The synthesis of a new series of Gd(III)-arylphosphonium complexes is described and the solution stability of selected compounds is reported. Their lipophilicity and uptake in human glial (SVG p12) and human glioblastoma multiforme (T98G) cell lines are presented. The in vitro cytotoxicity of all complexes was determined to be low at therapeutically-relevant concentrations. Selected Gd(III) complexes are potential candidates for further investigation as theranostic agents. Ψm), an attribute associated with the mitochondria of aggressive tumours, and have consequently shown promise as cancer targeting vectors3. When coupled with relevant lanthanoid chelators these phosphonium DLCs are able to selectively shuttle medically-relevant metal ions into the mitochondria of these cancer cells independent of endogenous transporters6. Initial investigations of these ligands have demonstrated their promise as candidates for the development of Gd(III)-based theranostics9.Phosphonium-based delocalized lipophilic cations (DLCs) demonstrate selective accumulation in mitochondria with a high membrane potential  and non-radioactive 157Gd isotope is also an excellent candidate for neutron capture therapy (NCT) as it possesses the largest neutron capture cross-section of any stable isotope. Upon thermal neutron capture by the 157Gd nucleus, the resulting excited 157Gd* nucleus undergoes a nuclear re-arrangement and subsequent production of a prompt γ-photon, internal-conversion electrons, and ca. 5 high-energy Auger and Coster-Kronig (ACK) electrons per neutron capture event. The ACK electrons appear to be the key therapeutically-relevant particles14. The potential use of 157Gd in the related neutron capture enhanced particle therapy (NCEPT) has recently been reported15. Due to its high atomic number (Z), Gd also shows great potential in photon activation therapy (PAT), more specifically synchrotron stereotactic radiotherapy (SSR), given its achievable K-edge of 50.2 keV, resulting in an average of ca. 8 ACK electrons per photon activation event16. Localisation within critical subcellular components is required for the high efficacy of Gd therapeutics that produce ACK electrons due to their high linear energy transfer (LET) nature, depositing their energy over very small path lengths (ca. 12.5 nm)17.Gd(III) has a number of unique properties that support its use in theranostic agents. Owing to the highly paramagnetic nature of Gd(III), this ion is a key component of contrast agents for magnetic resonance imaging (MRI)25. Radbruch et al. have proposed that differences in Gd deposition are attributable to the thermodynamic stability of the Gd agents administered to the patient, hence there is a critical need to ensure good stability of any Gd agents developed with a clinical application20.Recent studies demonstrating Gd deposition in the brain tissue of individuals who have received consecutive administrations of Gd-based MRI contrast agents have concerned radiologists from the US NIH, and these concerns have been recently highlighted by the US FDAN-acetic acid derivatives have demonstrated some of the highest stability constants with lanthanoids, facilitating Gd(III) complexation in the centre of a distorted square antiprism27. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetatogadolinium(III) (Gd-DOTA) complexes possesses the highest thermodynamic stability of these derivatives with a logK of ca. 25.3 resulting from their octadentate coordination28. Previous work investigating Gd(III)-phosphonium complexes has utilised 1,4,7,10-tetraazacyclododecane-1,4,7-tris(tert-butyl acetate) (DO3A) as the remaining unfunctionalised secondary amine within the cyclen structure allows for facile functionalisation of the complex, but it can result in a lower stability with logK values between 21.0 and 23.828. Herein we report the synthesis and a limited stability and SAR study of a novel series of Gd(III)-DOTA-phosphonium complexes. The uptake of these complexes in human glial (SVG p12) and human glioblastoma multiforme (T98G) cell lines is also presented.1,4,7,10-Tetraazacyclododecane (cyclen) is a versatile macrocycle and its 1–5) were synthesised utilising phosphonium mitochondrial-targeting vectors based upon related prototype Gd(III) complexes that we have reported previously9. Notably, the inclusion of an additional O-donor group attached to the DO3A macrocycle helps facilitate octadentate coordination of the Gd(III) ion and thus should provide enhanced solution stability for the complexes. In addition, a new series of complexes (6–10) featuring an alkyl linking group of varying chain length was synthesised with the primary aim of identifying any relationship between the nature of the bridging ligand and the degree of cellular uptake and tumour-cell selectivity.Novel Gd(III) complexes -10-acetic acid was performed using peptide coupling reagents (hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU) and N-methylmorpholine (NMM)) to afford the protected phosphonium ligands. Acid deprotection of the tert-butyl groups with trifluoroacetic acid (TFA) and subsequent purification by means of reverse-phase HPLC afforded the free macrocyclic ligands. Complexation of the Gd3+ ion was performed by employing a suspension of Gd2O3 in H2O to give the target Gd(III) complexes 1–10 in high yield and purity. The purity of each complex (> 95%) was confirmed by means of analytical reverse-phase HPLC, and their identities were confirmed by means of ESI-FTICR-MS or MALDI-TOF–MS.Complexes 1, 80 mg mL−1; 2, 70 mg mL−1; 4, 40 mg mL−1; 6, 60 mg mL−1 and 9, 250 mg mL−1. Complexes 1–10 also demonstrated excellent stability in aqueous media at pH 7.4 following incubation at 37 °C for prolonged periods (days), as confirmed by MALDI-TOF–MS. In addition, a 20 mM aqueous sample of complex 6 was analysed by HPLC and found to be > 95% intact after 3 years in solution, indicating excellent long-term stability. During this time, the pH of the solution decreased from 7.4 to ca. 5.5. However, decreased stability of complexes at lower pH values for the related DO3A-phosphonium complexes is well-documented29. This poor stability in acidic conditions is largely attributed to protonation of the macrocyclic ring nitrogen atoms which decreases the affinity of the ligand for Gd3+ ions29. Whilst the proposed biological application of these Gd(III) complexes would be dominated by physiological pH (7.4) and the slightly alkaline environment of the mitochondria (~ pH 8), one must also consider the most acidic cellular regions these complexes might encounter30. Lysosomes, for example, are acidic organelles that contain a battery of degradative enzymes. They are found in all cells, estimated to make up approximately 1% of the total cellular volume, and maintain a pH ~ 4.8 by proton pumps31. Whilst the Gd(III) complexes are unlikely to be actively taken up by the lysosomes, it is possible that some molecules may be passively exposed to the hostile acidic environment these organelles foster where they are more likely to undergo demetallation or transmetallation processes that might lead to the release of free Gd3+ ions.The Gd(III) complexes were found to be highly soluble in water, with selected examples as follows: 6 and 7 were compared to the previously-reported archetypal Gd(III)-DO3A triphenylphosphonium complex  between the control group  and the Gd(III)-DO3A analogue (11), as well as between 1 and 11 and at pH 5.0 and 5.8. These results demonstrate that under mild acidic conditions that are typically found in lysosomes, in the presence of the competitive chelator (xylenol orange), the stability of the Gd(III)-DOTA complex 1 is significantly greater (ca. 3 to 4 times) than that of the previously-reported DO3A analogue 11. For those experiments that were run at lower pH values (i.e. pH < 4.0), the readings did not exceed the baseline absorbance values (573 nm) at any meaningful levels, consistent with complex demetallation.Linear regression analysis of the data found a significant difference  which is somewhat higher than the value reported for the Gd-DO3A analogue 11 (5.1 mM−1 s−1)8. Furthermore, the relaxivity value of 1 lies at the higher end of clinical Gd MRI contrast agents such as Magnevist, probably related to the higher MW of 1 compared to that of clinical contrast agents. The complete relaxivity data are presented in Table Relaxivity studies were performed on the representative parent complex 34. Hence, it is important to consider and analyse the effect that the lipophilicity of these complexes has on their efficacy to assist in future Gd theranostic design. LogP (where P is the partition coefficient) is the most common measurement of lipophilicity and it was determined for 1–10 by means of a reverse-phase HPLC method involving a suite of standard reference compounds. A summary of the results is shown in Table Lipophilicity is a vital parameter to consider regarding DLCs as it underpins their membrane permeability, and also strongly influences absorption, distribution, metabolism and excretion of the Gd(III)-phophonium complex within the bodyP values ranging from 1.12 to 1.63. These values are consistent with those previously reported for a range of related DO3A-phosphonium complexes and lie within the pharmacologically optimal value of 1–3, as outlined by Waring et al.33. Furthermore, these logP values lie within the predictive model on the optimised uptake of lipophilic cations into the mitochondria reported by Trapp and Horobin35. This model indicates the optimal lipophilicity for DLCs lie above logP =  − 2, precluding any kinetically-limited uptake due to the high energy of desolvation, and also fall below logP = 2 in order to avoid non-specific lipid binding35.All of the Gd(III) complexes prepared in this work were shown to be reasonably lipophilic, with log50) of each of the assessed complexes for the human glial (SVG p12) and human glioblastoma multiforme (T98G) cell lines. Ideally, Gd theranostic agents should demonstrate relatively low cytotoxicity to ensure any uptake into healthy tissue causes only minimal damage, as the intended cytotoxic event for Gd-based binary agents results from either the thermal neutron (NCT/NCEPT) or X-ray photon (PAT) irradiation.In vitro cytotoxicity was determined for each of the new complexes by means of standard 3--2,5-diphenyltetrazolium bromide (MTT) colourimetric assays over a 62.5 μM–2 mM range. A summary of the results in Table 50 values for complexes 1–10 were found to lie in the low mM range, in agreement with those previously-reported for related DO3A complexes9. A one-tailed Mann–Whitney U test demonstrated that these complexes were slightly more cytotoxic toward the T98G cells over their healthy SVG p12 cell line, albeit the overall toxicity of the complexes was only marginal (mM).The IC1–10 with a known  Gd theranostic, Motexafin-Gd (MGd), was chosen. In vitro cell experiments involving MGd were reported to result in 50% cell arrest of the HF1 tumour cell line at concentrations of less than 100 μM, and the Gd agent exhibited significant cytotoxicity at concentrations of ca. 50 μM (in the absence of any X-ray irradiation)37. Evidently, complexes 1–10 appear to exhibit far lower in vitro cytotoxicities than the clinical agent MGd.In order to evaluate the relative cytotoxicities of complexes 1–10 using both SVG p12 and T98G cell lines at four different concentrations . The harvested cells were analysed for accumulated Gd by means of ICP-MS and these values were normalised to protein content, as determined by means of a modified Lowry protein assay. Representative cell uptake results are presented in Figs. As the therapeutic application of Gd(III) complexes relies upon maximising Gd accumulation within the mitochondria of the target tumour cells, comprehensive in vitro cellular uptake studies were performed on complexes 1–10 occurs in both tumour and healthy cell lines. Table 2 and 6–9 but no statistically significant difference was found between the two cell lines for complexes 1, 3–5 and 10. Indeed, for complexes 1–3, the tumour cell uptake and selectivity results are not as significant as our previous research reporting related DO3A complexes whereby a reduced delocalisation at the phosphonium centre in moving from, for example, R1 = Ph (1) to Me (2) was strongly correlated with a reduced tumour-cell selectivity and significantly increased Gd uptake9. Furthermore, statistical analysis demonstrated that at the lowest treatment dose for each Gd(III) complex (with the exception of complex 3), a significantly higher Gd uptake in the T98G tumour cell line over the SVG p12 cell line was found. This was also true at the second-lowest treatment dose for each complex, with the exception of the triethylene glycol (TEG) complex 10 which was found to exhibit high tumour cell selectivity (2.8) but only at the lowest treatment dose (62.5 μM). However, the propyl-bridged complex 6 exhibited the highest tumour cell selectivity (3.7) of all complexes studied in this work (at 125 μM). None of the complexes examined in this work demonstrated any significant selectivity for the T89G cell line at the two highest treatment doses (500 μM or 1 mM). The tendency for lower concentration treatment doses resulting in higher Gd uptake into the tumour cells over the healthy cells is consistent with the tumour cell’s elevated ΔΨm which is responsible for tumour-selective accumulation up to a point whereby high levels of Gd complex accumulation within mitochondria leads to depolarisation, after which passive Gd influx may become the dominant mechanism of mitochondrial accumulation39. This hypothesis is further supported by the tendency that the highest concentration treatment resulted in no significant difference in Gd uptake between the two different cell lines.Figures 4 and 5 led to a somewhat increased cell uptake compared to the parent 1 at lower treatment doses, but no significant improvement in tumour-cell selectivity was observed. Furthermore, replacement of the archetypal xylyl linker in 1 with various alkyl and TEG linkers (6–10) resulted in a significant increase in Gd uptake and tumour cell selectivity at lower treatment doses. In general, no statistically significant trend was established for complexes 6–10, and Gd uptake did not appear to be related to the relative distance between the phosphonium and Gd centres, although a very high Gd uptake was observed for 7 (5349.0 ± 478.1 ng Gd/mg protein), containing the n-butyl linker, at one of the assessed doses (250 μM). The incorporation of a hydrophilic TEG linker in complex 10, however, did lead to an increased Gd uptake at higher concentrations (250 and 500 μM) when compared to the more lipophilic alkyl linkers found in 6–9. However, the higher Gd uptake in 10 is countered by the reduced tumour cell selectivity when compared to most other Gd(III) complexes assessed in this study, except at the lowest treatment dose (62.5 μM).The electron-rich P-aryl rings in complexes 1–10 were synthesised in good to high yields and in high purity. Stability assays comparing the DOTA-based complex 1 to the previously-reported, archetypal DO3A-phosphonium complex 11 at mildly acidic pH conditions demonstrated a significant increase in the stability of 1, and these findings are consistent with those previously reported for Gd(III)-DOTA complexes by Caravan et al.26. Complexes 1–10 were all found to have low (mM) cytotoxicity at therapeutically-relevant concentrations, with significant in vitro tumour cell uptake and reasonable tumour cell selectivity at lower concentrations. The reduced tumour cell selectivity of the complexes at higher concentrations is consistent with previous findings which proposes a different mechanism for uptake beyond a certain threshold, although it may also be indicative of ΔΨm depolarisation9.Complexes 1–5), in direct contrast to previous studies involving Gd(III)-DO3A-phosphonium complexes where, for example, the DO3A analogue of 2 was found to have very high T98G cell uptake compared to the parent triphenylphosphonium complex but with a much lower tumour cell selectivity9. Similarly, a related trend was also observed for the alkyl and TEG linker complexes 6–10 which were found to have reduced tumour cell selectivities but showed significantly higher Gd uptake than the para-xylyl linker complexes 1–5 in both cell lines but even more so in the T98G tumour line. Indeed, the para-xylyl DOTA complexes 1–5 were not found to differ significantly from their previously-reported DO3A counterparts in terms of cytotoxicity and tumour cell uptake9, although, in general, tumour cell selectivity was found to be reduced for the DOTA family of complexes, a factor that might be related to a decreased lipophilicity and diminished ability to traverse both the cell and mitochondrial membranes owing to the presence of the amide linkage. However, the logP values in Table P difference between the archetypal complexes 1 and 11 is only marginal at best. In vivo uptake and biodistribution studies with selected Gd(III) complexes are currently underway and the results of this work will be reported in due course.No significant trends were observed in cell uptake when various phosphonium targeting vectors were used , a Bruker Avance III 400 , or a Bruker Avance III 500 . All NMR signals (δ) are reported in ppm. 1H and 13C NMR spectra were referenced according to their solvent residual peaks. 13C{1H} NMR spectra in D2O were referenced according to an internal standard of DMSO-d6 (39.39 ppm). 31P{1H} NMR spectra were referenced to external 1% TMS in CDCl3 using the unified reference scale41. Coupling constants are reported in Hz. Peak multiplicities have been abbreviated as s (singlet), d (doublet), t (triplet), q (quartet), qi (quintet), br (broad), and m (multiplet – unassignable multiplicity). Proton and carbon positions within aryl rings have been abbreviated i- , o- , m- , and p- . The xylyl linker has been abbreviated to ‘xy’ for clarity’s sake when assigning proton and carbon nuclei belonging to this linker group.All FT-IR spectra were run on a benchtop Bruker ALPHA FTIR spectrometer. All 96 and 384 well plates were read using a BMG LABTECH POLARstar Omega UV spectrometer. Low resolution ESI–MS were recorded on a Bruker amaZon SL mass spectrometer. MALDI-MS were recorded on a Bruker autoflex speed LRF MALDI-TOF mass spectrometer. High resolution ESI-FT-ICR-MS data were recorded on a Bruker Apex Qe 7 T FTICR mass spectrometer. Quantitative MS was recorded on a Perkin Elmer NexION 350X ICP-MS with a quadrupole analyser.v/v MeCN : 0.1% TFA in water) saturated with α-cyano-4-hydroxycinnamic acid (4CCA) matrix were prepared. The samples were prepared at a concentration of 2 mg mL−1 in TA30. MALDI-TOF MS samples were prepared by combining 20 μL of matrix solution with 4 μL of sample solution. 0.5 μL of this sample was deposited on a Ground Steel MALDI target plate and allowed to air dry. Stock solutions of trans-2-[3-(4-tert-butylphenyl)-2methyl-2-propenylidene]malononitrile (DCTB) (10 mg mL−1) in 50:50 DCM:MeCN were prepared. The samples were prepared at a concentration of 2 mg mL−1 in DCM. MALDI-TOF MS samples were prepared by combining 20 μL of matrix solution with 4 μL of sample solution. 0.3 μL of this sample was deposited on a Ground Steel MALDI target plate and allowed to air dry.Stock solutions of TA30 solvent  and a flow rate of 2 mL min−1. The mobile phase consisted of two solutions: H2O with 0.1% TFA and MeCN with 0.1% TFA, run in a gradient from 95% H2O/5% MeCN to 100% MeCN over 60 min. Analytical HPLC utilised a Waters Sunfire C18 analytical column  and a flow rate of 0.2 mL min−1. The mobile phase consisted of two solvent mixtures: H2O with 0.1% TFA and MeCN with 0.1% TFA, run in a gradient from 100% H2O to 100% MeCN over 45 min.All HPLC methods were performed using a variety of Waters HPLC systems equipped with Waters 2695 separation modules and tuneable absorbance UV/vis detectors (λ = 400–230 nm). Preparative separations utilised a Waters Sunfire C18 preparative column  and a flow rate of 7 mL minP values of the Gd(III) complexes were measured by means of a standard reverse-phase HPLC method on a Waters 2695 separations module equipped with the Waters Alliance column heater (set at 30 °C) and Waters 2996 Photodiode Array (PDA) detector using a Waters Sunfire C18 column . The mobile phase consisted of 65% (v/v) MeOH in a 50 mM sodium phosphate buffer adjusted to pH 7.4 by mixing 50 mM solutions of Na2HPO4 and NaH2PO4. The samples, along with a set of standards with known logP values, were determined using the solvent with an isocratic flow-rate of 0.2 mL min−1. The retention times of acetone (logD7.4 − 0.24), aniline (logD7.4 0.90), phenol (logD7.4 1.50), toluene (logD7.4 2.730), cumene (logD7.4 3.66), triphenylamine (logD7.4 5.74), and hexachlorobenzene (logD7.4 6.35) were plotted against their literature logD7.4 values, adjusting for the T0, to establish a calibration curve42. Fitting an exponential curve to the dataset generated the equation (y = 0.8822ln(x) + 1.8164; r2 = 0.984) from which the sample logP values could be derived using their retention times. The HPLC experiments were performed in triplicate.The logtert-butyl acetate) (DO3A-tBu3·HBr) was prepared using a method adapted from Moore et al.45. 1,4,7,10-Tetraazacyclododecane-1,4,7-tris(tert-butyl acetate)-10-acetic acid (DOTA-tBu3) was prepared using a method adapted from Strauch46. Complex 11 was prepared as previously described by Morrison et al8.1,4,7,10-Tetraazacyclododecane-1,4,7-tris. The crystallized product was filtered off and washed with toluene and diethyl ether to yield the phosphonium salt in high purity.Phosphonium salts of interest were prepared by means of dropwise addition of the precursor arylphosphines in toluene to a stirred solution of the relevant dibromo-3 in DMF at RT for 72 h. The DMF was removed under a flow of N2, and the residue was then dissolved in CHCl3 and any excess NaN3 filtered off. The filtrate was reduced in vacuo to yield the phosphonium azide in high purity (> 95%). The azide group was reduced to an amine using 10 mol% Pd/C stirred in MeOH under a H2 atmosphere at RT for 6 h. The catalyst was removed by means of filtration, and the solvent volume reduced to afford the phosphonium-amine in high purity. The phosphonium targeting vectors were coupled to the DOTA chelating component by means of a peptide coupling reaction performed in peptide-grade DMF with the HATU coupling reagent in the presence of excess NMM and stirred at RT for 24 h. The DMF and NMM were removed under N2, and the residue was deprotected by the addition of neat TFA upon stirring the mixture for a further 12 h at RT. The volatiles were removed in vacuo, and the crude residue dissolved in H2O and washed with CHCl3. The aqueous fraction was reduced in vacuo to give a hygroscopic solid which was purified by means of reverse-phase HPLC, and the product lyophilised to afford the pure ligand.The bromine functional groups of the phosphonium salts prepared in GP1 were replaced with azide groups by stirring with NaN2O3 in a minimum amount of H2O for 72 h at RT. Any remaining Gd2O3 was removed by means of centrifugation and subsequent filtration through a 0.2 μm filter. The filtrate was lyophilised to afford the purified Gd(III) complex as a colourless powder.The purified ligands were complexed with Gd(III) by stirring a suspension of excess Gd1): GP1 using triphenylphosphine  and α,α’-dibromo-p-xylene  in 30 mL of toluene heated at reflux for 4 h to yield 5.02 g (95.3%) of the phosphonium salt. By following GP2, the phosphonium salt  was added to excess NaN3  in DMF (50 mL) affording the colourless, crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 50 mg) in MeOH (20 mL) under a H2 atmosphere yielded the corresponding amine  as a colourless solid. Slow addition of this amine  in DMF (20 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (40 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (10 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . ESI–MS for [M+]: calculated m/z 768.35, observed m/z 768.32. 1H NMR (D2O) δ 7.85–7.80 , 7.61–7.54 , 7.12 , 6.92 , 4.33 , 4.20–3.00 . 13C NMR (D2O) δ 162.7  TFA, 2JCF = 35.7 Hz), 138.2 , 135.1 , 134.0 , 131.2 , 129.9 , 127.7 , 126.5 , 117.2 (m), 54.9, 53.3, 50.8–48.9 , 42.6 , 29.4 , C(O) of the acetate arms could not be located. 31P{1H} NMR (D2O) δ 22.1 (s). The purified ligand  was stirred with a suspension of Gd2O3  in water for 72 h at RT, and excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. ESI-FT-ICR-MS for [M]+: calculated m/z 923.25269, observed m/z 923.25249.2,2′,2″-(10-(2-((4-((Triphenylphosphonio)methyl)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetatogadolinium(III) trifluoroacetate (2): GP1 using methyldiphenylphosphine  and α,α’-dibromo-p-xylene  each in 20 mL of toluene at reflux for 2 h to yield 2.0 g (81.4%) of the phosphonium salt. Following GP2, the phosphonium salt  was added to excess NaN3  in DMF (15 mL) affording the colourless crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 100 mg) in MeOH (50 mL) under a H2 atmosphere yielded the corresponding amine  as a colourless solid. Slow addition of this amine  in DMF (10 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (30 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (12 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . ESI–MS for [M+·tBu3]: calculated m/z 874.52, observed m/z 874.55. ESI–MS for [M+]: calculated m/z 706.34, observed m/z 706.34. 1H NMR (CDCl3) δ 7.81–7.70 , 7.62–7.60 , 7.54–7.49 , 7.21–7.16 , 7.09 , 4.44 , 4.25–3.11 , 2.71 . 13C NMR (D2O) δ 166.1  TFA, 2JCF = 36.3 Hz), 138.4 , 135.2 , 133.6 , 130.9 , 129.9 , 127.4 , 126.7 , 117.9 (m), 54.7, 53.0, 51.4–48.3 , 42.1 , 29.9 , 5.4 , C(O) of the acetate arms could not be located. 31P{1H} NMR ((D2O) δ 22.2 (s). GP3 The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. ESI-FT-ICR-MS for [M]+: calculated m/z 861.23704, observed m/z 861.23663.2,2′,2″-(10-(2-((4-((Methyldiphenylphosphonio)methyl)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetatogadolinium(III) trifluoroacetate (3): GP1 using isopropyldiphenylphosphine  and α,α’-dibromo-p-xylene  each in 20 mL of toluene at RT for 24 h, ensuring the air sensitive phosphine precursor was kept out of contact with oxygen, to yield 1.7 g (73.9%) of the phosphonium salt. The phosphonium salt  was added to excess NaN3  in DMF (15 mL) affording the colourless crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 60 mg) in MeOH (20 mL) under a H2 atmosphere yielded the corresponding amine (506 mg (89.9%) as a colourless solid. Slow addition of this amine  in DMF (5 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (15 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (10 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . ESI–MS for [M+·tBu3]: calculated m/z 902.56, observed m/z 902.51. ESI–MS for [M+]: calculated m/z 734.37, observed m/z 734.40. 1H NMR (D2O) δ 7.71 , 7.60–7.45 , 6.99 , 6.79 , 4.25 , 4.10–2.7  1.19 2, 3JHP = 18.2 Hz, 3JHH = 7.1 Hz). 13C NMR (D2O) δ 168.1  TFA, 2JCF = 36.6 Hz), 138.6 , 135.1 , 134.1 , 131.5 , 129.7 , 126.5 , 123.7 (m), 116.1 (m), 56.6, 53.3, 51.8–47.0 , 42.3 , 27.3 , 20.3 , 15.2 (d), C(O) of the acetate arms could not be located. 31P{1H} NMR (D2O) δ 34.0 (s). GP3 The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. ESI-FT-ICR-MS for [M]+: calculated m/z 889.26927, observed m/z 889.26805.2,2′,2″-(10-(2-((4-((Isopropyldiphenylphosphonio)methyl)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetatogadolinium(III) trifluoroacetate (4): GP1 using diphenyl(p-tolyl)phosphine  and α,α’-dibromo-p-xylene  each in 20 mL of toluene at RT for 24 h to yield 1.0 g (89.4%) of the phosphonium salt. The phosphonium salt  was added to excess NaN3  in DMF (20 mL) affording the colourless, crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 30 mg) in MeOH (15 mL) under a H2 atmosphere yielded the corresponding amine (276 mg (88.1%) as a colourless solid. Slow addition of this amine  in DMF (5 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (10 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (5 mL), dissolved in H2O (100 mL), washed, purified by means of reversed-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . ESI–MS for [M·tBu3 + Na]2+: calculated m/z 486.77, observed m/z 486.75. ESI–MS for [M+]: calculated m/z 782.37, observed m/z 782.32. 1H NMR (D2O) δ 7.90–7.88 , 7.68–7.61 , 7.61–7.52 P+, m-Ph(Me)P+), 7.48 , 7.05 , 4.15 , 3.95–3.02 , 2.49 . 13C NMR (D2O) δ 162.8  TFA, 2JCF = 35.7 Hz), 147.1 P+), 135.2 , 133.7 P+, 3JCP = 9.1 Hz), 132.0 , 131.4 P+, 2JCP = 12.8 Hz), 130.7 , 129.3–129.1 , 116.6 (m), 112.8 (m), 55.2, 53.5, 51.2–49.7 , 48.4 , 29.4 , 20.9 , C(O) of the acetate arms could not be located. 31P{1H} NMR (D2O) δ 22.0 (s). GP3 The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. ESI-FT-ICR-MS for [M]+: calculated m/z 937.26834, observed m/z 937.26658.2,2′,2″-(10-(2-((4-((Diphenyl(p-tolyl)phosphonio)methyl)benzyl)amino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetatogadolinium(III) trifluoroacetate (5): GP1 using tris(4-methoxyphenyl)phosphine  and α,α’-dibromo-p-xylene  each in 20 mL of toluene at RT for 24 h to yield 1.4 g (81.4%) of the phosphonium salt. The phosphonium salt  was added to excess NaN3  in DMF (22 mL) affording the colourless crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 60 mg) in MeOH (25 mL) under a H2 atmosphere yielded the corresponding amine  as a colourless solid. Slow addition of this amine  in DMF (10 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (20 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (10 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . ESI–MS for [M+·tBu3]: calculated m/z 1026.57, observed m/z 1026.52. ESI–MS for [M+]: calculated m/z 858.38, observed m/z 858.35. 1H NMR (D2O) δ 7.52 , 7.25 , 7.12 , 7.00 , 4.50 , 4.23 , 3.95–2.73 . 13C NMR (D2O) δ 164.4 , 162.9  TFA, 2JCF = 34.8 Hz), 136.7 , 135.7 , 132.0 , 131.3 , 116.3 (m), 115.6 , 108.1 (d), 55.8 , 54.7, 53.4, 50.8–48.9 , 43.6 , 30.4 , C(O) of the acetate arms could not be located. 31P{1H} NMR (D2O) δ 20.4 (s). GP3 The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. ESI-FT-ICR-MS for [M]+: calculated m/z 1013.28438, observed m/z 1013.28328.2,2′,2″-(10-(2-Oxo-2-((4-((tris(4-methoxyphenyl)phosphonio)methyl)benzyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetatogadolinium(III) trifluoroacetate (6): The phosphonium salt  was added to excess NaN3  in DMF (25 mL) affording the colourless crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 30 mg) in MeOH (20 mL) under a H2 atmosphere yielded the corresponding amine  as a colourless solid. Slow addition of this amine  in DMF (10 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (15 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (10 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . MALDI-TOF–MS for [M+·tBu3]: calculated m/z 874.52, observed m/z 874.55. MALDI-TOF–MS for [M+]: calculated m/z 706.34, observed m/z 706.40.1H NMR (D2O) δ 7.81–7.53 , 3.85 , 3.57–3.12 , 1.56 . 13C NMR (D2O) δ 162.7, 134.3 , 133.1 , 129.8 , 118.1 (m), 57.3, 56.4, 50.4–48.7 , 42.8 , 24.9 , 19.9 . 31P{1H} NMR (D2O) δ 24.0 (s). The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. MALDI-TOF–MS for [M]+: calculated m/z 861.237, observed m/z 861.317.2,2′,2″-(10-(2-Oxo-2-((3-(triphenylphosphonio)propyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetato gadolinium(III) trifluoroacetate (7): The phosphonium salt  was added to excess NaN3  in DMF (25 mL) affording the colourless crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 50 mg) in MeOH (20 mL) under a H2 atmosphere yielded the corresponding amine  as a colourless solid. Slow addition of this amine  in DMF (10 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (20 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (15 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . MALDI-TOF–MS for [M+·tBu3]: calculated m/z 888.54, observed m/z 888.57. MALDI-TOF–MS for [M+]: calculated m/z 720.35, observed m/z 720.32. 1H NMR (D2O) δ 7.85–7.49 , 3.83 , 3.62–3.09 , 1.77 , 1.57 . 13C NMR (D2O) δ 162.6, 134.9 , 133.4 , 130.3 , 117.8 (m), 57.3, 56.4, 50.4–48.7 , 41.4 , 31.3 , 22.4 , 19.4 . 31P{1H} NMR (D2O) δ 23.9 (s). The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. MALDI-TOF–MS for [M]+: calculated m/z 875.253, observed m/z 875.213.2,2′,2″-(10-(2-Oxo-2-((4-(triphenylphosphonio)butyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetato gadolinium(III) trifluoroacetate (8): The phosphonium salt  was added to excess NaN3  in DMF (16 mL) affording the colourless crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 60 mg) in MeOH (20 mL) under a H2 atmosphere yielded the corresponding amine  as a colourless solid. Slow addition of this amine  in DMF (10 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (20 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (15 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . MALDI-TOF–MS for [M+·tBu3]: calculated m/z 902.56, observed m/z 902.52. MALDI-TOF–MS for [M+]: calculated m/z 734.37, observed m/z 734.37. 1H NMR (D2O) δ 7.72–7.56 , 3.65 , 3.55–3.03 , 1.65 , 1.55 , 1.51 . 13C NMR (D2O) δ 162.7, 135.7 , 133.9 , 130.8 , 117.4 (m), 58.8, 57.2, 51.6–50.2 , 43.8 , 32.2 (s 4-CH2), 24.3 , 20.1 , 19.5 . 31P{1H} NMR (D2O) δ 24.3 (s). GP3 The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. MALDI-TOF–MS for [M]+: calculated m/z 889.268, observed m/z 889.267.2,2′,2″-(10-(2-Oxo-2-((5-(triphenylphosphonio)pentyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetato gadolinium(III) trifluoroacetate (9): The phosphonium salt  was added to excess NaN3  in DMF (25 mL) affording the colourless crystalline azide . Hydrogenation of the azide  using 10% Pd/C (ca. 40 mg) in MeOH (20 mL) under a H2 atmosphere yielded the corresponding amine  as a colourless solid. Slow addition of this amine  in DMF (10 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (20 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (10 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . MALDI-TOF–MS for [M+·tBu3]: calculated m/z 916.57, observed m/z 916.55. MALDI-TOF–MS for [M+]: calculated m/z 748.38, observed m/z 748.41. 1H NMR (D2O) δ 7.82–7.51 , 3.44 , 3.38–3.02 , 1.62 , 1.41 , 1.24 , 1.17 . 13C NMR (D2O) δ 162.7, 134.4 , 132.9 , 129.7 , 117.0 (m), 58.1, 56.8, 51.4–48.3 , 42.8 , 32.4 , 29.0 , 28.9 , 28.0  25.7 . 31P{1H} NMR (D2O ) δ 24.0 (s). The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. MALDI-TOF–MS for [M]+: calculated m/z 903.284, observed m/z 903.306.2,2′,2″-(10-(2-Oxo-2-((6-(triphenylphosphonio)hexyl)amino)ethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetato gadolinium(III) trifluoroacetate (10): The phosphonium salt  was added to excess NaN3  in DMF (30 mL) affording a light yellow oil azide . Hydrogenation of the azide  using freshly prepared Raney nickel (ca. 100 mg) in MeOH (50 mL) under a H2 atmosphere (10 atm) at 50 °C for 72 h yielded the corresponding amine  as a slightly yellow oil. Slow addition of this amine  in DMF (5 mL) to a stirred solution of DOTA-tBu3 , HATU , and NMM  in DMF (15 mL) yielded the crude protected ligand, which was subsequently deprotected with neat TFA (10 mL), dissolved in H2O (100 mL), washed, purified by means of reverse-phase HPLC, and lyophilised to afford the pure ligand as a colourless powder . MALDI-TOF–MS for [M+·tBu3]: calculated m/z 992.59, observed m/z 992.36. MALDI-TOF–MS for [M+]: calculated m/z 824.40, observed m/z 824.42.1H NMR (D2O) δ 7.72- 7.51 , 3.81–3.12 . 13C NMR (D2O) δ 164.1, 135.1 , 133.6 , 130.3 , 116.9 (m), 69.8 , 69.2 , 68.6 , 58.0, 56.7, 50.8–49.3 , 41.6 ), 31.2 . 31P{1H} NMR (D2O) δ 24.5 (s). GP3 The purified ligand  was stirred with Gd2O3  in water, excess Gd2O3 removed and the solution lyophilised to afford the pure complex as a colourless powder . Purity > 95% by HPLC. MALDI-TOF–MS for [M]+: calculated m/z 979.300, observed m/z 979.272.2,2′,2″-(10-(2-Oxo-14-(triphenylphosphonio)-6,9,12-trioxa-3-azatetradecyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetatogadolinium(III) trifluoroacetate  was prepared, a portion of which was adjusted to pH 5.8 using NaOH, while other portions were adjusted to pH 5.0, 4.0, or 3.0 using glacial acetic acid. Xylenol orange (5.81 mg) was dissolved in the pH 5.8 acetate buffer (48.4 mL) to give a 10 × concentrated stock solution. These solutions were treated as light sensitive, work was conducted in minimal light and solutions were covered in foil.Solution stability assays were performed using the method described by Barge et al.3, 1 and 11 were prepared in the various buffer solutions. Two serial dilutions of each solution were freshly prepared, the first ranging from 25 mM to 19.5 μM, and the second ranging from 20 mM to 31.2 μM. To the wells of a 384 well plate were added 80 μL of the desired pH buffer and 10 μL of the selected serial dilution (in triplicate). The plates were agitated in a plate reader and allowed to equilibrate for 24 h. To each of the wells was added 10 μL of the 10 × concentrated xylenol orange stock solution in the absence of light. The plates were again agitated in a plate reader and allowed to equilibrate for 15 min at RT. The absorbance values were then determined at both 573 nm and 433 nm by means of a BMG LABTECH POLARstar Omega, UV Spectrometer, and the data plotted and processed using Graphpad Prism 7.25 mM stock solutions of each GdClv/v), penicillin (100 units mL−1), streptomycin (100 μg mL−1) and L-glutamine (2.5 mM), within incubators at 37 °C in a humidified 5% CO2 atmosphere. Centrifugation was performed at 3000 rpm for 5 min. Earls MEM, FCS, PBS, Trypsin, L-glutamine, and the antibiotic solutions were purchased from ThermoFisher scientific.The human glioblastoma multiforme (T98G) and human glial (SVG p12) cell lines were purchased from the ATCC and were maintained as monolayers in a minimum essential medium supplemented with foetal bovine serum , the trypsin diluted with complete medium, and cell pellets isolated via centrifugation. Any remaining traces of trypsin were removed from the pellet via a suspension/centrifugation step. The pellets were then completely re-suspended, the number of cells mL−1 was determined using a hemocytometer (Weber), and the solution diluted such that 90 μL aliquots of cell suspension per well of a 96-well plate yielded 1 × 104 cells per well. The cells were incubated for 24 h to allow them to adhere.The in vitro cytotoxicities of all Gd(III) complexes were assessed in both T98G and SVG p12 cell lines using the colourimetric 3--2,5-diphenyltetrazolium bromide (MTT) assay1–11) or the relevant vehicle (control). Each dilution/vehicle pair was repeated in triplicate within a plate and each plate was repeated in triplicate, for both cell lines. After 72 h incubation, MTT solution in phosphate-buffered saline  was added to all wells in the absence of light and the incubation was continued for a further 4 h. The culture medium and excess MTT solution were carefully removed so as not to disrupt any MTT–formazan crystals formed during the incubation and were subsequently dissolved in 150 μL of DMSO.The cells were subsequently dosed with 10 μL per well of a sterile 10 × stock of the serial dilution (2 mM–62.5 μM) of Gd(III) complex . Corresponding IC3 cell culture flasks were seeded with 3 mL of a cell suspension to yield 2 × 105 cells per flask and were incubated for 72 h until ~ 80% confluent. Sterile 20 mM stock solutions of the Gd(III) complexes 1–10 were diluted with warm (37 °C) culture medium to yield a series dilution of final concentrations including 1000 μM, 500 μM, 250 μM, 125 μM and 62.5 μM. The non-dosed culture medium in each flask was removed and replaced with Gd-dosed media or a relevant vehicle (control), each concentration was repeated in triplicate for both cell lines along with the relevant control. The cells were incubated for a further 48 h after which they were harvested.25 cmv/v). The trypsin was diluted with medium, and cell pellets isolated by means of centrifugation before the supernatant was removed and the pellets re-suspended in warm PBS. Further centrifugation produced washed pellets, eliminating the possibility of residual Gd(III) complex remaining outside of the cells, which were subsequently re-suspended in warm PBS (1 mL). From this suspension, 100 μL was set aside for protein analysis and the remaining 900 μL was centrifuged to isolate the cell pellets. The supernatant was removed, and the cells were digested in concentrated HNO3  at 40 °C in a heating block for 24 h. 100 μL of the digest solution was diluted to 10 mL to result in a 1% HNO3 solution which was measured for Gd content by means of ICP-MS. ICP-MS was run on a Perkin Elmer ELAN 6100 Inductively Coupled Plasma Emission Mass Spectrometer (ICP-MS). Gd uptake is reported as μg Gd/mg protein ± standard error.The culture medium was removed, and the monolayers washed with warm (37 °C) PBS (1 mL) prior to treatment with trypsin  protein standard curve was prepared each time the assay was performed, ranging from 1.0 to 0.1 mg mL−1 in PBS. Lysis of the 100 μL cell suspension was achieved using three snap freeze-heat cycles and thorough pipette mixing. The protein content of each solution was then analysed by pipetting 5 μL samples of the blanks, vehicle (control), and treated cell solutions into a 96-well plate, adding 25 μL of the alkaline copper tartrate solution and 200 μL of the Folin reagent solution included in the Bio-Rad kit in the absence of light. Each sample was prepared in triplicate. The plates were incubated at 37 °C for 15 min and the absorbance measured at 750 nm using a BMG LABTECH POLARstar Omega UV spectrometer. The protein concentration was determined by correcting all measurements with the background controls and comparing the absorbance with that of the BSA standard curve.A Bio-Rad DC protein assay kit was used to determine protein concentrationsSupplementary Information.


N-Acetyl glucosamine (GlcNAc) and N-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4–(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs.N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12–35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The Vmax, Km, Kcat, and Kcat/Km of CmNAGase for pNP-GlcNAc were 16,666.67 μmol min−1 mg−1, 0.50 μmol mL−1, 25,555.56 s−1, and 51,111.12 mL μmol−1 s−1, respectively. Analysis of the hydrolysis products of N-Acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-Acetyl COSs ((GlcNAc)3–(GlcNAc)7) from (GlcNAc)2–(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers.The gene encoding the novel β-CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.The observations recorded in this study that  N-Acetyl glucosamine (GlcNAc), is the second most abundant renewable source in nature behind cellulose and mainly exists in crustacean shells, fungal cell walls, and insect exoskeletons  , β-N-ace GlcNAc) , 17, andeactions . These eeactions –27. Howeeactions –34. Thereactions , 35, 36.Chitinolyticbacter meiyuanensis SYBC-H1 with a good ability to degrade chitin was isolated from soil [Escherichia coli BL21(DE3). The phylogenetic relationships and catalytic characteristics of the purified recombinant NAGase were described. Furthermore, its transglycosylation and reverse hydrolysis activities were also investigated.In our previous study, a chitinolytic bacterium rom soil . In thisC. meiyuanensis SYBC-H1 was performed. Following ammonium sulfate precipitation, anion exchange chromatography, and SDS-PAGE, a protein band with NAGase activity was obtained, as shown by zymogram analysis. The band with NAGase activity (named CmNAGase) had a molecular mass of between 75 and 100 kDa  analysis using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF MS/MS), and the results of PMF were interpreted by referencing the Mascot database . ProteinCmNAGase gene was cloned. As expected, a PCR product of 2.5 kb was obtained. Analysis of the PCR product showed that the CmNAGase gene was 2508 bp, encoding for a protein of 836 amino acids. The calculated molecular mass of CmNAGase was 91.6 kDa, and the isoelectric point (pI) was predicted to be 5.48. The sequence analysis suggested no putative signal peptide in the sequence of CmNAGase, which suggested that CmNAGase should be a non-secretory protein. The prediction using Gneg-mPLoc 2.0 also showed that the location of CmNAGase was in the periplasm. However, CmNAGase could be purified from the fermentation broth of the strain SYBC-H1. Furthermore, most reported NAGases are secretory proteins to date [CmNAGase location may be wrong.The  to date –23. Theshttp://www.cazy.org/), NAGases can be classified as part of the glycoside hydrolase (GH) families 3, 20, 73, 84, and 85 based on amino acid sequence homology. BLASTP analysis showed that CmNAGase belonged to GH family 20 (GH20) and shared the highest identity (81.68%) with the GH20 NAGase ChiI from Chitiniphilus shinanonensis (WP_018749679) [Chitiniphilus sp. HX-2-15 (WP_136772659). However, these coding genes have not been expressed and studied. Among characterized GH20 NAGases, CmNAGase showed the highest identity (77.58%) with GH20 NAGase from Aeromonas sp. 10S-24 (Accession no. BAA92145) [Serratia marcescens (PDB 1QBA) [Aeromonas caviae CB101 (Accession no. CAH55822) [Vibrio harveyi (PDB 6EZR) [Enterobacter sp. G-1 (Accession no. BAA74506) [Aeromonas hydrophila SUWA-9 (Accession no. BAF76001) [V. harveyi (Accession no. ADJ68332) [Arthrobacter sp. TAD20 (Accession no. CAB72127) [CmNAGase and verified GH20 NAGases were performed to construct the phylogenic tree, which also showed that CmNAGase exhibited a low sequence identity (12–35%) with most of functionally characterized bacterial GH20 NAGases  database (8749679) , followeAA92145) , followiDB 1QBA) , GH20 NAAH55822) , GH20 NADB 6EZR) , GH20 NAAA74506) , and GH2DJ68332) , and GH2AB72127) . The putCmNAGase) acts as the general acid/base residue for protonation, while Asp (D508 in CmNAGase) acts to orient the C2-acetamido group into position for correct nucleophilic attack by a water molecule, and subsequently provides the negatively charged carboxylate groups to stabilize the positively charged oxazolinium ion intermediate [CmNAGase with other GH20 NAGases from different sources indicated the substrate-binding residues  and catalytic residues (D508 and E509) in CmNAGase, which are highly conserved among GH20 members 8 fold in the GH20 catalytic domain ; Glyco_hydro_20b domain of residues 174–300 ; Glyco_hydro_20 domain of residues 304–719 ; and CHB_HEX C_1 domain of residues 753–828 (unknown function)  [CmNAGase  . The actCmNAGase gene was successfully expressed in E. coli BL 21 (DE3) at a high expression level . The recombinant CmNAGase with a C-terminal His6-tag was purified by Ni-NTA affinity chromatography with a yield of 80.5%. The reason for the high purification yield may be that the C-terminal His6-tag in the recombinant CmNAGase was well exposed, which led to the higher affinity with Ni-NTA resin. The specific activity of recombinant CmNAGase increased 1.5-fold from 3156.5 U/mg to 4878.6 U/mg after purification  [Paenibacillus sp. TS12 (6.0) [Vibrio harveyi 650 (7.5) [Enterobacter sp. G-1 (6.0) [Salmonella enterica (4.0) [Paraglaciecola hydrolytica S66 (6.0) [Cellulomonas fimi (7.3–8.7) [CmNAGase presented good activity after being stored at pH 4.0–8.5 for more than 84 h  , Paeniba12 (6.0) , Vibrio 50 (7.5) , Enterob-1 (6.0) , Salmoneca (4.0) , Paragla66 (6.0) , and Cels fimi 7.–8.7 [CmN NAGases , 51, 52.CmNAGase was 40 °C, which is different from that of NAGases from Shinella sp. (50 °C) [Trichoderma reesei (60 °C) [P. hydrolytica S66 (50 °C) [Enterobacter sp. G-1 (45 °C) [S. marcescens (52 °C) [CmNAGase was unstable at temperatures ˃ 40 °C  , Trichod (60 °C) , P. hydr (50 °C) , Enterob (45 °C) , and S.  (52 °C) . The CmN °C Fig. d. Half-lus casei , Sphingorium sp. , and AerCmNAGase were investigated. All counter-ions of the used metal ions were Cl−. As shown in Table CmNAGase is not metal-dependent. CmNAGase activity is completely inhibited by Zn2+, Cu2+, and Al3+, severely inhibited by Ba2+, Fe3+, and Cr3+. To date, many studies have shown that Zn2+, Cu2+, Fe3+, and Al3+ inhibit the activity of NAGases. For example, the GH20 NAGase from A. caviae is strongly inhibited by Cu2+ and Zn2+ [Paenibacillus sp. is strongly inhibited by Zn2+ [T. reesei is partially inhibited by Fe3+ [2+ enhanced the activity of CmNAGase, which is different from the GH20 NAGase from A. caviae (strongly inhibited by Mn2+) [2+ is unclear and the in-depth study is needed in the future.The effects of metal ions on and Zn2+ ; the GH2 by Zn2+ , and the by Fe3+ . Mn2+ enby Mn2+) . HoweverCmNAGase against various substrates were investigated using standard assay conditions. As shown in Table CmNAGase can hydrolyze pNP-GlcNAc, 4-MU-GlcNAc, and (GlcNAc)2–(GlcNAc)6. No activity was observed when pNP-glucose, pNP-acetyl galactosaminide, and cellobiose were used as the substrates. These results showed that CmNAGase represents the typical NAGase activity with strict substrate specificity. The specific activity of CmNAGase toward pNP-GlcNAc can reach 4878.6 U/mg, which is higher than most reported NAGases (< 2000 U/mg) [The specific activities of the recombinant 00 U/mg) , 48, 56.2 among natural substrates, and do not hydrolyze N-acetyl COSs and chitin polymer [CmNAGase showed good activities toward (GlcNAc)2–(GlcNAc)6 with the highest activity for (GlcNAc)2, followed by (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6. These results show that specific activity of CmNAGase toward N-Acetyl COSs decreased when the degree of polymerization increased, which is similar with other reports [LeHex20A from Lentinula edodes for (GlcNAc)6 was greater than for (GlcNAc)2 [VhNag2 from V. harveyi showed the highest activity against (GlcNAc)4, while the lowest activity was observed with (GlcNAc)2 [CmNAGase. Furthermore, CmNAGase showed some activity (0.02 U/mg) toward colloidal chitin. The result is similar with the GH20 NAGases from V. harveyi [Myceliopthora thermophila [L. edodes [M. anisopliae [Most of the GH20 NAGases have the highest catalytic efficiency for (GlcNAc) polymer , 56. How reports . The catGlcNAc)2 . The GH2GlcNAc)2 . These N harveyi  and the rmophila , L. edod. edodes , and M. isopliae , which wCmNAGase were also measured with pNP-GlcNAc as the substrate. The results showed that the Vmax, Km, Kcat, and Kcat/Km for CmNAGase were 16,666.67 μmol min−1 mg−1, 0.5 μmol mL−1, 25,555.56 s−1, and 51,111.12 mL μmol−1 s−1, respectively.In addition, the kinetic parameters for LeHex20A from L. edodes hydrolyzes colloidal chitin to various N-acetyl COSs at the start of the reaction, and these N-Acetyl COSs convert to GlcNAc after 3 h, which is different from CmNAGase.As shown in Fig. CmNAGase, (GlcNAc)2–(GlcNAc)6 were used as substrates. The overall rates of hydrolysis were in the order: (GlcNAc)2 > (GlcNAc)3 > (GlcNAc)4 > (GlcNAc)5 > (GlcNAc)6, which is consistent with the results of substrate specificities above. (GlcNAc)2 was degraded by CmNAGase to GlcNAc 3 2, and (GlcNAc)3 were produced 3, and (GlcNAc)4 were obtained from (GlcNAc)5 2, (GlcNAc)3, (GlcNAc)4, and (GlcNAc)5 were found 6 were both hydrolyzed to GlcNAc as the final product with the increase of hydrolysis time. Similar results were reported for GH20 NAGase (VhNag2) from V. harveyi [LeHex20A from L. edodes [VhNag2 and LeHex20A toward N-Acetyl COSs were in the order: (GlcNAc)4 > (GlcNAc)3 > (GlcNAc)5 > (GlcNAc)6 > (GlcNAc)2. Based on these results, we conclude that CmNAGase is a typical exo-acting NAGase.To evaluate the hydrolysis and transglycosylation activities of  harveyi  and LeHe. edodes . However3, (GlcNAc)4, (GlcNAc)5, (GlcNAc)6, and new peak generated after the peak of (GlcNAc)6 were also produced from (GlcNAc)2, (GlcNAc)3, (GlcNAc)4, (GlcNAc)5, and (GlcNAc)6 in short reaction times, respectively 7 and (GlcNAc)7 with a hydrogen adduct 3–(GlcNAc)7 from (GlcNAc)2–(GlcNAc)6, respectively. To date, most scholars all believe that production of (GlcNAc)n+1 from (GlcNAc)n (n=2–7) is a transglycosylation reaction [2 and (GlcNAc)n may form (GlcNAc)n+1 by transglycosylation. Meanwhile, GlcNAc and (GlcNAc)n could also form (GlcNAc)n+1 through reverse hydrolysis. It is very hard to tell the two reactions apart in this setup. Thus, we call this reaction transglycosylation for the time being.In addition, minor (GlcNAc)reaction , 29, 34.N-Acetyl COSs from shorter N-Acetyl COSs substrates via transglycosylation activity. For example, a chitinase from C. shinanonensis generated (GlcNAc)5 and (GlcNAc)6 when incubated with (GlcNAc)4 [T. reesei KDR-11 was shown to convert (GlcNAc)4 into (GlcNAc)6 [Flavobacterium johnsoniae synthesized (GlcNAc)6–(GlcNAc)8 and (GlcNAc)7–(GlcNAc)9 from (GlcNAc)5 and (GlcNAc)6, respectively [Microbulbifer thermotolerans DAU221 produced (GlcNAc)4 from (GlcNAc)3 [To date, some chitinases have been used to synthesize higher GlcNAc)4 . A chitiGlcNAc)6 . An endoectively . A chitiGlcNAc)3 .N-Acetyl COSs from shorter N-Acetyl COSs was often catalyzed by an auto-condensation reaction  [A. oryzae was used to catalyze the formation of (GlcNAc)3 and (GlcNAc)4 from (GlcNAc)2 [A. oryzae produces (GlcNAc)4–(GlcNAc)6 and (GlcNAc)5–(GlcNAc)6 from (GlcNAc)3 and (GlcNAc)4, respectively. GH20 NAGases SmHex from S. marcescens YS-1 [NoHex from N. orientalis IFO12806 [2 to (GlcNAc)3. GH20 NAGases AoHex from A. oryzae CCF1066 produce the mixture of (GlcNAc)2–(GlcNAc)8 from the mixture of GlcNAc–(GlcNAc)7 [CmNAGase is a novel bacteria-derived NAGase, which possesses transglycosylation activity toward (GlcNAc)2–(GlcNAc)6.For GH20 NAGases, production of higher cceptor) . For exaGlcNAc)2 . Singh eGlcNAc)2  reportedens YS-1  and NoHeIFO12806  were shoGlcNAc)7 . In comp3–(GlcNAc)7) from transglycosylation disappeared soon with the increase of reaction time 3–(GlcNAc)7.In addition, the products ((GlcNAc)ime Fig. b–f. This2–(GlcNAc)6, the reverse hydrolysis activity of CmNAGase was also investigated using GlcNAc as the substrate. As shown in Fig. 2 compared with the standard of GlcNAc–(GlcNAc)6. However, the retention time of peak 2 was between that of (GlcNAc)2 (8.2 min) and (GlcNAc)3 (12.3 min). To further identify peak 2, mass spectrum analysis was conducted. The m/z value of peak 2 was at 447.1586, which corresponds to (GlcNAc)2 (425.1766 Da) with a sodium adduct (22.9898 Da) GlcNAc)3 2.3 min. Acremonium persicinum CCF 1850, A. oryzae CCF 1066, Aspergillus flavipes CCF 2026, A. flavus CCF 1129, P. oxalicum CCF 2315, and A. terreus CCF 2539, which only produced β-GlcNAc-(1 → 6)-GlcNAc from GlcNAc [P. funiculosum CCF 1994, P. funiculosum CCF 2325, P. chrysogenum CCF 1269, and Trichoderma harzianum CCF 2687 synthesized β-GlcNAc-(1 → 3)-GlcNAc, β-GlcNAc-(1 → 4)-GlcNAc, and β-GlcNAc-(1 → 6)-GlcNAc from GlcNAc [A. fumigatus CCF 1059, P. pittii CCF 2277, and A. sojae CCF 3060 synthesized β-GlcNAc-(1 → 4)-GlcNAc and β-GlcNAc-(1 → 6)-GlcNAc from GlcNAc. Among these fungal NAGases, β-GlcNAc-(1 → 6)-GlcNAc is always the main product. Thus, the peak 1 in Fig. 2), and main peak 2 should be β-GlcNAc-(1 → 6)-GlcNAc. These results suggested that CmNAGase has reverse hydrolysis activity toward GlcNAc.The reverse hydrolysis reaction of GH20 NAGases toward GlcNAc can form many connection configurations of the GlcNAc dimer, for example by β(1 → 3), β(1 → 4), and β(1 → 6) glycosidic bonds, which is mainly determined by the regioselectivity of glycosidase . To datem GlcNAc , 31. Them GlcNAc . The GH2CmNAGase from C. meiyuanensis SYBC-H1. CmNAGase contains a GH20 family catalytic module and exhibits low similarity with reported GH20 NAGases. Analysis of the hydrolysis products from N-Acetyl COSs and colloidal chitin revealed that CmNAGase exhibited exo-acting activity. Interestingly, CmNAGase possesses transglycosylation activity toward (GlcNAc)2–(GlcNAc)6, which respectively leads to synthesis of (GlcNAc)3–(GlcNAc)7. In addition, CmNAGase also have reverse hydrolysis activity toward GlcNAc, which can produce its dimers with different linked. This is first report of a bacterial NAGase, which can produce GlcNAc dimers from GlcNAc via reverse hydrolysis activity.This study reports the isolation, cloning, and recombinant expression of the gene encoding N-Acetyl glucosaminide (4-MU-GlcNAc), p-Nitrophenyl N-Acetyl glucosaminide (pNP-GlcNAc), pNP-glucose, and pNP-acetyl galactosaminide were purchased from Aladdin reagent Co., Ltd . The standards of N-Acetyl chitooligosaccharides (N-Acetyl COSs) (purity: ≥ 95%) with degree of polymerization between 2 and 6 were acquired from Qingdao Bozhi Biotechnology Co., Ltd . Peptone and yeast extract were purchased from the Oxoid Co., Ltd. . All molecular reagents were purchased from TaKaRa . Colloidal chitin was prepared as described by Gao et al. [Chitin, 4-Methylumbelliferyl o et al. . Other cC. meiyuanensis strain SYBC-H1 (ATCC BAA-2140) used in this study was isolated previously [g at 4 °C and used for NAGase purification.The eviously . SYBC-H1eviously . The supE. coli strains were routinely cultivated aerobically at 37 °C in LB medium . E. coli transformants were grown in LB medium or on agar plates containing 50 μg/mL kanamycin.The strains, plasmids, and primers used in this study are listed in Additional file g for 30 min and dissolved in a suitable volume of 50 mM PBS (pH 7.0). The enzyme obtained in the previous step was further purified using a fast protein liquid chromatography (FPLC) system  with a DEAE Sepharose™ anion exchange column. The column was equilibrated with 50 mM Tris–HCl at pH 8.0, then protein was separated by gradient elution with NaCl solutions from 0.05 to 0.5 M. The purified NAGase was concentrated and collected using an ultrafiltration tube  at 4 °C. Then the purified enzyme was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with a 3% stacking gel and a 10% separating gel, according to the method described by Laemmli [NAGase was purified by saturation with ammonium sulfate, followed by anion exchange chromatography, and all purification procedures were carried out at 4 °C. The supernatant of the culture was used as a crude enzyme preparation and then fractionated at 40% to 60% saturation with ammonium sulfate. The precipitate was centrifuged at 12,000 Laemmli .CmNAGase, and the corresponding band was sliced for peptide mass fingerprinting (PMF) analysis.After electrophoresis, the gel was sliced vertically into two parts for staining and zymogram analysis. One part was stained using 0.1% Coomassie brilliant blue R-250 and then decolorized with a mixture of 10% acetic acid, 30% methanol, and 60% water. The other part was incubated with 2.5% (vol/vol) Triton X-100 for 30 min twice to refold, then was sprayed with 50 mM PBS (pH 7.0) containing 1 mM 4-MU-GlcNAc and incubated at 37 °C for 30 min; the NAGase strip became visible as fluorescence at 340 nm. Two parts were compared to determine the position of the http://www.matrixscience.com) to reveal the amino acid sequences of the peptide fragments. The peptide sequence was then aligned with the genome of C. meiyuanensis SYBC-H1  to find the NAGase and its coding gene.The gel sliced above was analyzed using the electrospray ionization quadrupole time-of-flight mass spectrometer (ESI-Q-TOF MS/MS) technique . These masses were then compared to theoretical mass values in the Mascot website databases (C. meiyuanensis SYBC-H1 was used as the template for polymerase chain reaction (PCR) amplification. According to the results of PMF and the complete genome of C. meiyuanensis SYBC-H1, the coding region of the CmNAGase gene was amplified by PCR with the primer pair CmNAGase-F-5′-GAATTCCATATGATGAGCCGTCCCGCCGGATC-3′ and CmNAGase-R-5′-TCCGCTCGAGTCAGGCGCCCACCTGCACCG-3′. The PCR conditions were as follows: 5 min at 94 °C, followed by 30 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 10 min. The amplified PCR product was purified by gel electrophoresis, digested with restriction enzymes NdeI and XhoI, and then ligated into pMD19-T Simple vector and sequenced by Invitrogen Corporation . The positive recombinant plasmids were digested with NdeI and XhoI, and the gene was inserted into the pET-28a(+) vector expression plasmid with a C-terminal His6-tag to generate the pET-28a(+)-CmNAGase.The genomic DNA of http://web.expasy.org/protparam/) [http://smart.embl-heidelberg.de/) [http://blast.ncbi.nlm.nih.gov/Blast.cgi) [http://www.cbs.dtu.dk/services/SignalP/) [http://www.csbio.sjtu.edu.cn/bioinf/Gneg-multi/) [http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi) [CmNAGase was predicted with RaptorX (http://raptorx.uchicago.edu/StructPredV2/predict/) [Nucleotide and amino acid sequences were analyzed using Snap Gene™ 1.1.3 software and the ExPASy Protparam tool (tparam/) . The conerg.de/) . The DNAast.cgi) , respectignalP/)  and Gneg-multi/) . Proteinipt.cgi) . Three-dredict/) .CmNAGase above was transformed into competent E. coli BL21(DE3) for protein expression. The E. coli BL21(DE3) harboring the pET-28a(+)-CmNAGase plasmid were cultured in LB medium (containing 50 μg/mL kanamycin) at 37 °C in a shaker with a rotation speed of 200 rpm. When the optical density (OD600) of the culture medium reached 0.6–0.8, isopropyl β-d-thiogalactoside (IPTG) was added at a final concentration of 1 mM for protein induction, and the culture was further grown at 25 °C for 12 h.The recombinant plasmid pET-28a(+)-g and 4 °C for 10 min, after which the cells were re-suspended with His6-tag binding buffer  and lysed by JY92-IIN ultrasonication . Cell debris was removed by centrifugation at 6000g for 10 min at 4 °C and the supernatant was retained as crude enzyme. The recombinant CmNAGase were purified using an FPLC system ) with a Ni-nitrilotriacetic acid affinity chromatography (Ni-NTA) column (His Trap™ FF 5 mL). The target protein was eluted with elution buffer . The eluted fractions were passed through an ultrafiltration tube of 10 kDa  to remove the imidazole with sodium phosphate buffer (pH7.0) and concentrate the enzyme solution.The cells were harvested by centrifugation at 6000Concentration of protein was quantified using the Bradford method . Bovine Reductive SDS-PAGE with a 3% stacking gel and 10% separating gel was performed to determine the molecular weight of purified recombinant protein according to purification part of wild-type NAGase above. A premixed protein marker  containing 180-, 140-, 100-, 75-, 60-, 45-, 35-, 25-, 15-, and 10-kDa bands was used as the molecular mass standard.CmNAGase used pNP-GlcNAc as the substrate [pNP-GlcNAc (0. 25 mM) in 50 mM sodium citrate buffer (pH 5.4) and incubated at 40 °C for 10 min. The reaction was terminated by adding 2 mL NaOH (0.5 M). The absorbance was measured at 405 nm to determine the amount of pNP produced using a standard curve. One unit of NAGase activity was defined as the amount of enzyme required to release 1 μmol pNP from the substrate per minute at 40 °C.The NAGase activity for ubstrate . A totalpNP-GlcNAc as the substrate, the optimum pH for activity of CmNAGase was determined using different buffers: 50 mM citrate buffer (pH 3.0–6.0), 50 mM phosphate buffer (pH 6.0–7.5), and Tris–HCl buffer (pH 7.0–9.0) at 40 °C for 30 min. To measure the pH stability, enzyme was incubated at 35 °C for 96 h in the different buffers and the residual activities were determined against 1 mM pNP-GlcNAc.With 1 mM CmNAGase activity was measured, and the reaction solutions were incubated at temperatures that ranged from 25 to 55 °C for 30 min. Enzyme thermostability was determined by measuring the residual activities after pre-incubation of the purified enzyme in 50 mM sodium citrate buffer (pH 5.4) at 20–40 °C without substrate for 12 h. The residual activities were performed at pH 5.4 and 40 °C according to enzymatic activity assay above.The optimum temperature of CmNAGase was treated with 10 mM EDTA for 4 h at 4 °C and then dialyzed against 50 mM Tris–HCl buffer (pH 7.0) to remove the EDTA. For reactivation, the metal-free enzyme was incubated with various metal salts containing Cr3+ (CrCl3), Fe3+ (FeCl3), Fe2+ (FeCl2), Ca2+ (CaCl2), Cu2+ (CuCl2), Mg2+ (MgCl2), Zn2+ (ZnCl2), Mn2+ (MnCl2), Ni2+ (NiCl2), Co2+ (CoCl2), K+ (KCl), or Na+ (NaCl) at final concentrations of 10 mM for 30 min, and the residual activities were then measured with pNP-GlcNAc at 40 °C and pH 7.0 (Tris–HCl buffer) for 10 min. The activity without addition of metal ions was used as the control (100%).The effects of metal ions on the activity were also determined. Purified recombinant CmNAGase was determined by measuring the activity of enzyme toward colloidal chitin, N-acetyl COSs ((GlcNAc)2–(GlcNAc)6), cellobiose, 4-MU-GlcNAc, and pNP-glycosides as substrates) at a concentration of 10 mg/mL. Reaction mixture (1 mL) containing enzyme (3 μg) and various substrates (10 g/L) was incubated at 40 °C and pH 5.4 for 10 min. The amount of reducing sugars from colloidal chitin and (GlcNAc)2–(GlcNAc)6 was quantified with HPLC. The amount of pNP released in the reaction mixture was determined by measuring the absorbance at 405 nm. Hydrolytic activity of the enzyme against N-acetyl COSs was assayed by measuring the amount of GlcNAc released during the enzymatic reaction. After incubation, the enzymatic reaction was stopped by heating the mixture in a boiling water bath for 5 min. One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 μmol of pNP or GlcNAc per minute under the assay conditions.The substrate specificity of pNP-GlcNAc as the substrate. The initial velocities were determined by incubating 17 ng purified CmNAGase with pNP-GlcNAc concentrations ranging from 50 to 1000 μM at 40 °C in a 1 mL reaction system  for 5 min. The values of Vmax, Km, and Kcat were estimated by linear regression from double-reciprocal plots according to the method of Lineweaver [Kinetics experiments were performed using neweaver .CmNAGase (3 μg) was conducted at 35 °C for various time intervals. Boiling (5 min) was used to stop the reaction.A 1 mL reaction system  containing colloidal chitin (10 g/L) and purified N-Acetyl COSs ((GlcNAc)2–(GlcNAc)6) and 0.3 μg purified CmNAGase was conducted at 35 °C for various time intervals. The enzyme reactions were stopped by boiling at 100 °C for 5 min.A 100 μL volume  with 10 g/L CmNAGase in a 100 μL volume at pH 5.4 and 35 °C for 1 h. The reactions were stopped by boiling at 100 °C for 5 min.The determination of reverse hydrolysis activity was performed with 10 g/L GlcNAc and 0.3 μg purified The resulting reaction products were analyzed with an Agilent 1260 series HPLC system according to our previous report . The molCmNAGase cloned from strain SYBC‑H1 was deposited in GenBank under Accession number no. MN548778.The sequence for the gene encoding Additional file 1: Table S1. Strains, plasmids, and primers used in this study. Table S2. Purification of recombinant CmNAGase. Table S3. Half-lives of recombinant CmNAGase. Fig. S1. Multiple alignments of the catalytic domain in CmNAGase with other GH20 NAGases. Fig. S2. The domain and structure prediction of CmNAGase. a) The conserved domain of CmNAGase. b) The prediction of the 3D structure of CmNAGase. c) The active site of CmNAGase. Fig. S3. SDS-PAGE analysis of recombinant CmNAGase. Fig. S4. Mass spectrum of new peak (~ 16.0 min) after (GlcNAc)6 in HPLC spectra. Fig. S5. Mass spectrum of peak 2 (~ 10.9 min) in HPLC spectra.