High Cell Density Cultivation of A Recombinant Bacillus Subtilis for Nattokinase Production

doi:10.21203/rs.3.rs-91180/v1

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High Cell Density Cultivation of A Recombinant Bacillus Subtilis for Nattokinase Production

https://doi.org/10.21203/rs.3.rs-91180/v1

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Background: Nattokinase (NK), a fibrinolytic enzyme, can be produced by culturing recombinant Bacillus subtilis in Luria-Bertani broth in a shaking flask. For use as a nutraceutical, however, a large-scale preparation and a simple purification process are required.

Results: The present study utilized a fed-batch process to cultivate a B. subtilis strain carrying a pHT01 plasmid with an NK-encoding gene (B. subtilis/pHT01-aprN1). For batch A (FB A), with a pH-stat two-stage fermentation strategy, we achieved an activity of 2910.5 ± 21.6 U mL^-1 and a specific activity of 30.32 U ml^-1 OD₆₀₀^-1. Then, we changed the strategy with a later induction and lower feeding rate to pursue higher cell density and thus higher enzyme activity, a 11.9-fold activity of 4521.8 ± 23.8 U mL^-1 was acquired, however, the specific activity was lower than FB A. For the third batch, low-glycerol-level-maintain feeding strategy was followed, and finally, a NK activity of 7778 ±17.28 U mL^-1 was obtained, according to our knowledge, it was the highest activity assayed by the fibrin plate method ever reported. Furthermore, fermentation supernatant was successively purified by ammonium sulfate precipitation and nickel column affinity chromatography with a total NK recovery rate of 65.2%.

Conclusions: Our results indicate that there is a balance between the cell growth rate and NK expression when recombinant Bacillus subtilis is cultured with a fed-batch process. The equilibrium state can be attained by optimizing the induction and feeding strategy, and thus a high cell density and enzyme activity can be achieved.

Applied & Industrial Microbiology

Nattokinase

Bacillus subtilis

recombinant protein expression

high cell density fermentation

induction and feeding strategy

Thrombosis, which is responsible for high morbidity and mortality in humans [1], can be effectively treated by thrombolytic drugs, but these are associated with adverse effects [2, 3]. Therefore, it is necessary to develop new biological substances, especially prophylactic food-source thrombolytic agents with low immunogenicity and preventative, long-term effects that are convenient for oral administration and stable in the gastrointestinal tract.

Nattokinase (NK), which decreases the ability of blood to clot, is traditionally taken from natto, a Japanese solid-state fermented soybean food [4, 5]. Currently, NK is used as a dietary supplement as well as a prophylactic or a curative thrombosis medicine [5, 6]; however, the process used to purify it from natto is complicated and accompanied by significant loss of bioactivity. Accordingly, fed-batch fermentation by adopting genetically modified bacteria may improve enzyme yield [7–10].

Several reports have shown that fed-batch culture led to increases in NK activity of 2.1–25-fold relative to batch culture and that the addition of glycerol during the cell growth phase increased NK production significantly [10–16]. Moreover, various protein purification methods have been utilized for NK purification [6, 16]. Taken together, these findings suggest that the expression of NK by genetically modified bacteria may reach a much higher level if efficient protocols for high cell density fermentation and subsequent purification are obtained.

We previously constructed a B. subtilis 168 strain containing a pHT01-aprN plasmid [17]. In this study, we further investigated the expression of NK by culturing this strain under different induction and feeding strategies. In addition, we investigated the efficiency of purification with ammonium sulfate precipitation and Ni–NTA affinity chromatography.

NK expression by flask culture and fed-batch experiment

At least five different NK activity assay methods have been reported to date [6, 11, 12, 18, 19]. Among these, the fibrin plate method and chromogenic method are the most widely used, but the casein-degradation and JBSL (Japan Bio Science Laboratory Co., Ltd) methods may show much higher values of enzyme activity [11, 12, 14].

The NK activity obtained at 24 h in ﬂask culture was 380.14 ± 5.71 U mL^-1, while the OD₆₀₀ reached 13.45 ± 0.45.

Many studies have demonstrated the significant contribution of media ingredients [20, 21] and nutrient feeding strategy to NK production [6, 15]. Berenjian confirmed that glycerol was a noteworthy carbon source influencing cell density during the fermentation of B. subtilis natto, and that the highest activity of NK was obtained by adding 3% glycerol as a carbon source [15]. It has also been reported that 2,6-pyridine dicarboxylic acid (PDCA) and metal ions such as Ca²⁺ and Mg²⁺ could improve osmotic pressure and help to maintain enzyme conformation, thereby improving the NK activity [17, 22]. Moreover, Wang demonstrated that glucose, K₂HPO_4·3H₂O and MgSO₄·7H₂O played key roles in the production of NK, and they obtained an activity of 12.34 FU mL^-1[23]. Taken together, the results of these studies showed that NK activity could be improved dozens of times by media optimization.

In addition to media, feeding solutions are critical factors that influence NK activity, which should support cell growth and recombinant protein production while avoiding substrate inhibition and other related problems [12, 15].

Based on this information, we selected a mixture of glycerol, yeast extract, PDCA and a concentrated inorganic mixture solution as the fermentation broth, and a mixture of glycerol and yeast extract as the feed broth.

The strategy of induction, including cell density at the time of induction, inducer concentration, pre-induction growth and post-induction incubation time, can also affect the efficiency of protein expression. The aim of this study was to investigate the effects of using pH-stat and low-glycerol-level-maintaining strategies on NK expression by B. subtilis 168/pHT01-aprN1.

Three experiments were performed to examine the effects of induction time, feeding time and feeding rate on the NK activity of fermentation broth. The results are presented in Figure 1 and Table 1.

Two-stage fermentation strategy has been used for production of recombinant protein and other metabolites [24-27]. For FB A, we proposed a two-stage fermentation strategy and expected to get a high production. In the first stage (cell growth stage), we fed 600 mL of media into the fermenter with a high flow rate at the eighth hour. Because the nutrition was not sufficient for cell growth, the glycerol concentration decreasing rapidly to about 70 mmol L^-1 at 12 h and the OD₆₀₀ not varying markedly after 13 h. However, the NK activity still increased significantly until 18 h. The final NK activity was 2910.5 ± 21.6 U mL^-1 and the specific activity was 30.32 U ml^-1 OD₆₀₀^-1.

Although we achieved a 7.7-fold activity compare to flask culture, the cell density and the total activity were not as high as reported previously [6, 12]. It suggested that later induction and lower feeding rate maybe to the benefit of higher cell density and thus higher enzyme activity [28, 29]. According to this hypothesis, for FB B, the expression was induced at the fifth hour, which was 1 h later than FB A, and the induced OD₆₀₀ was up to 27.3 ± 1.0, which was higher than that of FB A (17.6 ± 0.4). A three-stage feeding strategy was used, and the feeding of FB B started at 14 h, when the glycerol concentration was as low as 119.8 ± 1.3 mmol L^-1, which might have favored cell growth by reducing substrate inhibition. The glycerol content was sufficient to support the cell growth for 24 h. Although the OD₆₀₀ values from 15 h to 19 h were not varied significantly due to dilution and a new medium environment, it finally reached a high value of 208.8 ± 1.9 at 24 h, with a 11.9-fold activity of 4521.8 ± 23.8 U mL^-1. However, the specific activity was 21.66 U ml^-1 OD₆₀₀^-1 at 24 h, which was lower than that of FB A, and the NK activity did not synchronously increase following cell growth during the late fermentation stage, which implied that there should be a balance between the cell growth rate and enzyme expression.

The results of FB A and FB B suggested that maybe we should not unilaterally pursue high cell density, and instead the expression should be induced at an earlier time and kept a low glycerol content during the feeding period. Consequently, for FB C, continuous feeding was adapted, starting at 10 h when the OD₆₀₀ had reached 105.3 ± 1.1 and almost two-thirds of the initial glycerol had been consumed. The glycerol concentration was controlled to around 50 mmol L^-1 by adjusting the feeding rate.

As expected, an activity of 7778.0 ± 17.3 U mL^-1 and a specific activity of 44.86 U ml^-1OD₆₀₀^-1 was achieved, and these values were 1.7-, 2.6-, and 26-fold higher than those of batch B, batch A and the ﬂask culture, respectively. According to our knowledge, this was the highest ever reported activity value by the fibrin plate method [9, 10, 12, 15].

Recovery rate of NK by purification

The recovery rate of NK obtained by ammonium sulfate precipitation was 89.1% (Table 2), which was consistent with a study by Garg [16]. The high recovery rate and simple operation showed that this method could be used to purify NK on a large scale.

The purification was followed by Ni–NTA affinity chromatography, and the total recovery rate was 65.2% (Table 2). The high imidazole concentration in wash buffer B led to a high loss of NK when samples were purified using Ni–NTA affinity chromatography. Accordingly, the imidazole concentrations were adjusted to zero in wash buffer A and incubated supernatant, and to 10 mmol L^-1 in wash buffer B; thus, a recovery rate of 88% was obtained in this step. Different imidazole concentrations in elution buffer C were also investigated, but there were no significant differences in the range of 100 mmol L^-1to 500 mmol L^-1. According to the NK expression level and the recovery rate, the process developed here may be applied for large scale production of NK.

SDS-PAGE and Western blotting analysis of NK

SDS-PAGE analysis demonstrated that a 28 kDa protein was a crucial component in the supernatant from induced B. subtilis 168/pHT01-aprN1, but that it was not present in the supernatant from B. subtilis 168/pHT01 and non-induced B. subtilis 168/pHT01-aprN1 fermentation broth (Fig. 2 A/B), suggesting that recombinant NK could be expressed in a soluble form. Western blotting with a His-tag-specific monoclonal antibody also showed a specific signal at 28 kDa, whereas no cross-reaction occurred in the total soluble proteins from non-induced B. subtilis 168/pHT01-aprN1 broth, which confirmed that the 28 kDa protein was the recombinant NK, as expected (Fig. 2 C).

In view of these reports, NK was produced by fed-batch cultures of recombinant B. subtilis, and its production was improved to 7,778 U mL^-1 from 380 U mL^-1 of flask culture using pH-stat and low-glycerol-level strategies. Future studies will design a process and set a kinetic model for fermentation optimization.

Microorganism and inoculum preparation

A gene-modified strain (B. subtilis 168/pHT01-aprN1) was used to express NK. The medium for flask and fed-batch culture was as follows: yeast extract, 60 g L^-1; glucose, 40 g L^-1; glycerol, 20 mL L^-1; NH₄Cl, 3 g L^-1; K₂HPO₄·3H₂O, 1 g L^-1; MgSO₄·7H₂O, 2 g L^-1; MnSO₄·H₂O, 0.02 g L^-1; FeSO₄·7H₂O, 0.05 g L^-1; CoCl₂·6H₂O, 0.01 g L^-1; ZnCl₂, 0.01 g L^-1; 2,6-pyridinedicarboxylic acid, 0.167 g L^-1. Glucose and MgSO₄·7H₂O were autoclaved separately and aseptically added to the medium. Nutrient solution contained 100 g L^-1 yeast extract and 250 mL L^-1 glycerol was used as feed medium. To avoid plasmid loss and prevent microbial contamination, chloramphenicol (5 μg mL^-1) was added to all media after being dissolved in anhydrous ethanol and filtered through a 0.22 μm membrane.

Shake flask experiments

To prepare a stock culture, one colony of B. subtilis 168/pHT01-aprN1 was inoculated in 1 mL Luria-Bertani (LB) medium and then incubated overnight at 37°C in a shaking incubator at 200 revolutions per minute (rpm). A flask with 25 mL fed-batch culture media was then inoculated with 7.5% (v/v) of stock culture and incubated at 37°C until reaching mid-log phase (OD₆₀₀ of 0.8–1.0). Next, the temperature was adjusted to 28°C and held for 20 min, after which a 25-μL aliquot of 0.5 mmol L^-1 isopropyl β-D-1-thiogalactopyranoside (IPTG) was added and the culture was further incubated for 45 h at 28°C in a shaking incubator (200 rpm) for NK expression.

Fed-batch experiments

An in-situ sterilizable 15 L bioreactor (BIOSTAT^®B, Germany) equipped with pH and pO₂ probes was used for fed-batch experiments. One milliliter of glycerol stock bacteria was inoculated into 100 mL LB medium and cultured for 12 h at 37°C in a shaking incubator (200 rpm), after which the pre-culture solution was aseptically inoculated into 1 L LB medium and incubated under the same conditions for 12 h. Next, 700 ml of seed cultures were inoculated for 7-L fed-batch fermentation in the bioreactor. The value of oxygen dissolved was set at 20%, which was cascade controlled by agitation speed (300–1000 rpm) and air flow rate (3–20 L min^-1). The pH was set as 7.0 and automatically controlled by adding 2 mol L^-1 HCl or 2 mol L^-1 NaOH.

In the first fed-batch experiment (FB A), the temperature was adjusted to 28°C at 3.5 h, while IPTG was added 30 min later as the OD₆₀₀ value reached about 18. A total of 600 mL feed media was supplemented at 8 h with a flow rate of 30 mL min^-1. In the second fed-batch experiment (FB B), the expression was induced at 5 h when the OD₆₀₀ value had reached 27, and the feed media was constantly fed after 14 h of cultivation with a flow rate of 6 mL min^-1 for 1 h, then 3.6 mL min^-1 for 2 h, and finally 2.4 mL min^-1 for another hour. In the third experiment (FB C), the expression was induced at 3 h when the OD₆₀₀ reached about 9 and the feed media was fed at 10 h. The feeding rate depended on the content of glycerol, which was controlled at a concentration of 50 mmol L^-1. Cell growth was monitored at various times by measuring the OD₆₀₀ values.

NK activity assay

Quantitative analysis of NK activity was conducted by the fibrin plate method, with slight modification [18]. Briefly, bovine fibrinogen (Sigma, St. Louis, MO, USA) and thrombin (Sigma, St. Louis, MO, USA) were dissolved in 0.1 mol L^-1 sodium phosphate buffer (PBS) at pH 7.4. An equi-voluminal mixture of 5 g L^-1 bovine fibrinogen solution and 12 g L^-1 agarose solution was then warmed in a 45°C bath, after which 10 μL of 500 U thrombin was added to 15 mL of the mixture solution in a 90 mm petri dish and kept at room temperature for 1 h to form fibrin. Holes with a 2 mm diameter were made on the fibrin plate and 10 µL of each diluted supernatant of fermentation broth was then added. The plates were subsequently incubated at 25°C for 16 h, after which the areas of the lysis zones on the fibrin plates were measured and the fibrinolytic activities were determined according to the standard curve of urokinase.

Glycerol concentration measurement

The concentration of glycerol was determined enzymatically using a free glycerol assay kit (Sigma, St. Louis, MO, USA) according to the manufacturer’s procedures.

Purification and lyophilization of NK

The final fermentation broth was collected and centrifuged (GL-25M, Luxiangyi, Shanghai, China) immediately at 18,300 × g for 10 min. Solid (NH₄)₂SO₄ was then gradually added until the supernatant was 20% saturated, after which it was stored at 4°C overnight. Next, the broth was centrifuged at 18,300 × g for 10 min and the precipitate was discarded. Additional (NH₄)₂SO₄ was subsequently added to make the supernatant 60% saturated, after which the mixture was stored at 4°C overnight again. Following centrifugation at 18,300 × g for 30 min, the supernatant was removed and the precipitate was dissolved in 10 mmol L^-1 PBS (pH 7.4) as a crude enzyme. The crude enzyme was further purified by Ni–NTA affinity chromatography according to the manufacturer’s (Novagen, San Diego, CA, USA) protocols. After purification, 10 µl of eluted fractions of different dilution times were used to assay their fibrinolytic activities. Residual solution was desalted by dialysis and lyophilized using a GLZY-0.5B vacuum freeze drier (Pudong Freeze Drying Equipment, Shanghai, China) to obtain NK Lyophilized powder.

SDS-PAGE and Western blotting analysis

SDS-PAGE protein analysis was performed in a Mini-Protean Tetra system (BioRad, Hercules, CA, USA) by loading 30 μL of 1: 1 (v: v) boiled supernatant and dye buffer onto a 4–20% precast Mini protean TGX gel (BioRad) and then running the samples in 1 × Tris-glycine-SDS running buffer (BioRad, Shanghai, China) at 100 V for 15 min followed by 200 V for 25 min. A protein standard (10-250 kDa, Precision Plus Protein Kaleidoscope, Bio-Rad) was used as a ladder.

NK was identified by Western blotting analysis as described by Towbin, with slight modification [30]. Briefly, 10 μL aliquots of different supernatants were separated by 15% SDS–PAGE. After electrophoresis, the protein bands were transferred from the gel to an Amersham nitrocellulose (NC) membrane (GE Healthcare, Piscataway, NJ, USA) using a Mini Trans Blot electrophoretic transfer cell (BioRad). NK on the membrane was reacted with a mouse monoclonal antibody (Yisheng Biological Technology, Shanghai, China) against His-tag (1:1000 v/v), then incubated with anti-mouse IgG alkaline phosphatase (Sigma) conjugate (1:2000 v/v). After washing, the NC membrane was stained using Super ECL Detection Reagent (Yisheng Biology Technology, Shanghai, China) according to the manufacturer’s instructions, then covered by an X-ray film and exposed for 1 min. A Tanon v.3500 Gel Imaging System (Tanon Co., Shanghai, China) was used for the analysis of proteins.

Acknowledgment

This project was supported by the National Key Research and Development Program of China (No. 2017YFD0400205).

Conflict of interest

The authors have no conflicts of interest to declare.

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Table 1
Parameters for NK production process of flask fermentation and three fed-batch fermentation experiments with different induction and feeding strategies
	FB A	FB B	FB C	LB
Induction time	4	5	3	3
OD₆₀₀ at induction time	17.64 ± 0.44	27.28 ± 0.98	9.14 ± 0.34	0.85 ± 0.12
Feeding start time (h)	8	14	10	No feeding
Fermentation time (h)	24	24	22	24
Maximum value of OD₆₀₀	105.60 ± 0.85	208.80 ± 1.94	185.31 ± 0.85	13.45 ± 0.45
NK activity (U mL^− 1)	2910.53 ± 21.60	4521.78 ± 23.79	7778.02 ± 17.28	380.14 ± 5.71

Table 2
Recovery rates of NK by ammonium sulfate precipitation and nickel column purification
Sample	Recovery rate (%)
Fermentation supernatant	100
60% ammonium sulfate-precipitated protein	89.1 ± 3.2
Protein adsorbed in nickel column	78.6 ± 2.6
Nickel column elution buffer	65.2 ± 2.4

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High Cell Density Cultivation of A Recombinant Bacillus Subtilis for Nattokinase Production

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