*4.3. Cultivation of Recombinant E. coli Expressing Nitrilase*

A pure colony (*E. coli* pRSF-AfNit2 or *E. coli* pET21a-AfNit) was picked and cultivated overnight in the 50 mL LB medium with 50 µg/mL kanamycin at 37 ◦C and 200 rpm. Then, the 2 mL culture was incubated in 50 mL of culture medium (Soluble starch: 20 g/L, yeast extract powder: 15 g/L, NaCl: 5 g/L, K2HPO4: 4 g/L, MgSO4·7H2O 1 g/L) with 50 µg/mL kanamycin, and the cells were cultured at 30 ◦C and 200 rpm. When the OD<sup>600</sup> value reached 0.4 to 0.6, IPTG (0.4 mM) was added to the culture to induce enzyme expression at 30 ◦C for 4 h [23]. Harvested cells were suspended in 50 mM PBS buffer (pH 7.0) and then ultrasonicated on ice. Centrifugation was used to remove cell debris for 10 min at 12,000× *g* and 4 ◦C. The protein molecular mass was evaluated by SDS-PAGE [30,31].

#### *4.4. Enzyme Assay*

To assay the enzyme activity of AfNit, 0.5 mg of dry weight resting cells were suspended in 475 µL of phosphate buffer (pH 7.0, 100 mM). The cell suspension was preheated for 5 min on thermo-mixer compact at 30 ◦C and 1000 rpm, then 25 µL of 3-cyanopyridine solution (1 M) in ethanol were added to the cell suspension, corresponding to 50 mM of final substrate concentration. The reaction was performed at 30 ◦C for 15 min, and the reaction solution was immediately added to 100 µL of HCl (2 M) to terminate the reaction. Then, 1900 µL of anhydrous ethanol was added to the reaction mixture for dilution. After centrifugation at 12,000× *g* for 5 min, the supernatant was filtered with organic filter membrane (25 mm × 0.45 µm, sinopharm chemical reagent Co., Ltd., Shanghai, China.), and the filtrate was assessed by HPLC. One unit of the enzyme activity was defined as the amount of enzyme releasing 1 µmol of NA per minute under the described conditions [25].

#### *4.5. Analytical Methods*

The NA and 3-cyanopridine were quantitatively determined by HPLC (Shimadzu LC-20AT, Kyoto, Japan), equipped with UV detector and a reversed phase column (Diamonsil® Plus C18 5 µm 250 × 4.6 mm, Cat. No.: 99409, Dikma Co., Shanghai, China), and monitored under UV 217 nm at a column temperature of 25 ◦C. The mobile phase was acetonitrile: phosphate buffer (10 mM sodium dihydrogen phosphate, pH 2.5) = 15:85 (*v*:*v*). The sample was eluted at a flow rate of 1.0 mL/min. The retention time of the product and substrate are 3.2 min and 6.8 min, respectively.

The molar response factor of NA to 3-caynopyridine was 0.5345. It was determined by the analysis of equal molar amounts of NA and 3-cyanopyridine standards with HPLC, and the molar response factor was obtained by mapping the peak area of NA and 3-caynopyridine.

#### *4.6. Preparation of Immobilized Cells and Enzyme Assay*

General procedue for the immobilization of *E. coli* pRSF-AfNit2 whole cells (exampled as SA-GA/PEI): the *E. coli* fermentation broth was washed twice with 0.1 M phosphate buffer (pH 7.0). It was resuspended in certain amount of 0.8% NaCl solutions to 10 g/L. The cell suspension was then mixed with equivalent volume of alginate solution (30 g/L). The

mixture was added to 2% (*w*/*v*) CaCl<sup>2</sup> solution with a syringe or a peristaltic pump, and the curing time was 2 h, then a small ball having an approximately diameter of 3 mm can be obtained, and it is finally washed with deionized water. The prepared alginate immobilized pellets were crosslinked in 2 wt% glutaraldehyde aqueous solution for 1 h. The additional glutaraldehyde was washed away, and 3% of the polyethyleneimine solution was added for 1 h, and the surface residual polyethyleneimine was washed away. After curing for 12 h, it was finally washed three times with deionized water, and the enzyme activity of the immobilized cells was measured [25,26].

Immobilized cells containing 0.5 mg dry weight of free cells were placed in 475 µL of Tris-HCl (0.1 M, pH 7.0) buffer solution, and they were preheated in a constant temperature mixing instrument at 30 ◦C and 1000 rpm for 5 min, and then 25 µL of 3-cyanopyridine solution (1 M) in ethanol was added to the mixture (final substrate concentration: 50 mM). After reaction for 15 min, the product concentration was determined in the sample by HPLC, as described in Section 4.4, and the enzyme activity was calculated from the degree of conversion. One unit of the immobilized enzyme activity was defined as the amount of enzyme releasing 1 µmol of NA per minute under the described conditions.

#### *4.7. Determination of Mechanical Strength of Immobilized Cells*

The immobilized cells studied in this experiment were spherical gel particles. Therefore, when measuring the mechanical strength of immobilized cells, 100 balls of immobilized cells were directly counted and placed in Tris-HCl (0.1 M, pH 7.0) buffer at 30 ◦C and 180 rpm while shaking, and the gel breakage was observed at different times.

#### *4.8. Effect of Different Reaction Temperature and pH on the Activity of Free and Immobilized Cells*

To determine the effect of temperature, the activity of free cells (1 gdcw/L) or immobilized cells (containing 1 gdcw/L free cells) was assayed in Tris-HCl buffers (100 mM, pH 7.0) at various temperatures (20–70 ◦C). The relative activity referred to the ratio of the enzyme activity at different temperatures to their respective highest activity (free cell: 3076 U/gdcw, immobilized cell: 2462 U/gdcw). Experiments were independently performed in triplicate.

To determine the effect of pH value, the activity of free cells (1 gdcw/L) or immobilized cells (containing 1 gdcw/L free cells) was assayed at 30 ◦C in various buffers over a pH range of 3.0~11.0 (pH 3.0~6.0, citrate–citric acid buffer; pH 6.0~7.0, PBS buffer; pH 7.0~9.0, Tris-HCl buffer; pH 9.0~11.0, Gly-NaOH buffer). The relative activity was expressed as a percentage of their respective maximum activity of free or immobilized cells. Experiments were independently performed in triplicate.

#### *4.9. Semi-Continuous Packed-Bed Bioreactor*

The column bioreactor used in this experiment (Figure 5) was made by Chengxin Glass Technology Co., Ltd. (Shanghai, China). It was a jacketed glass column reactor, and the temperature of the bioreactor was controlled by an external circulating water bath. The height of column was 100 mm, and the inner diameter was 17 mm. The effective volume of the column was approximately 20 mL. There was a round sand core at the bottom of column to prevent the beads from leaking. The wet weight of immobilized cells packed into the bioreactor column was 7.8 g (~10 mL volume, containing 600 mg dry weight of cells). When the reaction begins, the substrate solution entered from the bottom at a constant flow rate, made contact with the immobilized cells, and reacted. The reaction solution flowed out from the top of the column and was pumped back into the substrate storage tank.

To investigate the effect of flow rate, 25 mL of 0.8 M substrate solution in (Tris-HCl buffer, 0.1 M, pH 7.0) was pumped into the column with different flow rate (from 0.5 mL/min to 3.0 mL/min). Substrate conversion was determined at different times to determine the best flow rate.

*Cells* 

in triplicate.

strate storage tank.

Experiments were independently performed in triplicate.

*4.9. Semi-Continuous Packed-Bed Bioreactor* 

**Figure 5.** Enzymatic synthesis of NA by immobilized cells in semi-continuous packed-bed biore-**Figure 5.** Enzymatic synthesis of NA by immobilized cells in semi-continuous packed-bed bioreactor.

*4.8. Effect of Different Reaction Temperature and pH on the Activity of Free and Immobilized* 

To determine the effect of temperature, the activity of free cells (1 gdcw/L) or immobilized cells (containing 1 gdcw/L free cells) was assayed in Tris-HCl buffers (100 mM, pH 7.0) at various temperatures (20–70 °C). The relative activity referred to the ratio of the enzyme activity at different temperatures to their respective highest activity (free cell: 3076 U/gdcw, immobilized cell: 2462 U/gdcw). Experiments were independently performed

To determine the effect of pH value, the activity of free cells (1 gdcw/L) or immobilized cells (containing 1 gdcw/L free cells) was assayed at 30 °C in various buffers over a pH range of 3.0~11.0 (pH 3.0~6.0, citrate–citric acid buffer; pH 6.0~7.0, PBS buffer; pH 7.0~9.0, Tris-HCl buffer; pH 9.0~11.0, Gly-NaOH buffer). The relative activity was expressed as a percentage of their respective maximum activity of free or immobilized cells.

The column bioreactor used in this experiment (Figure 5) was made by Chengxin Glass Technology Co., Ltd. (Shanghai, China). It was a jacketed glass column reactor, and the temperature of the bioreactor was controlled by an external circulating water bath. The height of column was 100 mm, and the inner diameter was 17 mm. The effective volume of the column was approximately 20 mL. There was a round sand core at the bottom of column to prevent the beads from leaking. The wet weight of immobilized cells packed into the bioreactor column was 7.8 g (~10 mL volume, containing 600 mg dry weight of cells). When the reaction begins, the substrate solution entered from the bottom at a constant flow rate, made contact with the immobilized cells, and reacted. The reaction solution flowed out from the top of the column and was pumped back into the sub-

To investigate the effect of flow rate, 25 mL of 0.8 M substrate solution in (Tris-HCl buffer, 0.1 M, pH 7.0) was pumped into the column with different flow rate (from 0.5 mL/min to 3.0 mL/min). Substrate conversion was determined at different times to de-To investigate the effect of substrate concentration, 25 mL of different concentrations of substrate (0.2 M, 0.5 M, 0.8 M, 1.0 M, 1.25 M) solutions were pumped into the column at 2.0 mL/min, and the conversion of the substrate was monitored at different time to determine the optimal substrate concentration.

termine the best flow rate. To study the operational stability of immobilized cells in sPBR, 25 mL of 0.8 M substrate solution (Tris-HCl buffer, 0.1 M, pH 7.0, with or without 30 mM CaCl2) were pumped into the column with a flow rate of 2 mL/min, and the conversion of the substrate was monitored by HPLC. When the reaction for each batch finished, the reaction solution was stored in a collection tank, and 25 mL of fresh substrate solution (0.8 M) was added into substrate storage tank to operate a new batch [28]. The batch reaction was repeated until the conversion at the same time decreased significantly. When the batch reaction finished, the immobilized cells were washed with 0.1 M Tris-HCl buffer (25 mL × 3, pH 7.0). The collected washing liquid was centrifuged (10,000 rpm, 5 min) to obtain the clear supernatant. The supernatant and the accumulated reaction solutions for each batch were combined together for the preparation of NA.

#### *4.10. Preparation of NA*

actor.

The combined solutions mentioned in Section 4.9 were heated at 70 ◦C for 30 min, and then they were centrifuged for 30 min at 10,000 rpm. The precipitate (mainly broken immobilized cells) was washed with normal saline (20 mL × 2) to dissolve NA absorbed in the precipitate. Finally, the supernatant and washing solution were collected, and concentrated hydrochloric acid was dropped into the collected solution at pH 3.7 (NA isoelectric points). After all the crystals were precipitated, they were heated to 70 ◦C, and the obtained solution was filtered while it was hot by a Büchner funnel to remove insoluble impurities. The filtrate was placed at room temperature for natural cooling for 30 min, and then it was placed in a refrigerator at 4 ◦C for low temperature recrystallization. The crystals were obtained by air pump filtration with a Büchner funnel. The filtrated liquid was concentrated by rotary evaporation, and the above operation of recrystallization was repeated to further obtain more NA and to increase the total yield of NA [32].

The NA product (see Figure S2a for its morphology) was analyzed qualitatively and quantitatively. Its melting point was determined by melting point analyzer (mp. 238.7–238.9 ◦C, ref. 236–239 ◦C), and its purity was analyzed by HPLC (Figure S2b). The chemical structure was determined by nuclear magnetic resonance (NMR) <sup>1</sup>H NMR

(501 MHz, DMSO-d6): δ 13.49 (s, 1H), 9.27–9.06 (m, 1H), 8.83 (dd, J = 4.8, 1.7 Hz, 1H), 8.32 (dt, J = 7.9, 2.0 Hz, 1H), 7.58 (ddd, J = 7.9, 4.8, 0.9 Hz, 1H) (Figure S2c) and Fourier infrared spectroscopy (Figure S2d).

#### **5. Conclusions**

A highly efficient biocatalytic process for NA production from 3-cyanopyridine by immobilized whole cells of recombinant strain *E. coli* pRSF-AfNit2 in sPBR was developed. The immobilized cells prepared by SA-GA/PEI method showed excellent activity recovery, improved stability, and the highest mechanical strength among the tested ones. The immobilized cells were used to catalyze the hydrolysis reaction of 3-cyanopyridine in sPBR. After optimization, the space–time yield of the reaction reached 1576 g/(L·d) during stable operation. The sPBR was repeatedly operated for 41 batches, keeping 100% conversion in the presence of 30 mM CaCl2, resulting in 95 g of NA (90% yield). It can be estimated that producing 1 ton of NA would require an immobilized biocatalyst containing only 6 kg of dry weight cells, which fufill the needs of practical applications. In summary, the developed method exhibits good application potential for the biosynthesis of NA.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/catal13020371/s1, Figure S1: SDS-PAGE of recombinant AfNit from *E. coli* pET21a-AfNit and *E. coli* pRSF-AfNit2; Figure S2: Characterization spectrum and picture of prepared nicotinic acid.

**Author Contributions:** Conceptualization, Y.X.; methodology, B.-D.M. and X.-J.L.; validation, X.-J.L. and Y.X.; formal analysis, X.-J.L.; data curation, X.-J.L. and X.-M.W.; writing—original draft preparation, X.-J.L.; writing—review and editing, X.-M.W., B.-D.M. and Y.X.; funding acquisition, Y.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Project of Leading Talents in Shandong Taishan Industry (Grant No. LJNY202019).

**Data Availability Statement:** Data are contained within the article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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