The modification of nisin with homocysteine thiolactone and its effect on antimicrobial activity

Abstract

The aim of the present study is to make an important contribution to the literature by focusing on the preparation of the N-homocysteine conjugate of nisin and evaluating the effect of the N-homocysteinylation reaction on its antimicriobial activity. The modification process was monitored using both acetic acid urea polyacrylamide gel electrophoresis (AAU-PAGE) and tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (tricine SDS-PAGE). The antibacterial effectiveness of modified nisin was assessed against Staphylococcus aureus ATCC 6538, Enterococcus faecium ATCC 9097, Bacillus subtilis ATCC 6633, Lactococcus lactis ssp. cremoris AÜ, Listeria monocytogenes NCTC 5348, and Escherichia coli RSKK. Optimal conditions for achieving the highest N-homocysteinylation degree (6.30%) were determined as 6 mg/mL nisin, 150 mM homocysteine thiolactone, 150 rpm shaking rate, pH of 3.0, and a reaction time of 6 h. The modified nisin obtained did not have a significant inhibitory effect on the strains tested except E. faecium. E. faecium was inhibited by the modified nisin and its antibacterial activity was determined as approximately 10% of the antibacterial activity of unmodified nisin. On the other hand, hydrolysis of nisin by trypsin and thermolysin resulted in significant specific side chain modifications induced by the homocysteine-thiolactone reaction, especially at Lys12 and Lys22. The results provide valuable insights into the potential of N-homocysteinylation to improve the antibacterial properties of nisin and also suggest that the effects of specific modifications identified during the modification process should be investigated.

Introduction

Bacteriocins that are bactericidal peptides and proteins synthesized and modified post-translationally by some bacteria can inhibit other bacteria. These show promise in the control of pathogenic bacteria due to their heat resistance, efficacy over a wide pH range, and ability to be digested by human intestinal proteases [1]. Nisin, predominantly produced by Lactococcus lactis, is a prominent commercial bacteriocin. It is a cationic peptide consisting of 34 amino acid residues and classified within the lantibiotic group due to its unusual amino acids (dehydroalanine, lanthionine, β-methyl-lanthionine, dehydrobutyrine) derived from threonine, serine, and cysteine by post-translational enzymatic changes [2, 3].

Eight native variants of nisin have been identified. Nisin A, Z, Q, and F are derived from Lactococcus lactis; nisin U and U2 originate from Streptococcus uberis, nisin P is derived from both Streptococcus suis and Streptococcus gallolyticus ssp. pasteurianus, and nisin H is produced by Streptococcus hyointestinalis [4–6].

Nisin demonstrates antibacterial properties against numerous Gram-positive bacteria such as Listeria and Staphylococcus which are food-borne pathogens while also preventing Clostridium and Bacillus bacteria from forming spores. Furthermore, it has a lengthy history of safe usage. This natural preservative has been approved by the FDA and widely adopted by the food industry, especially in products such as bakery items, eggs, dairy, vegetables, meat, and fish [7–9]. However, certain characteristics of nisin may restrict its application. It has low solubility in neutral and alkaline solutions, is unstable at physiological pH, and is rapidly degraded by proteolytic enzymes [8, 10, 11]. In addition, nisin has little or limited activity against most Gram-negative bacteria. This limited activity against most Gram-negative bacteria is attributed to their outer membrane, which prevents nisin from reaching lipid II in the inner membrane [12]. Therefore, the improvement of the mentioned properties of nisin by modifications such as chemical or biochemical is important. Modifications using protein engineering techniques can adversely impact both the efficiency of expression and purification [13]. As an alternative, nisin can be modified after expression, a process known as post-production modification. At this stage, nisin can be modified with a variety of both natural and artificial compounds to improve its characteristics.

Numerous studies have investigated the enhancement of nisin properties through structural alterations, specifically the manipulation of amino groups. However, the outcomes have been inconsistent. Some modifications enhance its antibacterial activity, while others decrease it. Conjugating nisin with thiol, treating with peroxide, PEGylation of the N-terminal amino group, and glycation with different sugars led to a reduction or complete loss of antibacterial activity [10, 14–18]. On the other hand, the antibacterial activity was preserved or enhanced by radiation-induced formation of glucose or nisin-dextran conjugates and C-terminal modifications with biotinylation or propargylamine [11, 19, 20]. The modification type and specific group incorporated into nisin are essential in determining its antimicrobial effects. Therefore, N-homocysteinylation could potentially enhance the physicochemical and biochemical properties of nisin, including its antimicrobial effectiveness. Homocysteine-thiolactone (HTL) forms amide bonds with protein-bound lysine residues via N-homocysteinylation, potentially introducing a free sulfhydryl group into the nisin molecule (Fig. 1). The reactive cyclic thioester of homocysteine can easily attach to free amino groups, leading to a change in protein function and structure [21, 22]. Moreover, modifications that generate dimers via disulfide bridging have augmented the antimicrobial efficacy of cationic peptides by 60 times when compared to their previous versions [23]. Therefore, it can be concluded that the N-homocysteinylation process has the potential to enhance the antimicrobial activity of nisin. However, the literature lacks information on the nisin conjugate formed through the N-homocysteinylation process. This study aims to produce the N-homocysteine nisin conjugate and evaluate its impact on nisin’s antibacterial activity.

Fig. 1.

N-homocysteinylation of a lysine residue bound to a protein

Materials and methods

Materials

Enterococcus faecium ATCC 9097, Lactococcus lactis ssp. cremoris AU (Ankara University), Staphylococcus aureus ATCC 6538, Escherichia coli RSKK (Refik Saydam National Type Culture Collection), Bacillus subtilis ATCC 6633, and Listeria monocytogenes NCTC 5348 from the collection of Department of Food Engineering, Nigde Omer Halisdemir University (Turkey) were used at study. High-purity nisin Z (95%, w/w) was obtained from an industrial producer (Handary SA, Brussels, Belgium).

Homocysteine thiolactone modification of nisin

The nisin concentration was used in the study kept constant concentration (6 mg/mL), which was defined in preliminary studies. The modification reaction was optimized for five factors: HTL concentration, temperature, reaction period, pH, and shaking rate.

The modification reaction was carried out as follows: nisin (6 mg/mL) dissolved in 0.1 M sodium phosphate buffer containing 4 M urea at 6 levels of pH (3.0, 4.0, 4.5, 5.0, 6.0, or 8.0). The mixture was then incubated at levels of 5 HTL concentrations (1, 10, 100, 150, 200 mM), 3 temperatures (45, 60, 80 °C), 2 mixing rates (150, 245 rpm), and 6 incubation times (2, 4, 6, 18, 24, or 48 h) levels. After the incubation period, the nisin that had been modified was separated through precipitation with trichloroacetic acid (TCA, 20%, w/v) for 30 min at 4 °C. It was then centrifuged at 1955 g for 40 min at 4 °C. The same procedure, but without HTL, was used for the control sample. The modified nisin was rinsed with purified water and analyzed to determine N-homocysteinylation degree. All samples were kept at −80 °C.

Evaluation of N-homocysteinylation degree

The reaction of homocysteinylation introduces free-SH groups into the nisin structure. Therefore, the degree of N-homocysteinylation was measured by analyzing the amount of free sulfhydryl (-SH) groups present [24].

Nisin was dissolved in 8 M urea and 2 mM β-mercaptoethanol at a concentration of 6 mg/mL and incubated at 37 °C for 1 h. After the incubation, the mixture was precipitated with TCA (20%) for 30 min at 4 °C. The obtained pellets were dissolved again in 0.1 M sodium phosphate buffer (pH 8.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA). Free-SH groups were measured in both control and modified nisin using the Ellman reagent, which is also commonly referred to as 5,5′-dithiobis-(2-nitrobenzoic acid). The samples’ absorbance was then taken at 412 nm. The quantity of free-SH group as cysteine was determined using the molar extinction coefficient (ε) of 14,290 1/M•cm. N-homocysteinylation degree was determined using Eq. 1;

$$\text{N}-\text{homocysteinylation}\text{degree}\left(\%\right)=\frac{\text{A}\times 100}{\text{M}}$$ 1

A: The quantity of free -SH groups as cysteine per g modified nisin

M: The maximum quantity of free-SH groups that can be formed in 1 g modified nisin as cysteine

Tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (tricine SDS-PAGE)

Tricine sodium dodecyl sulphate polyacrylamide gel electrophoresis (tricine SDS-PAGE) was conducted on a vertical gel electrophoresis unit. The samples were separated by using polyacrylamide gels that were 1 mm thick, containing 4.1% stacking and 16.5% separating gels [25]. Each lane was loaded with 10 μL (approximately 50 μg) of protein solution. A voltage of 30 V and 300 V was used for stacking and separating gel for about 9 h, respectively. Coomassie Brilliant Blue G-250 was employed for staining the gels, which were later de-stained with an acetic acid solution (10%).

Acetic acid urea polyacrylamide gel electrophoresis (AAU-PAGE)

Acetic acid urea polyacrylamide gel electrophoresis (AAU-PAGE) was conducted on a vertical gel electrophoresis unit. The samples were separated by using polyacrylamide gels that were 1 mm thick, containing 4.5% stacking and 22.5% separating gels [26]. Each lane was loaded with 10 μL of protein solution (approximately 50 μg). Electrophoresis analyses were conducted at 175 V as steady voltage for 18 h. A solution containing Coomassie Brilliant Blue G-250 (0.1%) in methanol (20%), acetic acid (7%), and distilled water (73%) was used to stain gels. Also, the identical solvent system, except for Coomassie Brilliant Blue G-250, was used for de-staining.

Determination of antibacterial activity

Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Lactococcus lactis ssp. cremoris AÜ, Enterococcus faecium ATCC 9097, Listeria monocytogenes NCTC 5348, and Escherichia coli RSKK were used as indicator microorganisms to determine the antibacterial activity of nisin. The spot-on lawn procedure was used to determine the antibacterial activity [27]. Briefly, a 10 μL solution of nisin (50 ppm) diluted in peptone water was spotted onto soft De Man Rogosa-Sharpe (MRS) agar seeded with Enterococcus faecium and Lactococcus lactis ssp. cremoris; or on soft brain heart infusion (BHI) agar seeded with Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes. After the plates were incubated at 35 °C for 24 h, the clear zone was determined to evaluate the antimicrobial activity of nisin.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration (MIC) of samples was determined according to the method described by Slootweg et al. [20]. Stock solutions for nisin with a concentration of 100 μM were prepared in acetic acid (0.01%) and bovine serum albumin (BSA, 0.2%). Nisin was subject to serial three-fold dilutions in BSA (0.2%) and acetic acid (0.01%) to determine the concentration.

Two tubes were filled with 500 μL of the test bacterium in an appropriate broth. MRS broth for Lactococcus lactis ssp. cremoris and Enterococcus faecium, BHI broth for Bacillus subtilis, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes were used in the present study. Lastly, a final bacterial concentration of 10⁷ CFU/mL and 500 μL of the nisin solution at several concentrations (0, 0.07, 0.21, 0.62, 1.85, 5.56, 16.7, and 50 μM) were added into the tubes, and the third tube had only a medium and was used as a negative control group. The tubes were then incubated for 24 h at 35 °C and 120 rpm in a shaker. After incubation, the optical density at 630 nm was measured, and the MIC in μM of nisin was determined as the lowest concentration capable of preventing observable bacterial growth.

Estimation of N-homocysteinylated lysine

Free forms of amino groups in proteins are only acylated by HTL. Therefore, modified nisin was subjected to enzymatic hydrolysis using thermolysin (30–175 U/mg) or trypsin (1000 BAEE U/mg) to state the modified Lys residue. Hydrolysis of modified and unmodified samples was carried out in 50 mM Tris-HCl (pH 7.5) buffer containing 5 mM CaCl₂ at an enzyme-to-substrate ratio of 1:5 and 35 °C for 3 h. After the hydrolysis reaction, separated peptide fragments were identified by tricin SDS-PAGE analysis to determine the modified Lys residue.

Statistical analysis

All data from three independent experiments are presented as mean and standard deviation. Statistical evaluation and data comparison were conducted with one-way ANOVA and Duncan’s post hoc tests (p < 0,05), using the statistics program (SPSS 22.0, IBM Corp., NY, USA).

Results and discussion

N-homocysteinylation of nisin

An important stage of the optimization process is to monitor the progress of the reaction. In the current study, the progress of the modification was evaluated by measurement of the free-SH groups inserted into the nisin structure in the N-homocysteinylation process (Fig. 1). Initially, the optimization of the N-homocysteinylation reaction was performed with regard to HTL concentration, temperature, reaction time, pH, and shaking rate. The progress of the reaction was investigated as a function of HTL concentration (1, 10, or 100 mM); pH (3.0, 6.0, or 8.0); and reaction time (6, 24, or 48 h) while keeping constant at the 6 mg/mL of nisin concentration, 45°C of temperature, and 150 rpm of shaking rate (Table 1). Resulting in, the highest degree of N-homocysteinylation (4.35%) was achieved using a concentration of 100 mM HTL at pH 3.0 for 6 h. Subsequently, the progress of the reaction was investigated as a function of HTL concentration (100, 150, or 200 mM) and pH (3.0, 4.0, 4.5, or 5.0) to further refine these parameters while keeping constant at 6 mg/mL of the nisin concentration, 60 °C of temperature, 6 h of process time, and 150 of shaker rate (Table 2). The most effective modification (6.30%) was obtained at the end of this experiment with a concentration of 150 mM HTL at pH 3.0. Finally, the reaction’s progress was investigated as a function of temperature (60 °C or 80 °C) and time (2, 4, or 6 h) while keeping constant at 6 mg/mL of nisin concentration, 150 mM of HTL concentration, 3.0 of pH, and 245 rpm of shaker rate (Table 3).

Table 1.

N-homocysteinylation degree of nisin under various reaction times, HTL concentrations, and pHs at 45 °C, 150 rpm shaker rate, and 6 mg/mL nisin concentration

Reaction time (h)	HTL concentration (mM)	pH	N-homocysteinylation degree (%)*
6	1	3.0	0.14±0.001
		6.0	0.05±0.016
		8.0	0.65±0.021
	10	3.0	0.39±0.089
		6.0	2.74±0.019
		8.0	1.89±0.024
	100	3.0	4.35±0.018
		6.0	4.30±0.024
		8.0	4.14±0.034
24	1	3.0	0.04±0.008
		6.0	0.12±0.019
		8.0	1.21±0.024
	10	3.0	1.85±0.025
		6.0	2.10±0.641
		8.0	3.07±0.084
	100	3.0	3.98±0.095
		6.0	4.34±0.010
		8.0	3.10±0.040
48	1	3.0	0.29±0.014
		6.0	0.61±0.010
		8.0	1.19±0.016
	10	3.0	2.66±0.001
		6.0	1.24±0.201
		8.0	3.13±0.214
	100	3.0	4.08±0.036
		6.0	4.31±0.018
		8.0	2.94±0.054

*Mean ± standard deviations, HTL homocysteine thiolactone

Table 2.

N-homocysteinylation degree of nisin under various HTL concentrations and pHs at 45 °C, 150 rpm shaker rate, and 6 mg/mL nisin concentration

HTL concentration (mM)	pH	N-homocysteinylation degree (%)*
100	3.0	6.07±0.002
	4.0	6.15±0.001
	4.5	5.53±0.002
	5.0	5.73±0.002
150	3.0	6.30±0.020
	4.0	6.22±0.021
	4.5	5.86±0.008
	5.0	5.74±0.050
200	3.0	5.96±0.035
	4.0	6.09±0.015
	4.5	5.85±0.030
	5.0	5.73±0.036

*Mean ± standard deviations, HTL homocysteine thiolactone

Table 3.

N-homocysteinylation degree of nisin under various reaction temperatures and times at 150 mM HTL concentration, 3.0 pH value, 245 rpm shaker rate, and 6 mg/mL nisin concentration

Reaction temperature (°C)	Reaction time (h)	N-homocysteinylation degree (%)*
60	2	1.46±0.006
	4	2.70±0.008
	6	5.35±0.023
80	2	4.31±0.050
	4	4.99±0.113
	6	5.23±0.224

*Mean ± standard deviations, HTL homocysteine thiolactone

The results showed that the maximum degree of modification (5.35%) can be achieved at 60 °C for 6 h incubation time. However, this result indicates that there is no significant increase in the degree of N-homocysteinylation. When evaluating all the experimental results, it was found that the maximum degree of N-homocysteinylation was obtained at 6 mg/mL of nisin concentration, 150 mM of HTL concentration, 245 rpm of shaker rate, pH 3, and 6 h incubation time. The maximum degree of N-homocysteinylation under the specified conditions was determined as 6.30%. It has been reported that a strong correlation exists between the lysine content of proteins and their reactivity with HTL [22]. In addition, studies have reported that Hcy modifies protein Lys residues by forming N-Hcy-protein adducts in which the carboxyl group of Hcy binds to the ε-amino group of a protein Lys residue [28, 29]. It is possible to consider that a correlation exists between protein solubility and consequent reactivity and modification. Some studies have reported a strong correlation between lysine content and HTL reactivity [30]. As the lysine content of nisin is relatively low compared to other antimicrobial peptides [31], it is thought that its reactivity with HTL may also be more limited. Targeting selective reactive lysines in nisin via site-specific modification techniques can increase the reactivity of nisin and thus the efficiency of the N-homocysteinylation reaction [32].

AAU-PAGE and tricine SDS-PAGE

The nisin samples were checked for N-homocysteinylation using AAU-PAGE, which separates proteins by size and charge differences at pH 3.0, and tricine SDS-PAGE, which separates proteins by size differences [25, 26]. At pH 3, both modified and unmodified nisin exhibited a positive charge, which was the main factor affecting its electrophoretic mobility. Significantly, nisin has two major bands on AAU-PAGE (Fig. 2a), but only one band on tricine SDS-PAGE (Fig. 2b). This suggests that unmodified nisin consisted of two components with differing mass/charge ratios. Similarly, the modified nisin samples also have two faint bands in the same location on the gel, indicating that the majority of nisin molecules disappeared due to modification. However, N-homocysteinylation is the process of creating an amide bond between the epsilon amine of lysine and the carboxylic group of homocysteine [33]. Furthermore, N-homocysteinylation causes lysine residues and the gain of sulfhydryl group to lose their positive charge and exhibit lower mobility in capillary zone electrophoresis [29, 34]. Similarly, Genoud, Quintana, Gionco, Baldessari, and Quintana [35] reported that N-homocysteinylated fibrinojen has a relative molecular mass that is significantly lower than that of Fbg and the charge/mass ratio is higher, resulting in an increase in electrophoretic mobility. This result is quite consistent with and can be explained by the fact that a very low degree of N-homocysteinylation (6.30%) was obtained.

Fig. 2.

Nisin (1) and N- homocysteinylated nisin (2) in acetic acid urea polyacrylamide gel (a) and tricine sodium dodecyl sulphate polyacrylamide gel (b)

The bands for nisin and modified nisin in the electrophoresis exhibited a comparable appearance and size (Fig. 2b). Furthermore, a slight increment in the higher mass fractions was found in the modified nisin line (Fig. 2b, line 2), indicating that the nisin was not extensively modified. Figure 2 demonstrates a significant disagreement between these two electrophoretic techniques. The lack of consistency detected in the AAU-PAGE method could be explained by inadequate dissolution of the modified nisin aggregates. In a similar, it is indicated that a simple reduction of disulfide bonds did not lead to the complete disintegration of homocysteinylated β-casein aggregates nor did the solubilization by 4 M guanidine hydrochloride [36]. In addition, this may be due to the fact that the changes in charge/mass ratio introduced by N-homocysteinylation are too small to be detected by this technique, as in Genoud, Quintana, Gionco, Baldessari, and Quintana’s [35] study.

Antibacterial activity of modified nisin

The antibacterial activity of nisin and modified nisin was evaluated by the spot-on-lawn method against Lactococcus lactis ssp. cremoris AÜ, Staphylococcus aureus ATCC 6538, Enterococcus faecium ATCC 9097, Listeria monocytogenes NCTC 5348, Bacillus subtilis ATCC 6633, and Escherichia coli RSKK. Only E. faecium ATCC 9097 growth was affected by modified nisin when compared to all the other bacteria that were tested. MIC values of pure nisin, unmodified nisin, and modified nisin were determined against E. faecium ATCC 9097 (Table 4). As shown in Table 4, the modification of nisin with HTL resulted in a significant (p < 0.05) increase in the MIC value (16.75 μM), almost 9 times higher than nisin (1.85 μM) and pure nisin (1.85 μM). This means that after modification, nisin had approximately 10% of the antibacterial activity against E. faecium ATCC 9097. These findings suggest that even a slight modification (6.30%) of nisin with HTL resulted in a considerable loss (approximately 90%) of its antibacterial activity. Jakubowski [22] reported that homocysteinylation reduces the positive charge for lysine residues. Consequently, the ε-amino group of lysine (pK 10.5) is more basic than the ε-amino group of homocysteine in ε-N-homocysteine-lysine (pK 7.1). It is well known that the antibacterial activity of nisin is due to the positive charges provided by lysine residues, which are also the main sites for the N-homocysteinylation reaction [18]. Therefore, it is thought that after homocysteinylation of nisin, the decrement of positive charges reduces the antibacterial activity. In addition to all of this, it is believed that newly added free-SH groups may react with dehydroalanine or dehydrobutyrine to produce an intermolecular lanthionine bond, causing a considerable reduction in antibacterial activity [37]. Finally, after modification of nisin, the reduction in antibacterial activity may also be explained by the modification resulting in an important change in the structure of nisin by the addition of free-SH groups. Due to these considerations, the reaction of N-homocysteinylation may not be a suitable method for improving the antibacterial activity or broadening the antibacterial spectrum of nisin. The antimicrobial activity of nisin is limited in high-salt environments due to its lysine and arginine content. Additionally, its mechanisms against Gram-positive and Gram-negative bacteria differ. This is explained by the cytoplasmic permeability of nisin, which varies depending on its cationic content [38]. Therefore, homocysteinylation’s effect on lysine residues may alter nisin’s known characteristics.

Table 4.

MIC values for modified and unmodified nisin against Enterococcus faecium

Nisin samples	MIC (μM)
Pure nisin (95%)	1.85±0.00a
HTL-control nisin	1.85±0.00a
HTL-modified nisin	16.75±0.00b

^a,bMeans followed by different letters within the same column represent significant differences (p < 0.05)

HTL homocysteine thiolactone

HTL-modified nisin: nisin modified under optimum conditions with homocysteine thiolactone

HTL-control nisin: nisin prepared by the same procedure just without homocysteine thiolactone

Estimation of N-homocysteinylated lysine

The reaction of N-homocysteinylation selectively modifies lysine side chains; thus, it is hypothesized that specific enzymatic hydrolysis could be utilized to identify modified lysines in nisin molecules. Therefore, thermolysin and trypsin enzymes were selected for hydrolysis of nisin. The cleavage sites of nisin by thermolysin and trypsin are shown in Fig. 3.

Fig. 3.

Sites of cleavage of nisin by trypsin and by thermolysin

Trypsin exclusively cleaves C-terminal to Arg and Lys residues, if there are no steric hindrances. It is clear that there is no Arg residue on the nisin molecule, so it can cleave the Lys22-Abu23 or Lys12-Abu13 bonds, as shown in Fig. 3, to give 1–12, 1–22, 13–22, 13–34, and 23–34 fragments. If there is an alteration in the side chains of the Lys residues, trypsin will not recognize and hydrolyze nisin. Thermolysin cleaves N-terminal to Met, Ile, Trp, Tyr, Phe, and Val residues. It is, therefore, able to hydrolyze nisin even when the side chain of the Lys residues is modified.

The results of the hydrolysis of nisin using the two enzymes showed a high degree of agreement with the expected results. As demonstrated in Fig. 4, if the control nisin sample was treated with trypsin or thermolysin (lines 2 and 3), the nisin band disappeared entirely, showing that the nisin was completely hydrolyzed by the two enzymes. Nevertheless, exposing N-homocysteinylated nisin to trypsin (line 5) did not alter the appearance of the modified nisin band, showing that the modified nisin was insensitive to trypsin activity. Considering these findings, it is concluded that homocysteine thiolactone modified both the Lys12 and Lys22 side chains. This hypothesis is confirmed by the entire hydrolysis of the modified nisin molecule by thermolysin. The complete hydrolysis of modified nisin with thermolysin suggested that when no steric hindrance is present, the stability of modified nisin to trypsin activity is most probably due to side chain modification of Lys.

Fig. 4.

Nisin (1), nisin treated with trypsin (2), nisin treated with thermolysin (3), N- homocysteinylated nisin (4), N-homocysteinylated nisin treated with trypsin (5), and N-homocysteinylated nisin treated with thermolysin (6) in tricine sodium dodecyl sulphate polyacrylamide gel

Conclusion

This study has shown significant improvement in the production of the homocysteine-thiolactone conjugate, one of the techniques that has not been previously focused on to improve the properties of nisin. In addition, the optimal conditions for the N-homocysteinylation process were identified as a reaction temperature of 60 °C, a reaction time of 6 h, a concentration of 150 mM for HTL, a shaker rate of 245 rpm, and a pH of 3, all while maintaining a nisin concentration of 6 mg/mL.

In the optimal specified conditions, the highest degree of N-homocysteinylation was determined to be 6.30%. The modified nisin obtained an inhibitory effect on E. faecium. The antibacterial activity of modified nisin was determined as approximately 10% of the antibacterial activity of unmodified nisin. The electrophoresis analysis provided compelling evidence of the significant specific modifications induced by the homocysteine-thiolactone reaction, particularly at Lys12 and Lys22, following hydrolysis of nisin by trypsin and thermolysin. These results not only demonstrate the success of our N-homocysteinylation approach but also suggest new possibilities for enhancing the antibacterial properties of nisin.

This study offers valuable insights into the effectiveness of N-homocysteinylation in enhancing the antibacterial activity and range of action of nisin. The findings strongly encourage further investigation of the specific modifications identified during the modification process and suggest that these modifications can be investigated for other properties than the antimicrobial activity of nisin.

Funding

The Scientific Research Projects of Nigde Omer Halisdemir University funded the present study (Project No: 2014/21-BAGEP).

Declarations

Conflict of interest

The authors declare no competing interests.