**1. Introduction**

Nicotinic acid (NA) and nicotinamide are two main forms of vitamin B3, and they are collectively referred to as vitamin PP, and they are also known as anti-sculpturial factors. Named 3-pyridinic acid in the chemistry of NA, commonly known as Nicotinic acid or vitamin B3, is an indispensable nutrient component in the human body [1,2]. NA, usually in the form of coenzymes (coenzyme I and coenzyme II) in the human body, participate in the oxidative reactions in the body, thereby promoting the metabolic activity of the human body, maintaining the normal function of the human organs [3].

Traditional chemical synthetic methods of NA typically require harsh conditions, such as strong alkali, high temperatures, etc., which not only increase the process costs, but also produce certain pollution to the environment. Therefore, biocatalytic methods have been increasingly applied to NA synthesis [4–9]. There are two main routes (Scheme 1) for the biocatalytic synthesis of NA. One is the direct hydrolysis of 3-cyanopyridine to NA by nitrilase, releasing the ammonia molecule at the same time. So far, there were many reports on the synthesis of NA by wild nitrile hydrolase from different sources [10–13]. Mathew, C.D. et al. reported the production of NA from 3-cyanopyridine by resting whole cells of *Rhodococcus rhodochrous* J1, and the highest yield was achieved, being 172 mg of NA per 1.0 mL of reaction mixture containing 2.89 mg (dry weight) of cells in 26 h according to feed batch addition of substrate [8]. Another route is that the 3-cyanopyridine is hydrolyzed by nitrile hydratase to nicotinamide, which is then hydrolyzed by amidase to NA and ammonia. Cantarella, L. et al. reported a cascade bioconversion of 3-cyanopyridine to NA using a nitrile hydratase–amidase containing resting cells of *Microbacterium imperiale*

**Citation:** Liu, X.-J.; Ma, B.-D.; Wu, X.-M.; Xu, Y. Highly Efficient Biosynthesis of Nicotinic Acid by Immobilized Whole Cells of *E. coli* Expressing Nitrilase in Semi-Continuous Packed-Bed Bioreactor. *Catalysts* **2023**, *13*, 371. https://doi.org/10.3390/ catal13020371

Academic Editors: Zhilong Wang and Tao Pan

Received: 27 December 2022 Revised: 4 February 2023 Accepted: 6 February 2023 Published: 8 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

CBS 498-74 in an ultrafiltration-membrane reactor, operated in either batch or continuous mode [9]. It is worth mentioning that the Swiss company Lonza commecialized the bioconversion of 3-cyanopyridine to nicotinamide by immobilized cells of *Rhodococcus rhodochrous* J1 at >10,000 t/year scale [14]. for the hydrolysis of 3-cyanopyridine to nicotinic acid, cell immobilization, and the development of an efficient process in a semi-continuous packed-bed bioreactor (sPBR). Finally, the preparation of NA by an immobilized whole cell of nitrilase at hectogram scale in sPBR was firstly reported.

Therefore, the development of a highly efficient bioprocess by high active and stable nitrilase under high substrate concentration in a suitable kind of bioreactor will facilitate the practical application process. The current research was mainly focused on the construction of recombinant *E. coli* pRSF-AfNit2 capable of two-site expression of nitrilase

route is that the 3-cyanopyridine is hydrolyzed by nitrile hydratase to nicotinamide, which is then hydrolyzed by amidase to NA and ammonia. Cantarella, L. et al. reported a cascade bioconversion of 3-cyanopyridine to NA using a nitrile hydratase–amidase containing resting cells of *Microbacterium imperiale* CBS 498-74 in an ultrafiltration-membrane reactor, operated in either batch or continuous mode [9]. It is worth mentioning that the Swiss company Lonza commecialized the bioconversion of 3-cyanopyridine to nicotinamide by immobilized cells of *Rhodococcus rhodochrous* J1 at >10,000 t/year scale [14].

Although the production of NA by recombinant nitrilases were reported, there were several shortcomings, which restricted the practical application of nitrilase, such as low substrate concentration, low catalytic efficiency, and low stability of the enzyme [15,16]. In addition, most of such reaction was carried out in a batch reactor [17–19]. There were few reports on using packed-bed bioreactor for the production of NA by recombinant nitrilase. Therefore, we constructed the recombinant nitrile with dual-site expression. In order to achieve the industrial production of niacin, cell immobilization of recombinant bacteria was studied and constructed a semi-continuous packed-bed bioreactor (sPBR). The vector pRSFDuet-1 contains two multiple cloning sites, and each of them is preceded by a T7 promoter and ribosome binding site. There were several reports on the successful co-expression of two enzymes [20–22]. The use of pRSFDuet as recombinant

*Catalysts* **2022**, *12*, x 2 of 12

plasmid may improve the expression level of nitrilase in *E. coli*.

**Scheme 1.** Enzymatic synthesis of nicotinic acid via nitrilase or nitrile hydratase–amidase route. **Scheme 1.** Enzymatic synthesis of nicotinic acid via nitrilase or nitrile hydratase–amidase route.

**2. Results**  *2.1. Expression of Recombinant Bacteria*  After the strains of *E. coli* pRSF-AfNit2 were constructed, the strains of *E. coli* pRSF-AfNit2 and *E. coli* pET21a-AfNit were cultured [23,24]. SDS-PAGE was conducted in order to verify the expression level of nitrile hydrolase from different recombinant strain *E. coli*. The size of the band was similar to the target band and the theoretical values were consistent. It can be seen, from Figure S1, that the strains of *E. coli* pRSF-AfNit2 had Although the production of NA by recombinant nitrilases were reported, there were several shortcomings, which restricted the practical application of nitrilase, such as low substrate concentration, low catalytic efficiency, and low stability of the enzyme [15,16]. In addition, most of such reaction was carried out in a batch reactor [17–19]. There were few reports on using packed-bed bioreactor for the production of NA by recombinant nitrilase. Therefore, we constructed the recombinant nitrile with dual-site expression. In order to achieve the industrial production of niacin, cell immobilization of recombinant bacteria was studied and constructed a semi-continuous packed-bed bioreactor (sPBR).

higher expression of soluble nitrilase. Under the condition of optimal flask level, the cell amount and enzyme activitiy of *E. coli* pRSF-AfNit2 reached 4.8 gdcw/L and 3218 U/L, respectively (the enzyme activity was determined according to the hydroysis of 3-cyanopyridine at 30 °C and pH 7.0, see Section 4.4 for details). The vector pRSFDuet-1 contains two multiple cloning sites, and each of them is preceded by a T7 promoter and ribosome binding site. There were several reports on the successful co-expression of two enzymes [20–22]. The use of pRSFDuet as recombinant plasmid may improve the expression level of nitrilase in *E. coli*.

Therefore, the development of a highly efficient bioprocess by high active and stable nitrilase under high substrate concentration in a suitable kind of bioreactor will facilitate the practical application process. The current research was mainly focused on the construction of recombinant *E. coli* pRSF-AfNit2 capable of two-site expression of nitrilase for the hydrolysis of 3-cyanopyridine to nicotinic acid, cell immobilization, and the development of an efficient process in a semi-continuous packed-bed bioreactor (sPBR). Finally, the preparation of NA by an immobilized whole cell of nitrilase at hectogram scale in sPBR was firstly reported.

#### **2. Results**

#### *2.1. Expression of Recombinant Bacteria*

After the strains of *E. coli* pRSF-AfNit2 were constructed, the strains of *E. coli* pRSF-AfNit2 and *E. coli* pET21a-AfNit were cultured [23,24]. SDS-PAGE was conducted in order to verify the expression level of nitrile hydrolase from different recombinant strain *E. coli*. The size of the band was similar to the target band and the theoretical values were consistent. It can be seen, from Figure S1, that the strains of *E. coli* pRSF-AfNit2 had higher expression of soluble nitrilase. Under the condition of optimal flask level, the cell amount and enzyme activitiy of *E. coli* pRSF-AfNit2 reached 4.8 gdcw/L and 3218 U/L, respectively (the enzyme activity was determined according to the hydroysis of 3-cyanopyridine at 30 ◦C and pH 7.0, see Section 4.4 for details).

#### *2.2. Immobilization of Whole Cells of E. coli pRSF-AfNit2 by Different Entrapment Method*

This experiment was mainly based on the sodium alginate (SA) gel carrier, selecting other different complexes to further combine with calcium alginate to immobilize the cells [25]. The beads prepared using different immobilization methods were between

3~5 mm in size. Immobilized cells were prepared by different methods, and their enzyme activity and mechanical strength were compared to judge which immobilization method was better (Table 1).


**Table 1.** Effect of different cellular immobilization methods on stability of nitrile hydrolase.

<sup>a</sup> The mechanical strength is expressed by the time (*t*) required for the breakage rate of the immobilized pellets to reach 25%. One star (\*): *t* < 30 min; two stars (\*\*): 30 min < *t* < 60 min; three stars (\*\*\*): *t* > 60 min.

It can be seen that, because the polyvinyl alcohol (PVA) solution was too viscous and the solubility was too poor, it was not easy to prepare the polyvinyl alcohol–calcium alginate (PVA-SA) immobilized cells, and other methods were relatively easy to prepare; the mechanical strength test results showed that the calcium alginate, Fe3O4-calcium alginate pellets, and activated carbon–calcium alginate pellets had poor mechanical strength and were easy to swell and break. Calcium alginate–glutaraldehyde (SA–GA) and calcium alginate–glutaraldehyde-polyethyleneimine (SA–GA/PEI) had moderate to high mechnical strength, with both having over 90% activity recovery. Therefore, the best method was the SA–GA/PEI method [25].

## *2.3. Enzymatic Properties of SA-GA/PEI Immobilized Cells and Free Cells*

Temperature has always been an important factor affecting an enzymatic reaction. Too high of a temperature will cause an enzyme to denature and inactivate; if the temperature is too low, the catalytic reaction rate will be slower, and the enzyme activity will not be high. This experiment compared the enzyme activity of free cells and immobilized cells at different temperatures to determine the optimal temperature for free cells and immobilized cells. It can be seen from Figure 1a that the optimum temperature for free cells was 55 ◦C, and the optimum temperature for immobilized cells was 60 ◦C. When the temperature was greater than the optimum temperature, as the temperature rised, the enzyme activity of both free cells and immobilized cells all have decreased, but the enzyme activity of immobilized cells was higher than that of free cells at the same temperature. For example, the relative enzyme activity of free cells was only 18% at 70 ◦C, while the relative enzyme activity of immobilized cells was 50%. This showed that its heat resistance was improved, and the temperature range has been broadened [26]. *Catalysts* **2022**, *12*, x 4 of 12

**Figure 1.** Effect of reaction temperature (**a**) and pH (**b**) on the activity of free and SA–GA/PEI immobilized cells.

In practical industrial applications, an important evaluation criterion for the application value of a catalyst was its storage stability. In this experiment, the storage stability of free cells and immobilized cells was studied, and the results were shown in Figure 2. The residual enzyme activity of free cells was only about 15% after being stored in a refrigerator at 4 °C after 55 days. However, it was still more than 80% for immobilized cells after nearly two months, which showed a much better storage stability of immobilized

**Figure 1.** Effect of reaction temperature (**a**) and pH (**b**) on the activity of free and SA–GA/PEI im-

*2.5. Research on Semi-Continuous Synthesis of NA in a Packed-Bed Reactor with Immobilized* 

The packed-bed bioreactor can also be called a fixed-bed bioreactor, because the biocatalyst in the reactor was always in a static state, and the reaction happened when substrate solution flowed through the biocatalyst [28]. Since different methods of immobilizing cells produced different cell shapes, the catalyst in the reactor can be filled in different forms. When there existed product inhibition, the use of this type of reactor had certain advantages. Since the catalyst in the reactor is always static, the fluid flow pattern can be regarded as a plug flow. Compared with the continuous stirred tank bioreactor

**Figure 2.** Storage stability of free and immobilized cells.

immobilized cell

free cell

0 20 40 60

**Time (days)**

mobilized cells.

cells than free cells [27].

*Cells* 

0

20

40

60

**Residual activity (%)**

80

100

120

**Temperature (℃)**

free cell immobilized

**Relative activity (%)**

In the enzymatic reaction, only when the catalyst exists in an appropriate pH buffer system can the enzyme achieve higher catalytic activity. As shown in Figure 1b, the optimal pH for free cells was 8.0, while it was 7.0 for immobilized cells. The activity of immoilized cells decreased less than that of free cells with the increase in pH value under alkaline conditions, which indicated that immobilized cells had a higher pH tolerance than free cells in the alkaline range [26]. **Figure 1.** Effect of reaction temperature (**a**) and pH (**b**) on the activity of free and SA–GA/PEI immobilized cells.

free cell immobilized cell

**a b** 

0 2 4 6 8 10 1

**pH value**

**Relative activity (%)**

#### *2.4. Storage Stability of Immobilized Cells 2.4. Storage Stability of Immobilized Cells*  In practical industrial applications, an important evaluation criterion for the appli-

*Catalysts* **2022**, *12*, x 4 of 12

In practical industrial applications, an important evaluation criterion for the application value of a catalyst was its storage stability. In this experiment, the storage stability of free cells and immobilized cells was studied, and the results were shown in Figure 2. The residual enzyme activity of free cells was only about 15% after being stored in a refrigerator at 4 ◦C after 55 days. However, it was still more than 80% for immobilized cells after nearly two months, which showed a much better storage stability of immobilized cells than free cells [27]. cation value of a catalyst was its storage stability. In this experiment, the storage stability of free cells and immobilized cells was studied, and the results were shown in Figure 2. The residual enzyme activity of free cells was only about 15% after being stored in a refrigerator at 4 °C after 55 days. However, it was still more than 80% for immobilized cells after nearly two months, which showed a much better storage stability of immobilized cells than free cells [27].

**Figure 2.** Storage stability of free and immobilized cells.

#### **Figure 2.** Storage stability of free and immobilized cells. *2.5. Research on Semi-Continuous Synthesis of NA in a Packed-Bed Reactor with Immobilized 2.5. Research on Semi-Continuous Synthesis of NA in a Packed-Bed Reactor with Immobilized Cells*

*Cells*  The packed-bed bioreactor can also be called a fixed-bed bioreactor, because the biocatalyst in the reactor was always in a static state, and the reaction happened when substrate solution flowed through the biocatalyst [28]. Since different methods of immobilizing cells produced different cell shapes, the catalyst in the reactor can be filled in different forms. When there existed product inhibition, the use of this type of reactor had certain advantages. Since the catalyst in the reactor is always static, the fluid flow pattern can be regarded as a plug flow. Compared with the continuous stirred tank bioreactor The packed-bed bioreactor can also be called a fixed-bed bioreactor, because the biocatalyst in the reactor was always in a static state, and the reaction happened when substrate solution flowed through the biocatalyst [28]. Since different methods of immobilizing cells produced different cell shapes, the catalyst in the reactor can be filled in different forms. When there existed product inhibition, the use of this type of reactor had certain advantages. Since the catalyst in the reactor is always static, the fluid flow pattern can be regarded as a plug flow. Compared with the continuous stirred tank bioreactor (CSTR), the shear force was smaller, which facilitated keeping the stability of the biocatalyst. Up to now, only scarce research was focused on the biosynthesis of NA in packed-bed bioreactors. In the current research, we studied the effect of flow rate of substrate solution and substrate concentration on the efficiency of catalytic reaction.

It can be seen, from Figure 3a, that the reaction rate increased with the increase in the flow rate when it was increased from 0.5 to 2.0 mL/min. Further increasing the flow rate from 2.0 to 3.0 mL/min, it was maintained in a stable state, which means the influence of external diffusion could be negligible. Therefore, the optimal flow rate was 2.0 mL/min.

After determining the optimal flow rate, the substrate concentration was investigated. It can be seen from Figure 3b that as the substrate concentration was increased, and the conversion rate was decreased at the same time. When the substrate concentration was greater than 0.8 M, the time for full conversion of the substrate was gradually increased. The space–time yield at different substrate concentrations was shown in Table 2. When the substrate concentration was 0.8 M, the space–time yield reached the highest value of 1576 g/(L·d). Therefore, the optimal substrate concentration was 0.8 M.

**Figure 3.** Study on different parameters on the conversion of NA in semi-continuous packed-bed bioreactor. (**a**) Flow rate (after operation for 90 min); (**b**) substrate concentration.

(CSTR), the shear force was smaller, which facilitated keeping the stability of the biocatalyst. Up to now, only scarce research was focused on the biosynthesis of NA in packed-bed bioreactors. In the current research, we studied the effect of flow rate of substrate solution and substrate concentration on the efficiency of catalytic reaction.

It can be seen, from Figure 3a, that the reaction rate increased with the increase in the flow rate when it was increased from 0.5 to 2.0 mL/min. Further increasing the flow rate from 2.0 to 3.0 mL/min, it was maintained in a stable state, which means the influence of external diffusion could be negligible. Therefore, the optimal flow rate was 2.0 mL/min.



#### increased. The space–time yield at different substrate concentrations was shown in Table *2.6. Reusability of Immobilized Cells in the Bioreactor*

2. When the substrate concentration was 0.8 M, the space–time yield reached the highest value of 1576 g/(L·d). Therefore, the optimal substrate concentration was 0.8 M. **Table 2.** The space–time yield at different substrate concentrations. **Substrate Concentration (M) 0.2 0.5 0.8 1.0 1.25**  Full conversion time (h) 1.5 1.5 1.5 3.0 3.0 According to the optimal substrate flow rate and substrate concentration, repeated batch experiments were performed in the reactor to verify the operational stability of the immobilized cells. The effect of calcium ions on the stability of immobilized cells was also investigated (Figure 4). When no calcium ions were added to the substrate solution, the immobilized cells began to swell and broke after only five batches, resulting in a sharp decrease in the conversion. However, the immobilized cells with CaCl<sup>2</sup> (30 mM) in solution can be reused for at least 41 batches, keeping 100% of conversion. After 41 batches, the immobilized cells started to swell and partially break, leading to a decrease in conversion.

tion was greater than 0.8 M, the time for full conversion of the substrate was gradually

When the reaction finished, the immobilized cells in the bioreactor were washed. The washing liquid was combined with all the product solutions for each batch, and NA was furthur seperated and purified. Finally, 95 g of pure NA were obtained from 43 batches, corresponding to 90% of isolated yield [28]. Since the immobilized cells packed in sPBR contained around 0.6 g dry cell weight of cells, it can be estimated that preparing 1 ton of NA requires an immobilized biocatalyst containing only 6 kg of dry weight cells.

Space–time yield (g/(L·d)) 393 ± 1.0 984 ± 0.5 1576 ± 0.7 981 ± 0.5 1227 ± 0.5

*2.6. Reusability of Immobilized Cells in the Bioreactor* 

**Figure 4.** Repeated use of immobilized cells in sPBR for the production of nicotinic acid.

#### **3. Discussion**

version.

**Figure 4.** Repeated use of immobilized cells in sPBR for the production of nicotinic acid. When the reaction finished, the immobilized cells in the bioreactor were washed. The washing liquid was combined with all the product solutions for each batch, and NA In this paper, by molecular cloning, the nitrilase gene derived from *Acidovorax facilis* was ligated to a vector with two independent multiple cloning sites for two-site expression, and the nitrilase-producing recombinant strain *E. coli* pRSF-AfNit2 showed higher soluble expression of nitrilase than that of *E. coli* pET21a-AfNit2.

According to the optimal substrate flow rate and substrate concentration, repeated batch experiments were performed in the reactor to verify the operational stability of the immobilized cells. The effect of calcium ions on the stability of immobilized cells was also investigated (Figure 4). When no calcium ions were added to the substrate solution, the immobilized cells began to swell and broke after only five batches, resulting in a sharp decrease in the conversion. However, the immobilized cells with CaCl2 (30 mM) in solution can be reused for at least 41 batches, keeping 100% of conversion. After 41 batches, the immobilized cells started to swell and partially break, leading to a decrease in con-

was furthur seperated and purified. Finally, 95 g of pure NA were obtained from 43 batches, corresponding to 90% of isolated yield [28]. Since the immobilized cells packed in sPBR contained around 0.6 g dry cell weight of cells, it can be estimated that preparing 1 ton of NA requires an immobilized biocatalyst containing only 6 kg of dry weight cells. **3. Discussion**  In this paper, by molecular cloning, the nitrilase gene derived from *Acidovorax facilis* was ligated to a vector with two independent multiple cloning sites for two-site expression, and the nitrilase-producing recombinant strain *E. coli* pRSF-AfNit2 showed higher soluble expression of nitrilase than that of *E. coli* pET21a-AfNit2. In order to further improve the stability of nitrilase, the whole cell immobilization of nitrilase was systematically studied, and the immobilized cells were used to catalyze the semi-continuous hydrolysis reaction of 3-cyanopyridine in a packed bed column bioreactor. The results of the study were the following: the best immobilization method was the calcium alginate–glutaraldehyde–polyethyleneimine cross-linking method, and the enzyme activity of immobilized cells was 95% of free cells, and the residual enzyme activity of immobilized cells was still more than 80% after nearly two months storage at 4 In order to further improve the stability of nitrilase, the whole cell immobilization of nitrilase was systematically studied, and the immobilized cells were used to catalyze the semi-continuous hydrolysis reaction of 3-cyanopyridine in a packed bed column bioreactor. The results of the study were the following: the best immobilization method was the calcium alginate–glutaraldehyde–polyethyleneimine cross-linking method, and the enzyme activity of immobilized cells was 95% of free cells, and the residual enzyme activity of immobilized cells was still more than 80% after nearly two months storage at 4 ◦C. In the semi-continuous packed-bed column reactor, it was found that both the substrate solution flow rate and the substrate concentration affected the catalytic efficiency. Regarding this, the catalytic efficiency reached its highest when the substrate flow rate was 2.0 mL/min and the substrate concentration was 0.8 M. The space–time yield of the reactor during stable operation can reach 1576 g/(L·d). When the substrate solution contains 30 mM CaCl2, the immobilized cells can be reused for at least 41 batches, and the conversion was still kept at 100%. Finally, 95 g of NA were obtained in 90% isolated yield through seperation and purification. It can be estimated that producing 1 ton of NA would require immobilized biocatalyst containing only 6 kg of dry weight cells, which fufill the needs of pactical applications.

#### °C. In the semi-continuous packed-bed column reactor, it was found that both the sub-**4. Materials and Methods**

#### strate solution flow rate and the substrate concentration affected the catalytic efficiency. *4.1. Chemicals, Plasmids and Strains*

Regarding this, the catalytic efficiency reached its highest when the substrate flow rate was 2.0 mL/min and the substrate concentration was 0.8 M. The space–time yield of the 3-Cyanopridine, nicotinic acid, protein marker, and soluble starch were from the Aladdin reagent company (Shanghai, China); NaCl and yeast extract powder were from sinopharm chemical reagent Co., Ltd. (Shanghai, China); anhydrous ethanol, CaCl2, K2HPO4, KH2PO<sup>4</sup> and MgSO4·7H2O were purchased from Shanghai Taitan Technology Co., Ltd. (Shanghai, China); kanamycin was from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China).

Plasmids pET-21a(+) (Novagen, Merck, Darmstadt, Germany) and pRSFDuet-1 (Novagen, Merck, Darmstadt, Germany) were preserved in the Biocatalysis and Biopharmaceutical Laboratory (Shanghai Institute of Technology, Shanghai, China). Restriction endonucleases (*Nde*I, *Xho*I, *Pst*I, *Nco*I) were purchased from Takara Biotechnology (Dalian, China). *E. coli* BL21 (DE3) and *E. coli* DH5α were purchased from Friction Biological Engineering (Shanghai) Co., Ltd. (Shanghai, China). The recombinant plasmid pET21a-AfNit was constructed in our previous work [23].

## *4.2. Construction of Recombinant E. coli Strains Expressing Nitrilase*

The nitrilase gene (FJ851547) [29] from *Acidovorax facilis* 72W (AfNit) was synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China). AfNit was ligated into pET21a via *Nde*I/*Xho*I restriction sites to generate expression plasmid pET21a-AfNit. The pET21a-AfNit was transformed into *E. coli* BL21 (DE3) to construct the recombinant strain *E. coli* pET21a-AfNit. For the construction of *E. coli* pRSF-AfNit2, the AfNit gene was ligated into the first multi clone site (MCS1) via *Pst*I/*Nco*I restriction sites, and the second MCS (MCS2) was ligated via the *Nde*I/*Xho*I restriction sites to generate the expression plasmid pRSF-AfNit2. The pRSF-AfNit2 was transformed into *E. coli* BL21 (DE3) to construct the recombinant strain *E. coli* pRSF-AfNit2. The construction of recombinant pRSFDuet-1 plasmids and their transformation into *E. coli* were performed according to standard protocols.
