Characterization of a Nattokinase from the Newly Isolated Bile Salt-Resistant Bacillus mojavensis LY-06

Abstract

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.

Combined Computer-Aided Predictors to Improve the Thermostability of Nattokinase

“…A recombinant plasmid pET-28a-AprY (resistance to kanamycin) containing a nattokinase full peptide (signal peptide + propeptide + mature peptide, recombinant AprY, rAprY) from Bacillus mojavensis LY-06 was used as the expression vector [ 33 ]. Escherichia coli BL21 (DE3; E. coli BL21) was used as the expression host for rAprY [ 33 ]. Luria-Bertani (LB) medium (5 g/L yeast extract, 10 g/L tryptone, 10 g/L NaCl, pH 7.0) was used for plasmid propagation and overexpression.…”

“…Since the primary structure of AprY is consistent with the sequence of nattokinase AprN (PDB number: 4DWW, deposition time: 27 February 2012), the 3D structure of AprN was used as the object of the thermostability-prediction algorithms [ 33 ]. (1) FoldX: The RepairPDB module was used to identify and repair residues with poor torsion angles or van der Waals force conflicts, and then the high-resolution structure of the target proteins were submitted for ΔΔG values calculation [ 28 ].…”

“…The expression and purification method of rAprY and its variants refers to the previous research of Li et al [ 33 ]. Escherichia coli BL21 (DE3) was used as host for protein induction, and their overexpression was achieved after 20 h of incubation at 18 °C by the addition of 0.1 mM IPTG.…”

“…The thrombolytic activity and thermostability of the crude enzyme of each rAprY variant were determined by the fibrin plate method [ 33 ]. The thrombolytic activity of rAprY and its variants was obtained by calculating the area of the transparent circle of the fibrin plate after 18 h of incubation at 37 °C, with a urokinase standard used as control.…”

Microbial nattokinase: from synthesis to potential application

“…22,[37][38][39] In addition, milk, seawater, rust and soil are also sound resources for obtaining NK-producing strains (Table 1). [14][15][16]40 Among them, B. subtilis, B. amyloliquefaciens, B. mojavensis, and B. cereus are all affiliated to the Bacilli, 16,[41][42][43] and Pseudomonas aeruginosa CMSS and Pseudomonas sp. TKU015 are both affiliated to the γ-Proteobacteria.…”