3.1 Strategy of ligating the α- and β-NHase subunits by SpyTag/Spycatcher
NHase, which catalyzes the formation of AM, is one of the most important industrial enzymes[1, 22]. The stress, heat release, and solvent immerse present in this reaction can cause conformational changes or damage to the amino acids of NHase. Therefore, enhancing thermostability or solvent tolerance is crucial [5, 10].
In this study, the mutant NHase gene [9], containing a disulfide bond to stabilize the binding between subunits and exhibiting better thermal stability and AM tolerance than the natural gene, was selected as the parent gene. To further enhance the interaction between the α- and β-NHase subunits, SpyTag and SpyCatcher were fused to the N- and C- termini of the α- and β-NHase subunits in different combinations. They were then overexpressed using the R. ruber-E.coli shuttle plasmid pNV-Pa2 [23] to obtain the engineered R. ruber strains NBSt, StNB, NASt and StNA (Fig. 1). NBSt and StNB were generated by fusing SpyCatcher to the C-terminus of α-NHase, and then SpyTag was fused to either the N- or C-terminus, respectively, of β-NHase. NASt and StNA were generated by fusing SpyCatcher to the C-terminus of β-NHase, and then SpyTag was fused to either the N- or C-terminus, respectively, of α-NHase. Meanwhile, R. ruber TH8 [9] engineered by overexpressing the template NHase gene in R. ruber TH3 [22], served as the control.
3.2 Verification of the ligation conformation of α- and β-NHase subunits
Parallel cultures of the engineered R. ruber NBSt, StNB, NASt, StNA, and the control strain R. ruber TH8 were cultivated in shake flasks to assess the NHase expression via SDS-PAGE (Fig. 2). The protein sample from TH8 was separated, and the α- and β-NHase subunits were identified after SDS-PAGE; their known molecular weights are 22.8 and 26.4 kDa, respectively. The ligation of the α- and β-NHase subunits by SpyTag/SpyCatcher fusion in all engineered strains was successful, as indicated by the band at ~ 60 kDa, even after sample boiling. The molecular weights were determined using AAT Bioquest (https://www.aatbio.com/tools/calculate-peptide-and-protein-molecular-weight-mw); the molecular weights of SpyTag and SpyCatcher are 1.5 and 12.3 kDa, respectively.
This experiment further demonstrated the successful application of SpyTag/SpyCatcher. It showed that the strategy of cyclizing two termini of one enzyme or binding two different subunits by introducing SpyTag/SpyCatcher is universal, as supported by other literature reports [12, 15, 18, 20, 24]. The percentage of total protein represented by ligated NHase was calculated using Gel-Pro Analyzer software and varied based on the ligation strategy: 9.5%, 9.6%, 6.4% and 8.2% for NBSt, StNB, NASt and StNA, respectively. Additionally, SDS-PAGE analysis revealed a single subunit NHase band in all engineered strains, attributed to the native NHase encoded by the genome of the host strain TH3 [22].
3.3 Effect of introduced SpyTag/SpyCatcher on NHase activity
The biomass and NHase activity of the engineered R. ruber strains NBSt, StNB, NASt, StNA, and the control R. ruber TH8 were measured and compared in Fig. 3. SpyTag/SpyCatcher ligation did not reduce the biomass of NHase (Fig. 3A), but it did reduce NHase activity (Fig. 3B). Previous reports have also documented a reduction in enzyme activity caused by SpyTag/SpyCatcher-mediated cyclization of lichenase [20]. NHase activity decreased from 15.4–37.9% depending on the fusion strategy. R. ruber strain NBSt, engineered by fusing SpyTag to the C-terminus of β-NHase and fusing SpyCatcher to the C-terminus of α-NHase, exhibited the highest activity at 2801.7 U/mL, representing a 15.4% decrease compared to the TH8 control. The only difference in fusion strategy between NBSt and StNB was the β-NHase terminus fused to SpyTag; the C-terminus was fused in NBSt, whereas the N-terminus was fused in StNB. The different fusion termini led to similar ligated-NHase biomass, but NHase activity was further reduced to 2057 U/mL in StNB, a decrease of 37.9% compared to the control. This is in accordance with previous studies showing that decreasing NHase activity occurred by introducing salt bridges [1, 6] or reducing NHase solubility by inserting assembly peptides [25] at the N-terminus of β-NHase.
The fusion of SpyCatcher to the C-terminus of β-NHase in the NASt engineered strain also resulted in reduced NHase activity, 2154 U/mL, relative to the control. This activity was similar to that of StNA (2213 U/mL), in which SpyTag was fused to the N- terminus of α-NHase. Comparing NBSt and NASt, NHase activity was higher in NBSt, suggesting that the larger SpyCatcher protein should be fused to the α-NHase subunit rather than the β-subunit [6, 10]. Based on these results, we conclude that the C-terminus of β-NHase is a better fusion site than the N-terminus, the C-terminus of α-NHase is more suitable for fusion with a larger protein. fusion of a peptide to the N- or C-terminus of α-NHase yields similar results, and the N-terminus of β-NHase is more sensitive to peptide fusion than the N-terminus of α-NHase.
3.4 Stress resistance of ligated-NHase
The stress resistance (thermostability and solvent tolerant) of the different ligated-NHase strains and the original NHase was tested using TH8, NBSt, StNB, NASt, and StNA cells (Fig. 4).
As shown in Fig. 4A, the control TH8 cells retained 62.2% of their NHase activity after incubating at 60 ℃ for 10 min. The residual NHase activity of the engineered strains was 63.2%, 14.4%, 34.6%, and 20.3% in NBSt, StNB, NASt, and StNA, respectively. Among all the engineered strains, NBSt exhibited superior thermal-tolerance. The 9.5% ligated-NHase mediated by SpyTag/SpyCatcher in NBSt showed a 1% increase in heat shock resistance. By fusing SpyTag and SpyCatcher to the C-terminus of β- and α-NHase, respectively, NHase thermostability was improved, but the effect was not significant. The results obtained here show little enhanced thermostability achieved by introducing SpyTag/SpyCatcher-mediated enzyme ligation or cyclization, similar to the situation of cyclizated l-phenylalanine aldolase caused by SpyTag/SpyCatcher [18]. Additionally, a few studies have reported that introducing SpyTag/SpyCatcher cyclization did enhance the thermostability of Lichenase [20], Firefly Luciferase [26], β-lactamase [27], and phytase [28]. This suggests that the strategy of cyclizing one enzyme or ligating two subunits to fix the structure or conformation of the enzyme to enhance thermostability is not universal, and this needs to be examined in each specific case.
Thermostability of all mutants assessedbefore and after heat shock treatment at 60 ℃ for 10 min(A), and the solvent tolerance of all mutants evaluated after immersion in 30, 40, and 50% acrylamide (AM) solutions for 20 min (B).
Industrially, different applications require the production of 30%, 40% and 50% AM solutions. Thus, was assessed the effect of these AM concentrations on ligated-NHase activity by immersing the respective cells for 20 min (Fig. 4B). NHase activity decreased as the AM concentration increased in both the control and all engineered strains. The engineered strain NBSt exhibited better AM tolerance than the other engineered strains. Upon immersion of the cells in 30%, 40%, and 50% AM solution, approximately 40.7%, 32.8% and 22.1% of the NHase activity remained in the control cells, while 48.2%, 38.6% and 24.1% remained in NBSt, respectively. The residual NHase activity of NBSt was higher than that of the control in all AM concentrations. This indicates that the fusion of SpyTag and SpyCatcher to the C-terminus of β- and α-NHase, respectively, in NBSt results in higher AM tolerance compared to the control NHase and other ligated-NHase strains. The improved AM tolerance achieved by stabilizing the enzyme structure through the introduction of a covalent bond between two subunits using SpyTag/SpyCatcher is similar to the results of our previous study, which focused on enhancing enzyme structure through the introduction of salt bridges [6] or a disulfide bond [9] between two subunits. All these results indicate that the SpyTag/SpyCatcher-mediated ligation between subunits of an enzyme is an effective strategy to enhance the toxic AM tolerance of NHase.
To investigate the effect of introducing SpyTag/SpyCatcher on NHase, the native NHase gene in the genome of R. ruber TH8 and NBSt was knocked out using CRISPR/cas9 [29] to obtain the engineered R. ruber TH and TH-NBSt. The kinetic parameters of intracellular NHase produced by these two engineered strains were measured. The Km value of ligated-NHase mediated by introducing SpyTag/Spycatcher is 0.311 mM, which is higher than that of the control TH (0.204 mM). However, the Vmax of ligated-NHase (5.956 µmol/min/mg DCW) is lower than that of the control TH (1.034 µmol/min/mg DCW). This result indicates that the ligated NHase mediated by SpyTag and SpyCatcher decrease the substrate affinity but increases the structural stability of the enzyme stability of the enzyme to some extent. These results are not consistent with the application of SpyTag/SpyCatcher on L-phenylalanine aldolase and lichenase. Overall, introducing SpyTag/SpyCatcher to cyclize mono-subunit or ligate different subunits of the enzyme can improve the structural stability, but enhancing it is a case-by-case trial.