:5.4), MU-PFPS has a more balanced composition of Glu:Man:Gal in the ratio of 39.4:27.8:32.8 and contains more branched chains. Furthermore, antioxidant analysis revealed that high concentration (at concentrations above 600 μg/mL) MU-PFPS demonstrated stronger protective effects against oxidative damage in RAW264.7 cells than U-PFPS did. Collectively, these data suggest that lower molecular weight and higher branching degree of polysaccharides at appropriate concentrations may correlate with enhanced antioxidant enzyme activities. Our work provides a method for isolating polysaccharides with higher antioxidant activity and offers insights into the structure–activity relationship of polysaccharides, laying the foundation for future applications in polysaccharide modification and structural characterization.

Nattokinase is a potential new thrombolytic drug because of its strong thrombolytic effect, high safety, and low cost. However, there is no research reporting on bile salt-tolerant nattokinase-producing probiotics. In this study, the bile salt-tolerant nattokinase-producing strain Bacillus mojavensis LY-06 was isolated from local Xinjiang douchi, and the fermentation yield of nattokinase of 1434.64 U/mL was obtained by both a single factor experiment and an orthogonal experiment. A gene responsible for fibrinolysis (aprY) was cloned from the genome of strain Bacillus mojavensis LY-06, and the soluble expression of this gene in Escherichia coli (rAprY, fused with His-tag at C-terminus) was achieved; molecular docking elucidates the cause of insoluble expression of rAprY. The optimal pH and temperature for the fibrinolysis activity of nattokinase AprY fermented by Bacillus mojavensis LY-06 were determined to be pH 6.0 and 50 °C, respectively. However, the optimal pH of rAprY expressed in Escherichia coli was 8, and its acid stability, thermal stability, and fibrinolytic activity were lower than those of AprY. Bioinformatics analysis found that the His-tag carried at the C-terminus of rAprY could affect its acidic stability by changing the isoelectric point and surface charge of the enzyme; in contrast to AprY, changes in the number of internal hydrogen bonds and the flexibility of the loop region in the structure of rAprY resulted in lower fibrinolytic activity and poorer thermal stability.