High and Economical Nattokinase Production with Acetoin as a Useful Byproduct from Soybean Milk and Glucose.

Nattokinase (NK) is a potent fibrinolytic enzyme with wide pharmaceutical and nutraceutical applications. Safe and high NK-yielding strains are urgently needed. In this study, the best strain NDF was isolated from one of the 11 natto samples and then identified as Bacillus subtilis. The effects of carbon and nitrogen sources on NK production were investigated, and glucose and soybean milk were finally selected as the optimal carbon and nitrogen sources, respectively. Acetoin, a valuable compound with versatile usages, was detected as the main byproduct of carbon overflow. In a 6-L fermenter, NK and acetoin reached their peak concentrations simultaneously (10,220 IU/mL and 25.9 g/L, respectively) at 25 h in a culture medium containing 180 g/L of soybean milk and 105 g/L of glucose. The NK product was verified by sequencing of the aprN gene and SDS-PAGE analysis. Only very limited kinds of proteins were found in the supernatant of the fermentation broth, and NK was one of the main bands. This study has developed an economical and high NK production method with acetoin as a useful byproduct.

Study on the mechanism of production of γ-PGA and nattokinase in Bacillus subtilis natto based on RNA-seq analysis.

Poly-γ-glutamic acid (γ-PGA) and nattokinase (NK) are the main substances produced by Bacillus subtilis natto in solid-state fermentation and have wide application prospects. We found that our strains had higher activity of nattokinase when soybeans were used as substrate to increase the yield of γ-PGA. Commercial production of γ-PGA and nattokinase requires an understanding of the mechanism of co-production. Here, we obtained the maximum γ-PGA yield (358.5 g/kg, w/w) and highest activity of NK during fermentation and analyzed the transcriptome of Bacillus subtilis natto during co-production of γ-PGA and NK. By comparing changes in expression of genes encoding key enzymes and the metabolic pathways associated with the products in genetic engineering, the mechanism of co-production of γ-PGA and nattokinase can be summarized based on RNA-seq analysis. This study firstly provides new insights into the mechanism of co-production of γ-PGA and nattokinase by Bacillus subtilis natto and reveals potential molecular targets to promote the co-production of γ-PGA and nattokinase.

Recombinant Production and Characterization of an Extracellular Subtilisin-Like Serine Protease from Acinetobacter baumannii of Fermented Food Origin.

Acinetobacter baumannii is a ubiquitous bacteria that is increasingly becoming a formidable nosocomial pathogen. Due to its clinical relevance, studies on the bacteria's secretory molecules especially extracellular proteases are of interest primarily in relation to the enzyme's role in virulence. Besides, favorable properties that extracellular proteases possess may be exploited for commercial use thus there is a need to investigate extracellular proteases from Acinetobacter baumannii to gain insights into their catalytic properties. In this study, an extracellular subtilisin-like serine protease from Acinetobacter baumannii designated as SPSFQ that was isolated from fermented food was recombinantly expressed and characterized. The mature catalytically active form of SPSFQ shared a high percentage sequence identity of 99% to extracellular proteases from clinical isolates of Acinetobacter baumannii and Klebsiella pneumoniae as well as a moderately high percentage identity to other bacterial proteases with known keratinolytic and collagenolytic activity. The homology model of mature SPSFQ revealed its structure is composed of 10 ß-strands, 8 α-helices, and connecting loops resembling a typical architecture of subtilisin-like α/ß motif. SPSFQ is catalytically active at an optimum temperature of 40 °C and pH 9. Its activity is stimulated in the presence of Ca2+ and severely inhibited in the presence of PMSF. SPSFQ also displayed the ability to degrade several tissue-associated protein substrates such as keratin, collagen, and fibrin. Accordingly, our study shed light on the catalytic properties of a previously uncharacterized extracellular serine protease from Acinetobacter baumannii that warrants further investigations into its potential role as a virulence factor in pathogenicity and commercial applications.

High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters.

BACKGROUND: Nattokinase (NK), which is a member of the subtilisin family, is a potent fibrinolytic enzyme that might be useful for thrombosis therapy. Extensive work has been done to improve its production for the food industry. The aim of our study was to enhance NK production by tandem promoters in Bacillus subtilis WB800. RESULTS: Six recombinant strains harboring different plasmids with a single promoter (PP43, PHpaII, PBcaprE, PgsiB, PyxiE or PluxS) were constructed, and the analysis of the fibrinolytic activity showed that PP43 and PHpaII exhibited a higher expression activity than that of the others. The NK yield that was mediated by PP43 and PHpaII reached 140.5 ± 3.9 FU/ml and 110.8 ± 3.6 FU/ml, respectively. These promoters were arranged in tandem to enhance the expression level of NK, and our results indicated that the arrangement of promoters in tandem has intrinsic effects on the NK expression level. As the number of repetitive PP43 or PHpaII increased, the expression level of NK was enhanced up to the triple-promoter, but did not increase unconditionally. In addition, the repetitive core region of PP43 or PHpaII could effectively enhance NK production. Eight triple-promoters with PP43 and PHpaII in different orders were constructed, and the highest yield of NK finally reached 264.2 ± 7.0 FU/ml, which was mediated by the promoter PHpaII-PHpaII-PP43. The scale-up production of NK that was promoted by PHpaII-PHpaII-PP43 was also carried out in a 5-L fermenter, and the NK activity reached 816.7 ± 30.0 FU/mL. CONCLUSIONS: Our studies demonstrated that NK was efficiently overproduced by tandem promoters in Bacillus subtilis. The highest fibrinolytic activity was promoted by PHpaII-PHpaII-PP43, which was much higher than that had been reported in previous studies. These multiple tandem promoters were used successfully to control NK expression and might be useful for improving the expression level of the other genes.

Strain Screening from Traditional Fermented Soybean Foods and Induction of Nattokinase Production in Bacillus subtilis MX-6.

The plasminogen-free fibrin plate assay method was used to isolate Bacillus subtilis MX-6, a strain with high production of nattokinase from Chinese douchi. The presence of aprN, a gene-encoding nattokinase, was verified with PCR method. The predicted amino acid sequence was aligned with homologous sequences, and a phylogenetic tree was constructed. Nattokinase was sublimated with ammonium sulfate, using a DEAE-Sepharose Fast Flow column, a CM-Sepharose Fast Flow column and a Sephadex G-75 gel filtration column. SDS-PAGE analysis indicated that the molecular weight of the purified nattokinase from Bacillus subtilis MX-6 was about 28 kDa. Fermentation of Bacillus subtilis MX-6 nattokinase showed that nattokinase production was maximized after 72 h; the diameter of clear zone reached 21.60 mm on the plasminogen-free fibrin plate. Nattokinase production by Bacillus subtilis MX-6 increased significantly after supplementation with supernatant I, supernatant II and soy peptone but decreased substantially after the addition of amino acids. This result indicated that the nattokinase production by B. subtilis MX-6 might be induced by soybean polypeptides. The addition of MgSO4 and CaCl2 increased B. subtilis MX-6 nattokinase production.

Extracellular Production of a Potent and Chemically Resistant Nattokinase in Immobilized Escherichia coli Using Response Surface Methodology.

BACKGROUND: Nattokinase is a potent fibrinolytic protease, which is used as a drug for treatment or a supplement for preventing thrombosis besides various industrial applications. The present study aimed to produce a soluble nattokinase in low cost media, which has with high activity and resistance to metal ions, detergents, and organic solvents, and can be easily used in medicines or as detergents. METHODS: Generally, most of the native extracellular proteins, such as nattokinase from Bacillus subtilis, are lysed by secretory proteases. One way for solving this problem is to employ other hosts for nattokinase production. For producing secretory form of nattokinase from Bacillus subtilis, different factors such as a suitable host, optimized media for the maximum enzyme activity and easy purification are important. RESULTS: These factors are studied in this investigation. Escherichia coli BL21 (DE3), as a reliable host was selected for a high yield production of an extracellular recombinant nattokinase. A mature nattokinase gene from Bacillus subtilis 1023, was cloned in the expression vector pET22b by which the host was transformed. The recombinant nattokinase was expressed through induction with IPTG. The expressed protein was confirmed by SDS-PAGE, and its fibrinolytic activity was assayed on the fibrin plates. Afterwards, the enzyme was purified by Ni-NTA native affinity column. Different media components were evaluated for maximum nattokinase production and activity. The highest enzyme activity of 883.107 U/ml was obtained, when a medium approximately consisting of yeast extract (4.38 g/L), tryptone (4 g/L), K2HPO4 (1.61 g/L) and CaCl2 (0.01 g/L) was used. CONCLUSION: Entrapping the transformed host in calcium alginate could lead to more enzyme activity and decrease media cost.

A New Bacillus licheniformis Mutant Strain Producing Serine Protease Efficient for Hvdrolvqis of Sov Meal Proteins.

Induced mutagenesis with y-irradiation of the industrial strain Bacillus licheniformis-60 VKM B-2366,D was used to obtain a new highly active producer of an extracellular serine protease, Bacillus licheni- formis7 145. Samples of dry.concentrated preparations of serine protease produced by the original and mutant strains were obtained, and identity of their protein composition was'established. Alkaline serine protease sub- tilisin DY was the main component of the preparations. The biochemical and physicochemical properties of the Protolkheterm-145 enzyme preparation obtained from the mutant strain were studied. It exhibited pro- teolytic activity (1.5 times higher than the preparation from the initial strain) within broad ranges of pH (5- 11) and temperature (30-70'C).-Efficient hydrolysis of extruded soy meal protein at high concentrations (2 to 50%) in-the reaction mixture was.the main advantage of the Protolikheterm 145 preparation. Compared to,. the preparation obtained using the initial strain, the new preparation with increased proteolytic-activity pro- vided for more complete hydrolysis of the main non-nutritious soy,proteins.(glycinin and 0-conglycinin) with the yield of soluble protein increased by 19-28%, which decreased the cost of bioconversion of the protein- aceous material and indicated promise of the new preparation in resource-saving technologies for processing soy meals and cakes.

A genome-wide expression quantitative trait loci analysis of proprotein convertase subtilisin/kexin enzymes identifies a novel regulatory gene variant for FURIN expression and blood pressure.

Proprotein convertase subtilisin/kexin (PCSK) enzymes cleave and convert their immature substrates into biologically active forms. Polymorphisms in the PCSK genes have been reported to associate with human diseases and phenotypes, including hypercholesterolemia and blood pressure (BP), and targeting PCSKs is considered a promising future form of drug therapy. PCSK processing is readily induced upon upregulation of the enzyme, but the genetic factors contributing to PCSK expression have not been thoroughly characterized. To gain a comprehensive understanding of the genetic regulation of PCSK expression, we performed, for the first time, a genome-wide expression quantitative trait loci (eQTL) analysis using mRNA expression in >1400 human peripheral blood samples from the Cardiovascular Risk in Young Finns Study and ca. ten million single-nucleotide polymorphisms (SNPs). The expression data showed clear expression for FURIN, PCSK5, PCSK7 and MBTPS1 (membrane-bound transcription factor peptidase, site 1) mRNAs in virtually all tested samples. A discovery analysis demonstrated a genome-wide significant (p < 5 × 10(-8)) association with the selected PCSK probes for 1024 variants, which were located at ten independent loci. Of these loci, 5/10 could be confirmed to regulate PCSK expression in two additional and independent sample sets. Finally, a phenotypic analysis demonstrated that a novel cis-eQTL SNP rs4702 for FURIN is strongly associated with both diastolic (p = 0.012) and systolic (p = 0.035) BP levels, as well as peripheral vascular resistance (p = 0.003). These findings indicate that the expression of the PCSK enzymes is regulated by genetic factors, which have biological roles in health and disease.

Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization.

Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography. Second, the gene fragments of P43 promoter, Svpr, nattokinase and TamyL were combined into pHY300PLK to form the expression vector pP43SNT. In BL10 (pP43SNT), the fermentation activity and product activity per unit of biomass of nattokinase reached 14.33 FU/mL and 2,187.71 FU/g respectively, which increased by 39 and 156 % compared to WX-02 (pP43SNT). Last, Svpr was replaced with SsacC and SbprA, and the maximum fermentation activity (33.83 FU/mL) was achieved using SsacC, which was 229 % higher than that of WX-02 (pP43SNT). The maximum NK fermentation activity in this study reaches the commercial production level of solid state fermentation, and this study provides a promising engineered strain for industrial production of nattokinase, as well as a potential platform host for expression of other target proteins.

Deleting multiple lytic genes enhances biomass yield and production of recombinant proteins by Bacillus subtilis.

BACKGROUND: Bacillus subtilis is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins. RESULTS: Five genes or genetic elements representing three different functional categories were studied as follows: lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGlA), and skfA and sdpC that encode cannibalism factors. Cell lysis was reduced and biomass was enhanced by deleting individually skfA, sdpC, xpf, and lytC. We constructed the multiple deletion mutant LM2531 (skfA sdpC lytC xpf) and found that after 4 h of culture, its biomass yield was significantly increased compared with that of prototypical B. subtilis 168 (wild-type) strain and that 15% and 92% of the cells were lysed in cultures of LM2531 and wild-type, respectively. Moreover, two expression vectors were constructed for producing recombinant proteins (ß-galactosidase and nattokinase) under the control of the P43 promoter. Cultures of LM2531 and wild-type transformants produced 13741 U/ml and 7991 U/ml of intracellular ß-galactosidase, respectively (1.72-fold increase). Further, the level of secreted nattokinase produced by strain LM2531 increased by 2.6-fold compared with wild-type (5226 IU/ml vs. 2028 IU/ml, respectively). CONCLUSIONS: Our novel, systematic multigene deletion approach designed to inhibit cell lysis significantly increased the biomass yield and the production of recombinant proteins by B. subtilis. These findings show promise for guiding efforts to manipulate the genomes of other B. subtilis strains that are used for industrial purposes.

Self-processing of a barley subtilase expressed in E. coli.

The barley protease BAJ93208 belongs to the subtilase family of serine proteases. We have expressed BAJ93208 in the cytoplasm of the Escherichiacoli strain SHuffle C3030 using a rhamnose-inducible promoter. The expression construct included a (His)6-tag at the N-terminus and a strep-tag at the C-terminus. Western blot analysis revealed that the protein was processed at the N- and C-terminus. To exclude that this processing was due to contaminating E. coli proteases, a mutated BAJ93208 protease was constructed. This inactive mutant was not processed, demonstrating that the processing was an autocatalytic process. To define the exact cleavage sites mass spectrometry was used which detected four differently processed versions of the protease. At the N-terminus, the self-processing removed the internal inhibitor and an additional 19 amino acids. At the C-terminus there was a cleavage site after Ala(765) which also removed the strep-tag. This explained the inability to detect the purified (His)6-BAJ93208-strep protease with an anti-strep-tag antibody. Finally, an additional alanine was removed either at the N-terminus (Ala(119)) or at the C-terminus (Ala(764)).

Production, optimization and characterization of fibrinolytic enzyme by Bacillus subtilis RJAS19.

The present study aimed at the production, purification and characterization of fibrinolytic nattokinase enzyme from the bacteria isolated from natto food. For the purpose, a fibrinolytic bacterium was isolated and identified as Bacillus subtilis based on 16S rDNA sequence analysis. The strain was employed for the production and optimization of fibrinolytic enzyme. The strain showed better enzyme production during 72nd h of incubation time with 50 degrees C at the pH 9. The lactose and peptone were found to be increasing the enzyme production rate. The enzyme produced was purified and also characterized with the help of SDS-PAGE analysis. The activity and stability profile of the purified enzyme was tested against different temperature and pH. The observations suggesting that the potential of fibrinolytic enzyme produced by Bacillus subtilis RJAS 19 for its applications in preventive medicines.

Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800.

BACKGROUND: Nattokinases/Subtilisins (EC 3.4.21.62) belong to the second large family of serine proteases, which gain significant attention and play important role in many biotechnology processes. Thus, a number of nattokinases/subtilisins from various Bacillus species, especially from B. subtilis strains, extensively have been investigated to understand their biochemical and physical properties as well as to improve the production for industrial application. The purpose of this study was to clone a nattokinase gene from Bacillus subtilis strain VTCC-DVN-12-01, enhance its production in B. subtilis WB800, which is deficient in eight extracellular proteases and characterize its physicochemical properties for potential application in organic synthesis and detergent production. RESULTS: A gene coding for the nattokinase (Nk) from B. subtilis strain VTCC-DVN-12-01 consisted of an ORF of 1146 nucleotides, encoding a pre-pro-protein enzyme (30-aa pre-signal peptide, 76-aa pro-peptide and 275-aa mature protein with a predicted molecular mass of 27.7 kDa and pI 6.6). The nattokinase showed 98-99% identity with other nattokinases/subtilisins from B. subtilis strains in GenBank. Nk was expressed in B. subtilis WB800 under the control of acoA promoter at a high level of 600 mg protein per liter culture medium which is highest yield of proteins expressed in any extracellular-protease-deficient B. subtilis system till date. Nk was purified to homogeneity with 3.25 fold purification, a specific activity of 12.7 U/mg, and a recovery of 54.17%. The purified Nk was identified by MALDI-TOF mass spectrometry through three peptides, which showed 100% identity to corresponding peptides of the B. subtilis nattokinase (CAC41625). An optimal activity for Nk was observed at 65 °C and pH 9. The nattokinase was stable at temperature up to 50 °C and in pH range of 5-11 and retained more than 85% of its initial activity after incubation for 1 h. Mg2+ activated Nk up to 162% of its activity. The addition of Triton X-100, Tween 20, and Tween 80 showed an activation of Nk up to 141% of its initial activity but SDS strongly inhibited. The enzyme was highly resistant to organic solvents. CONCLUSIONS: Our findings demonstrated that an eight-protease-gene-deficient Bacillus subtilis WB800 could overproduce the nattokinase from B. subtilis VTCC-DVN-12-01. Due to high resistance to detergents and organic solvents of this nattokinase, it could be potentially applied in organic synthesis and detergent production.

[Studies on the mode of action of bacterial AB5 toxins].

Bacterial AB5 toxins are proteins, produced by pathogenic bacteria including of Vibrio cholerae, Shigella dysenteriae, and enterohaemorrhagic Escherichia coli, which are usually released into the extracellular medium and cause disease by killing or altering the metabolism of target eukaryotic cells. The toxins are usually composed of one A subunit (a toxic domain) and five B subunits (a receptor-binding domain). This article overviews the characteristics and mode of actions of AB5 toxins including cholera toxin, Shiga-like toxin, and subtilase cytotoxin, and highlights current topics related to the roles of the effectors in promoting bacterial infection.

An extracellular subtilase switch for immune priming in Arabidopsis.

In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity.

The H-NS protein silences the pyp regulatory network of Yersinia enterocolitica and is involved in controlling biofilm formation.

Horizontal gene transfer plays an important role in bacterial evolution. DNA acquired by horizontal gene transfer has to be incorporated into existing regulatory networks. The histone-like nucleoid structuring protein H-NS acts as a silencer of horizontally acquired genes to avoid potential damage. However, specific regulators can overcome H-NS repression, resulting in the integration of newly acquired genes into existing regulatory networks. Here, we analyzed the influence of H-NS on the transcription of the Yersinia enterocolitica hreP gene and its regulators pypA, pypB, and pypC by establishing a dominant-negative H-NS version. Using transcriptional fusions and electrophoretic mobility shift assays, we show that H-NS silences hreP, pypA, pypB, and pypC by direct interactions. While the H-NS antagonist RovA activates pypC, it has no effect on pypA and pypB. Furthermore, H-NS affects biofilm formation in Y. enterocolitica.

Metabolic pathway analysis and kinetic studies for production of nattokinase in Bacillus subtilis.

We have constructed a reaction network model of Bacillus subtilis. The model was analyzed using a pathway analysis tool called elementary mode analysis (EMA). The analysis tool was used to study the network capabilities and the possible effects of altered culturing conditions on the production of a fibrinolytic enzyme, nattokinase (NK) by B. subtilis. Based on all existing metabolic pathways, the maximum theoretical yield for NK synthesis in B. subtilis under different substrates and oxygen availability was predicted and the optimal culturing condition for NK production was identified. To confirm model predictions, experiments were conducted by testing these culture conditions for their influence on NK activity. The optimal culturing conditions were then applied to batch fermentation, resulting in high NK activity. The EMA approach was also applied for engineering B. subtilis metabolism towards the most efficient pathway for NK synthesis by identifying target genes for deletion and overexpression that enable the cell to produce NK at the maximum theoretical yield. The consistency between experiments and model predictions proves the feasibility of EMA being used to rationally design culture conditions and genetic manipulations for the efficient production of desired products.

Characterization of two novel subtilases from common bean (Phaseolus vulgaris L.) and their responses to drought.

Protein breakdown by proteases is basic to the plant response to abiotic stresses such as drought. A large number of genes encoding proteases or putative proteases exist in plants. Only a few of those involved in the response to drought have been characterized, and their regulation is poorly understood. We have identified two new subtilases from leaves of Phaseolus vulgaris L. cultivar Zorin, PvSLP1 and PvSLP2. PvSLP1 was identified at the gene level, using primers based on the gene sequence of the putative drought induced serine protease from Arachis hypogaea L. In P. vulgaris, expression of the PvSLP1 transcript did not change on water withdrawal. PvSLP2 was isolated and characterized at the protein level, together with complete gene and cDNA sequences. The deduced amino acid sequences of both PvSLP1 and PvSLP2 are characteristic of plant subtilases of the S8 family of clan SB. PvSLP2 shows 33% sequence identity to PvSLP1. Expression of the PvSLP2 transcript did not change on withdrawal of water, but its proteolytic activity in leaves increased, depending on the age and position of the leaf. In addition, the level of activity in senescent leaves of well watered plants was higher than in mature or young leaves. These results, together with the fact that PvSLP2 cleaves peptide bonds following an Arg residue, point to regulation of PvSLP2 subtilase activity at translational and/or post-translational levels and suggest a specific role in the response to drought and senescence.

Strain screening, fermentation, separation, and encapsulation for production of nattokinase functional food.

This study presents a novel and integrated preparation technology for nattokinase functional food, including strain screening, fermentation, separation, and encapsulation. To rapidly screen a nattokinase-productive strain, PCR-based screening method was combined with fibrinolytic activity-based method, and a high productive strain, Bacillus subtilis LSSE-22, was isolated from Chinese soybean paste. Reduction of poly-γ-glutamic acid (γ-PGA) concentration may contribute to separation of nattokinase and reduction of late-onset anaphylaxis risk. Chickpeas were confirmed as the favorable substrate for enhancement of nattokinase production and reduction of γ-PGA yield. Using cracked chickpeas, the nattokinase activity reached 356.25 ± 17.18 FU/g (dry weight), which is much higher than previous reports. To further reduce γ-PGA concentration, ethanol fractional extraction and precipitation were applied for separation of nattokinase. By extraction with 50 % and precipitation with 75 % ethanol solution, 4,000.58 ± 192.98 FU/g of nattokinase powders were obtained, and the activity recovery reached 89 ± 1 %, while γ-PGA recovery was reduced to 21 ± 2 %. To improve the nattokinase stability at acidic pH condition, the nattokinase powders were encapsulated, and then coated with methacrylic acid-ethyl acrylate copolymer. After encapsulation, the nattokinase was protected from being denatured under various acid conditions, and pH-responsible controlled release at simulated intestinal fluid was realized.

Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis.

Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.