Abstract
BackgroundNattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative.ResultsThis report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity.ConclusionsThis study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.
Highlights
Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion
This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, lowcost preparation of nattokinase
The results of the present study indicate that nattokinase from B. subtilis natto is able to be expressed at high titers using the P. pastoris system, and the recombinant nattokinase exhibits obvious thrombolytic activity along with strong pH- and thermo-stability
Summary
Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. Nattokinase has been expressed using several heterologous expression systems. Nattokinase (E.C 3.4.21.62) was first found in natto, a traditional Japanese fermented soybean food product [1]. Full length nattokinase includes 381 amino acids and is encoded by the aprN gene of B. subtilis natto [2]. A three-dimensional structural model of nattokinase from B. subtilis natto (NK-Bs) was constructed using homology modeling in 2005, and the crystal structure of the enzyme was published later (PDB: 4DWW, 3vyv) [3,4,5]. The catalytic center of nattokinase consists of Ser-His-Asp (D32, H64, S221), and Ser221 is the key residue for substrate recognition
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