Table 5.

Enzyme engineering to improve the enzymatic properties of nattokinase.

Strains	Molecular Modification Strategy	Enzyme Activity Assay Method	Effect	References
Bacillus subtilis var. natto	Mutation based on the literature	Tetrapeptide substrate method	The I31L mutant increased catalytic efficiency	[57]
Bacillus subtilis var. natto	Mutation based on the literature	Tetrapeptide substrate method	The M222A/I31L mutant increased oxidative stability	[57]
Bacillus subtilis QK02	Surface charge engineering, sequence alignment, and mutation based on the literature	Tetrapeptide substrate method	The Q59E mutant increased specific activity	[72]
Bacillus subtilis QK02	Surface charge engineering, sequence alignment, and mutation based on the literature	Tetrapeptide substrate method	The S78T mutant improved acid stability	[72]
Bacillus subtilis QK02	Surface charge engineering, sequence alignment, and mutation based on the literature	Tetrapeptide substrate method	The Y217K mutant enhanced acid and thermal stability	[72]
Bacillus subtilis QK02	Surface charge engineering, sequence alignment, and mutation based on the literature	Tetrapeptide substrate method	The N218D mutant improved thermal stability	[72]
Bacillus subtilis QK02	Surface charge engineering, sequence alignment, and mutation based on the literature	Tetrapeptide substrate method	The S78T/Y217K mutant improved acid stability	[72]
Bacillus subtilis var. natto strain AS 1.107	Non-oxidative mutation of amino acid residues surrounding the catalytic residue Ser221	Fibrin plate method and tetrapeptide substrate method	The T220S mutant increased oxidative stability	[43]
Bacillus subtilis var. natto strain AS 1.107	Non-oxidative mutation of amino acid residues surrounding the catalytic residue Ser221	Fibrin plate method and tetrapeptide substrate method	The M222A mutant increased oxidative stability	[43]
Bacillus licheniformis WX-02	Deletion of protease genes and construction of high-efficiency expression system of nattokinase (PHY300PLK + P43 + SsacC + aprN + TamyL)	UV spectrophotometer method	Improved the synthesis efficiency of nattokinase in Bacillus licheniformis WX-02	[73]
Bacillus licheniformis DW-02	Manipulation of signal peptides and signal peptidases (signal peptide of AprE and signal peptidase SipV)	UV spectrophotometer method	Enhanced secretion efficiency of nattokinase in Bacillus licheniformis	[74]
Bacillus subtilis WB800	Tandem promoter (PHpaII-PHpaII-PP43)	Fibrin plate method and UV spectrophotometer method	Improved the synthesis efficiency of nattokinase in Bacillus subtilis WB800	[75]
Bacillus subtilis 168	Sequence trimming and nucleotide optimization of the conserved region of the promoter PsrfA	HPLC method	High-efficiency self-inducible expression of nattokinase in Bacillus subtilis	[58]
strain Bacillus licheniformis DW2	An optimized single-stranded Shine–Dalgarno (SD) sequence was inserted into the hairpin loop for better ribosome recognition and recruitment.	Milk plate method	Increased the fermentation yield of nattokinase in Bacillus licheniformis	[59]
Bacillus subtilis WB 800	Deletion of protease genes	Tetrapeptide substrate method	Increased the fermentation yield of nattokinase in Bacillus subtilis WB 800	[65]