Table of Contents  
Year : 2020  |  Volume : 19  |  Issue : 1  |  Page : 25-28

Gene amplification and overexpression of Bacillus subtilis L-asparaginase

1 Department of Microbial Genetics, National Research Centre, Cairo, Egypt
2 Department of Microbial Chemistry, National Research Centre, Cairo, Egypt

Date of Submission30-Jul-2019
Date of Decision21-Aug-2019
Date of Acceptance29-Aug-2019
Date of Web Publication24-Mar-2020

Correspondence Address:
Wafaa K Hegazy
Department of Microbial Genetics, National Research Centre, Cairo
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/epj.epj_38_19

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Background and objectives The aim of this paper was to focus on improving production level of l-asparaginase by developing recombinant strains.
Materials and methods Asp gene was cloned into the shuttle vector pNW33N, and the recombinant plasmid was used to transform Bacillus subtilis protoplast. Asp gene was expressed into both Escherichia coli and B. subtilis. Enzyme activity of the recombinant strains was measured as compared with wild-type strains.
Results Asp gene was successfully subcloned into the recombinant plasmid named S-ASP-NRC-27. The gene was expressed efficiently in both host strains: E. coli and B. subtilis. The enzyme activity of the transformants was increased up to threefold under control of Lac Z promoter.
Conclusion From the previous results, the shuttle vector pNW33N seemed to be a very useful plasmid as a cloning vector in a wide variety of the genus Bacillus. Both of Asp gene amplification and the control of Lac Z promoter had direct effects on producing the super Asp-expression strains.

Keywords: antitumor, Bacillus subtilis, cloning, l-asparaginase

How to cite this article:
Hegazy WK, Abdel-Salam MS, Moharam ME. Gene amplification and overexpression of Bacillus subtilis L-asparaginase. Egypt Pharmaceut J 2020;19:25-8

How to cite this URL:
Hegazy WK, Abdel-Salam MS, Moharam ME. Gene amplification and overexpression of Bacillus subtilis L-asparaginase. Egypt Pharmaceut J [serial online] 2020 [cited 2023 Feb 9];19:25-8. Available from:

  Introduction Top

Asparaginase catalyzes the hydrolysis of l-asparagine to l-aspartic acid and ammonia. Bacterial l-asparaginases are classified into subtypes I and II, which are defined by their intracellular or extracellular localization [1]. The application of asparaginase I from Bacillus subtilis was extensively used in food processing industry, whereas type II l-asparaginases, in particular, had tumor inhibitory activity [2],[3],[4]. Asparaginase gene from different bacterial sources, such as Helicobacter pylori [5], Erwinia chrysanthemi [6], and B. subtilis [7], have been cloned and expressed into different bacterial hosts.

There are several methods to introduce plasmid DNA into Bacillus spp., such as transformation of competent cells, polyethylene glycol (PEG)-mediated transformation of protoplasts, electroporation, transduction, and conjugation [8]. Preliminary studies have reported that plasmid transformation procedures effective with Escherichia coli are unsatisfactory for transformation in B. subtilis. Plasmid transformation of protoplasts appeared to be an attractive approach as it does not require cell competence for DNA uptake [9]. Many reports indicated that protoplast transformation is a useful method for gene cloning of B. subtilis [10].

The objective of our study was to improve l-asparaginase production of indigenous B. subtilis strain through increasing the corresponding gene copies.

  Materials and methods Top

Bacterial strains, plasmids, and medium

Bacterial strains and plasmids used in the present study are presented in [Table 1]. B. subtilis Alazhar, local isolate, and E. coli JM 107 were used as the cloning and expression hosts for l-asparaginase gene in transformation trials.
Table 1 Bacterial strains and plasmids used in the present study

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ASP-NRC-1 is a recombinant plasmid harboring B. subtilis l-asparaginase gene [11]. It was used to subclone the l-asparaginase gene.

The cloning plasmid pNW33N is a shuttle vector that shows stable replication in B. subtilis, Geobacillus stearothermophilus, and E. coli. It contains a large multiple cloning site and encodes a chloramphenicol acetyltransferase resistant gene (Cmr). It was used for cloning Asp gene and transformation of both E. coli and B. subtilis Alazhar strains.

All molecular biology manipulations were performed according to standard protocols [12] and kit supplier’s instructions, unless specified. DNA bands were purified from the gel using MEGAquick-spin T.


Luria-Bertani (LB) medium [13] was used for bacterial growth. Regeneration medium was used for B. subtilis protoplast transformation. It contains 5 g glucose, 1.0 g NH4PO4, 3.5 g K2HPO4, 1.5 g KH2PO4, 0.33 mol/l sodium succinate, 5 g gelatin, 4.07 g MgCl2. 6H2O, 5 g casamino acids, and 20 g agar/l [14].

E. coli and B. subtilis transformants were selected on media supplemented with the antibiotic chloramphenicol (Cm) (25 μg/ml and 5 μg/ml, respectively).

Construction of recombinant plasmid

The two plasmids, ASP-NRC-1 and pNW33N, were isolated, purified, and digested with Bam HI and Xba I (the two restriction enzymes flanking the l-asparaginase gene in ASP-NRC-1 plasmid). Both digested fragments were purified from agarose gel electrophoreses and ligated. The resulting recombinant plasmid designated as S-ASP-NRC-27 was transformed to both B. subtilis protoplast and E. coli host strains.

Escherichia coli transformation

The genetic transformation procedure of competent cells and the selection of recombinants were performed according to Sambrock et al. [12].

A volume of 5 μl of recombinant DNA (S-ASP-NRC-27, harboring Asp gene) was used to transform E. coli JM 107 competent cells using heat shock technique. Transformants (cmr) were selected on LB agar medium containing chloramphenicol (25 μg/ml).

The recombinant plasmid from E. coli JM 107 transformants was isolated using DNA-spin plasmid DNA purification Kit (INtRON Biotechnology, Korea) and characterized by its digestion with different restriction enzymes.

Bacillus subtilis protoplast preparation

B. subtilis protoplast induction was performed according to Akamatsu and Sekiguchi [14] method with some modifications.

B. subtilis Alazhar was grown in LB medium with shaking (120 rpm) up to OD570=0.4, and then 15 ml was centrifuged (6000 rpm for 15 min) and suspended in 2 ml of SMM buffer (0.5 mol/l sucrose, 0.02 mol/l maleic acid, 0.02 mol MgCl2, pH 6.5). A volume of 2 mg of lysozyme (1 mg/ml) was added and incubated at 37°C for 1 h. After centrifugation (4000 rpm, 10 min), they were suspended in 2 ml of SMM buffer.

Protoplast transformation of Bacillus subtilis by constructed plasmid DNA

A volume of 1 ml of B. subtilis protoplasts was mixed with 100 μl of recombinant S-ASP-27 plasmid DNA and 3 ml of 40% PEG 4000 in SMM buffer. The mixture was kept at 0°C for 2 min and then incubated at 30°C for 2 h. The protoplast mixture was then centrifuged at 4000 rpm for 10 min and suspended in 2 ml SMM. Overall, 100 μl was added to overlay tube of regeneration medium (0.7% agar) and poured into regeneration medium supplemented with 5 μl/ml chloramphenicol and incubated at 30°C up to 3 days. The transformants (cmr) were selected, and their plasmid content was isolated.

L-asparaginase assay

l-asparaginase activity was assayed according to Wriston [15]. The reaction mixture contained 0.1 ml culture supernatant and 0.9 ml of 0.01 mol/l l-asparagine prepared in 0.05 mol/l Tris-HCl buffer, pH 8.6, and incubated for 30 min at 37°C. The reaction mixture was centrifuged at 6000g for 10 min, and the ammonia released in the supernatant was determined by Nesslerization reaction. In brief, to 0.5 ml of supernatant, 1.75 ml distilled H2O, 0.25 ml of Nessler reagent was mixed. After 10 min, absorbance at 480 nm was read with appropriate control. One enzyme unit (U) is defined as the amount of enzyme that liberates 1 µmol of ammonia per min at 37°C. Standard curve of ammonium sulfate was used for calculating ammonia concentrations.

  Results Top

Construction of recombinant plasmid

Asp gene in ASP-NRC-1plasmid is located on Bam HI/Xba I restriction fragment; the purified Bam HI/Xba I fragment, which contains Asp gene, was successfully ligated to Bam HI/Xba I-digested pNW33N plasmid. The two ligated DNA fragments formed a recombinant plasmid named S-ASP-NRC-27 ([Figure 1]). It was used to transform each of E. coli JM 107 and B. subtilis strains.
Figure 1 S-ASP-27 plasmid.

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Bacterial transformation

Following transformation trial of E. coli and B. subtilis using pS-ASP-NRC-27 plasmid, E. coli transformants were selected on LB supplemented with 25 μg/ml chloramphenicol and B. subtilis transformants on regeneration medium supplemented with 5 μl/ml chloramphenicol.

Plasmids were isolated from (cmr) transformants, and agarose gel electrophoresis pattern of some obtained plasmids has been shown in [Figure 2]. The restriction pattern of the digested plasmid with Bam HI+Xba I (lane 1), Bam HI+Pest I (lane 2), and Xba I+Pest I (lane 3) is illustrated in [Figure 3] to characterize its physical mapping. The data obtained confirmed the same expected DNA fragment lengths of [Figure 1] and that the Asp gene is under control of lac Z promoter of the recombinant plasmid.
Figure 2 Agarose gel electrophoresis of isolated S-ASP-NRC-27 plasmids from Escherichia coli and Bacillus. Subtilis transformants. Lane 1: Escherichia coli (pS-ASP-NRC-27); lane 2: Escherichia coli; lanes 3,4,5: Bacillus subtilis (pS-ASP-NRC-27) 2, 3 and 7, respectively, lane 6: Bacillus subtilis Alazhar strain.

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Figure 3 Agarose gel electrophoresis of S-ASP-NRC-27 plasmid cut with different DNA restriction enzymes. Lane M: DNA marker − Axygen DNA marker (300–10 000 bp); lane 1: S-ASP-NRC-27+BamHI+XbaI; lane 2: S-ASP-NRC-27+BamHI+PestI; lane 3: S-ASP-NRC-27+XbaI+PestI.

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Expression of Asp gene in Bacillus subtilis and Escherichia coli transformants

L-asparaginase activity of both recombinant strains, E. coli (S-ASP-NRC-27) and B. subtilis (S-ASP-NRC-27), was determined compared with their host strain: E. coli JM107 and B. subtilis. The data in [Table 2] revealed that cloned Asp gene from B. subtilis was successfully expressed in B. subtilis as well as in E. coli JM107.; E. coli transformant harboring recombinant plasmid produced threefold increase of asparaginase yield, whereas B. subtilis transformant produced 2.9-fold increase as compared with parent strain.
Table 2 L-asparaginase activity of Escherichia coli

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  Discussion Top

Cloning of Asp gene from B. subtilis strain and its nucleotide sequence was described by our team. The gene expressed efficiently in E. coli JM107, and the expression level is controlled by one of the two promoters T7and lacUV5 [11]. In the present study, we try to find an efficient strategy to transfer and express Asp gene into B. subtilis strain as well as E. coli JM107. For this purpose, two-step approach has been studied; protoplast transformation in Bacillus spp. and the usefulness of plasmid pNW33N DNA as a cloning vector in Bacillus spp. Many research studies described conditions of the plasmid transformation [10]. Transformation by plasmid DNA which is an essential step in most cloning experiments was improved by the use of protoplasts in PEG solution. However, protoplast transformation of Bacillus spp. has been reported [12].

Gene expression is dependent mainly on its promoter efficiency. In the present study, l-asparaginase-specific activity of E. coli JM 107 (pS-ASP-NRC-27) the gene was under control of lac Z promoter. These results reflect the superior efficiency of the lac Z promoter than both promoters studied before [14].

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Duval M, Suciu S, Ferster A, Rialland X, Neblen B, Lutz P et al. Comparison of Escherichia coli–asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment of Cancer—Children’s Leukemia Group phase 3. Blood 2002; 99:2734–2739.  Back to cited text no. 3
Avramis VI, Tiwari PN. Asparaginase (native ASNase or pegylated ASNase) in the treatment of acute lymphoblastic leukemia. Int J Nanomedicine 2006; 1:I241–I254.  Back to cited text no. 4
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Bron S. Plasmids. In: Harwood CR, Cutting SM (editor). Molecular biological methods for Bacillus. Chichester: John Wiley 1990. 75–174  Back to cited text no. 8
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Hegazy WK, Abdel-Salam MS, Maysa Moharam E. Biotechnological approach for production of L-asparaginase from Bacillus subtilis local isolate. EPJ 2019; (Unpublished data in EPJ). (in press).  Back to cited text no. 11
Sambrock J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual. 2nd ed. Cold Spring Harbor, New York, NY: Cold Spring Harbor Laboratory Press 1989.  Back to cited text no. 12
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  [Figure 1], [Figure 2], [Figure 3]

  [Table 1], [Table 2]


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