Strains and culture conditions
Microbacterium sp. XT11 was cultured at 30 °C in xanthan medium (3 g xanthan, 0.5 g glucose, 3 g yeast extract, 0.025 g MgSO4·7H2O, 0.05 g K2HPO4, 0.8 g NaCl and 0.7 g KNO3 dissolved in 1 L deionized water, pH 7.0). Escherichia coli strains were cultivated at 37 °C in 1 L Luria–Bertani (LB) medium (10 g NaCl, 5 g yeast extract, and 10 g tryptone, pH 7.0), and 100 μg/mL ampicillin or 30 μg/mL kanamycin was added if necessary.
Cloning, expression and purification of the gene encoding xanthan lyase
The xanthan lyase expressed in E. coli (EcXly) was obtained according to the following method. The gene encoding xanthan lyase was cloned from Microbacterium sp. XT11 (Yang et al. 2014) genome by PCR amplification using the primes Fwd and Rev (Additional file 1: Table S1). The amplification products were ligated to the vector pET-32a by restriction-free cloning method (RF-cloning) (Van den Ent et al. 2006). The methylated plasmid pET-32a among the PCR mixtures was digested with 0.5 μL of DpnI at 37 °C for 1 h, and the products were then transducted into E. coli DH5α. The resulted plasmid pET-EcXly verified by DNA sequencing was transformed into E. coli Rosetta-gami (DE3) pLysS. The recombinants were selected on LB plates containing 100 μg/mL ampicillin and 30 μg/mL kanamycin.
For protein expression, E. coli Rosetta-gami (DE3) pLysS containing plasmid pET-EcXly was first cultured in LB low salt medium containing 100 μg/mL ampicillin and 30 μg/mL kanamycin at 37 °C. When the OD600 reached 0.6, a final concentration of 1 mM IPTG was added and the culture was further grown at 16 °C for 16–20 h. The cells were harvested by centrifugation at 8000 g for 5 min. The precipitate was collected, resuspended in NaH2PO4–NaCl buffer, and ultrasonicated. The debris was removed by centrifuging at 12,000 g for 20 min. The supernatant was used as the crude extract for enzyme purification by Ni–NTA affinity chromatography due to the ability of the protein with His-tag to bind to nickel (Crowe et al. 1994). The purity of the fraction was confirmed by SDS-PAGE. The protein concentration was measured using Bio-Rad protein assay kit (Bio-Rad, USA). The xanthan lyase derived from Microbacterium sp. XT11 (MiXly) was purified by ammonium sulfate fractionation, hydrophobic interaction chromatography and anion exchange chromatography in sequence according to the procedure of Yang et al. (2014).
Analysis of glycosylation site
Based on specific capture of glycoproteins by hydrazide resin, the N-glycosylation sites were analyzed and identified using the LC–MS/MS (Thermo Scientific, MA, USA) (Zhang et al. 2003) (Fig. 1). 1 mg EcXly were dissolved in coupling buffer (100 mM NaAc, 150 mM NaCl), and 15 mM NaIO4 was added to oxidize the proteins for 1 h. NaIO4 was then removed using the ultrafiltration tube, and the proteins were coupled with hydrazide resin (Bio-Rad, USA) at room temperature for 10–24 h. To remove the nonglycoproteins, the sample was washed six times using an equal volume of buffer. The proteins were reduced by adding 20 mM dithiothreitol at 60 °C for 1 h and subsequently alkylated by the addition of 20 mM iodoacetamide in the dark for 40 min. The trypsin was used to hydrolyze proteins into peptides, and the PNGase F was used to release the enriched glycopeptides from hydrazide resin. Finally, the released peptides were desalinated and resuspended in an appropriate amount of 0.1% formic acid for further LC–MS/MS analysis. The peptides of MiXly and the mutants of EcXly were obtained by in-gel digestion and used for LC–MS/MS analysis (De Godoy et al 2006). The acquired MS data were analyzed using Proteome Discoverer 2.2.1 software as previously described (Yuan et al. 2022).
MALDI-TOF MS analysis of xanthan lyase
MALDI-TOF MS analyses were performed using a Bruker Microflex LRF MALDI-TOF mass spectrometer (Bruker Daltonics; Billerica, MA, USA) equipped with a nitrogen laser (337 nm) under the control of FlexControl software (version 3.0; Bruker Daltonics; Billerica, MA, USA). Mass spectra were manually collected in positive linear mode within a mass range from 100 to 150 kDa. Ion source voltages 1 and 2 were set at 20 and 18.15 kV, respectively. The lens voltage was set to 9.05 kV. Each spectrum was obtained by the accumulation of 200 laser shots in 100 shot increments.
Sequence analysis and homology modeling
Sequence alignment among EcXly and other proteins with high sequence identities was performed by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) and the result was visualized with online software ESPript (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The three-dimensional structure of EcXly was predicted by utilizing the I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I -TASSER/) for homology modeling (Roy et al. 2010). The predicted three-dimensional structure of the protein was examined and shown using PyMOL software. Developmental tree mapping and analysis were performed using MEGA 7.0 (Kumar et al. 2016).
Construction of EcXly deglycosylation mutants
In order to probe the effect of N-glycosylation on enzymatic properties of EcXly, the glycosylation site N599 was then, respectively, mutated to alanine (A) and aspartic acid (D) and glycine (G) to obtain deglycosylation mutants by RF-cloning using pET-EcXly as template. The primers for construction of mutants are given in Additional file 1: Table S1. The deglycosylated mutants were constructed using the same method for the construction of pET-EcXly mentioned above. The verified plasmids were then transformed into electrocompetent E. coli Rosetta-gami (DE3) pLysS and expressed. The recombinant proteins EcXlyN599A, EcXlyN599D and EcXlyN599G were purified as described above for the EcXly.
Characterization of EcXly and its mutants
To determine the specific activity of the xanthan lyase, 1 mg/mL xanthan was incubated with 0.1 mg/mL EcXly or its mutants in phosphate buffer (pH 6.0). The enzymatic reaction was performed at 30 °C for 20 min, and was ended immediately by heating in boiling water for 5 min. The denatured protein was removed by centrifuging at 12,000 g for 5 min. The supernatant absorbance was measured at 235 nm due to the formation of the double bond between glucuronic acid and terminal mannose of side chain. The definition of a unit of enzyme activity is the amount of enzyme required to increase 1.0 absorbance per minute at 235 nm.
The optimum temperature was assayed by incubating the enzyme–xanthan mixture at various temperatures (25–55 °C, with 5 °C intervals) for 20 min as described above. For the thermostability of xanthan lyase, the proteins were incubated at different temperatures (20–70 °C, with 5 °C intervals) for 2 h, then the reaction was conducted as mentioned above.
For the optimum pH for the enzymatic reaction, the enzyme–substrate mixture was incubated in various buffers (pH 4.0–9.0) at 30 °C for 20 min. To determine the pH stability of proteins, the wild type EcXly and its mutants were incubated in various buffers (pH 4.0–9.0) at 30 °C for 12 h, respectively, and the residual enzyme activity was tested with the same method described above. The different pH buffers contained citric acid–sodium citrate buffer (pH 4.0–6.0), Na2HPO4–NaH2PO4 buffer (pH 6.0–8.0), Tris–HCl buffer (pH 8.0–9.0).
Determination of kinetic parameter
To investigate the kinetic parameters (Km and Vmax), the wild type EcXly and its mutants were incubated with different concentrations of xanthan (0.6, 0.8, 1, 1.2, 1.5, 2, 2.5, 3, 4, 5 mg/mL) for 20 min under the optimum conditions, respectively. Perkin Elmer Lambda 35 UV/VIS Spectrometer/PTP Peltier Temperature Programmer (PerkinElmer, Shanghai, China) was used to monitor Vmax. Due to the formation of unsaturated glucuronic acid (the extinction coefficient is 6150 M−1 cm−1), the molar concentration of the products was transformed by the Lambert–Beer law (Stender et al. 2019). Finally, according to the time course for the formation of unsaturated glucuronic acid, the Michaelis–Menten equation was applied for the calculation of Km and Vmax of the wild type EcXly and its mutants.
Circular dichroism spectra
Circular dichroism is used to analyze the secondary structure of proteins through the circular dichroism of proteins and the different absorption of left and right circularly polarized light by asymmetric molecules. To monitor the secondary structure of EcXly and its mutants, circular dichroism (CD) spectra were performed using a JASCO J-815 CD spectrometer (JASCO, Tokyo, Japan). The sample, with a final concentration of 0.2 mg/mL, was prepared in 20 mM Na2HPO4–NaH2PO4 buffer (pH 6.0). The data were recorded from 200 to 260 nm at 1 nm intervals at room temperature using a quartz cuvette with 1 mm path length. The value of scan speed and response time was 500 nm/min and 1.0 s, respectively. The CD data were submitted to BeStSel (Beta Structure Selection) online server (http://bestsel.elte.hu.) to analyze the relative proportion of secondary structure (Micsonai et al. 2018).
Fluorescence spectra
Fluorescence spectroscopy is used to study the spatial conformation of proteins by the characteristics that the side chain groups of aromatic amino acid residues in proteins absorb the incident light in the ultraviolet region and emit fluorescence. Fluorescence spectrometer (F-4600, Hitachi, Japan) was used to characterize the change of tertiary structure of EcXly and its mutants. The sample was prepared in Na2HPO4–NaH2PO4 buffer with a final concentration of 0.05 mg/mL. The fluorescence spectra from emission wavelength of 300 nm to 400 nm were recorded under an excitation wavelength of 280 nm. Each sample was tested three times.
Statistical analysis
All tests and determinations were carried out in triplicate unless otherwise stated. Data were expressed as the average values ± standard deviation. The significant difference (P < 0.05) was analyzed by the Statistical software SPSS 11 (SPSS Inc, Chicago, USA).