NK expression by flask culture and fed-batch experiment
At least five different NK activity assay methods have been reported to date [4, 11, 12, 17, 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 [4, 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 [10, 22]. Moreover, Wang demonstrated that glucose, K2HPO3H2O 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-apr N1.
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.
For FB A, the OD600 increased rapidly from the second hour. Although we fed 600 mL of media into the fermenter at the eighth hour, the nutrition was not sufficient for cell growth as indicated by the glycerol concentration decreasing rapidly to about 70 mmol L-1 from 4 to 12 h and the OD600not varying markedly after 13 h. However, the NK activity still increased significantly until 17 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.
For FB B, we attempted to achieve higher enzyme production via higher cell density; hence, 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). The feeding time of FB B was 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, and the OD600 reached a high value of 208.8 ± 1.9 at 24 h, with the highest activity reaching 4521.8 ± 23.8 U mL-1. Interestingly, the specific activity was 21.66 U ml-1OD600-1 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. Consequently, we did not unilaterally pursue high cell density in FBC, and instead induced at an earlier time and kept the glycerol content low during the feeding period in FB C.
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 at 20 h, and these values were 1.7-, 2.6-, and 26-fold higher than those of batch B, batch A and the flask culture, respectively. These values were also higher than those of other studies in which the reported NK production levels were 587 U mL-1 [15], 7100 U mL-1 [12], and 3194.3 U mL-1[4], respectively.