Introduction

l-Asparaginase (l-ASNase, EC 3.5.1.1) hydrolyses l-asparagine into aspartic acid and ammonia. The enzyme can deplete free l-asparagine from the bloodstream, hence finding its applications in treating acute lymphoblastic leukemia (ALL) (Kidd 1953; Hill et al. 1967; Silverman et al. 2001; Pui et al. 2009; Costa et al. 2022). Usually, blood cells can produce their l-asparagine due to l-asparagine synthetase activity, but cancerous blood cells cannot synthesize l-asparagine. Hence, l-ASNase treatment deprived cancerous blood cells of nutrition, leading to their apoptosis (Kelo et al. 2009; Soncini et al. 2020). Besides l-ASNase role in ALL, different strategies are underway to find a new role for l-ASNase as an anticancer medicine for lung and breast cancers (Baskar et al. 2018; Knott et al. 2018). It has application in the food industry to mitigate acrylamide formation in baked or fried foods (Kornbrust et al. 2009; Meghavarnam and Janakiraman 2018; Paul and Tiwary 2020). Additionally, l-ASNase-based biosensors have been designed to measure the concentration of free l-asparagine from the serum samples (Verma et al. 2007).

l-ASNase is widely distributed in microbes, plants, and animals (Batool et al. 2016). However, microorganisms offer a convenient alternative for extracellular l-ASNase production and purification (Vimal and Kumar 2017; Tundisi et al. 2017). Microbes possess two different variants of l-ASNases based on their cellular localization with a variable affinity for l-asparagine. The cytoplasmic (type I) and periplasmic (type II) variants of l-ASNase have low and high specific activity for l-asparagine, respectively (Yun et al. 2007; Schalk et al. 2014). It has been suggested that the Km value of l-ASNase must be in a lower micromolar range for efficient hydrolysis of ~50 µM blood l-asparagine concentration (Cooney et al. 1970). Currently, E. coli and Erwinia chrysanthemi (E. chrysanthemi) based commercial formulations of l-ASNase II are used in the chemotherapeutic treatment of ALL. However, the commercial formulations show hypersensitive and immunogenic responses during and after the treatment (Wang et al. 2009).

The evident limitations of currently used l-ASNase-based commercial drugs have shifted the researcher’s focus on the periplasmic enzyme. The periplasmic enzymes are endotoxin-free and protease deficient, thus, helping in minimizing the side effects. Additionally, extracellular enzymes are promoted to proper folding by the suitable redox potential of periplasmic space (Singh et al. 2013; Wingfield 2015; Tundisi et al. 2017). Also, l-ASNase associated l-GLNase activity is thought to be partially responsible for the side effects (Batool et al. 2016; Hijiya and Van Der Sluis 2016). Therefore, overcoming the side effects of microbial l-ASNase treatment requires scientific interventions. The advances in biotechnology, and improved developmental plans for the existing products, bioprospecting for new and novel sources of the enzymes with lesser or no side effects are of prime importance for future l-ASNase-based drug commercialization.

Our lab has explored several bacteria from high-altitude regions for l-ASNase in the past few years (Kumar et al. 2019). Amongst, l-ASNase II of Pseudomonas sp. PCH44 (Ps44-ASNase II) has higher activity for l-glutamine than l-asparagine, reflecting the enzyme’s specificity and uniqueness. The l-GLNase activity has also been reported from bacterial species such as E. coli, Bacillus licheniformis, Rhizomucor miehei (R. miehei), and Erwinia carotovora (E. carotovora) but to a lesser extent than l-ASNase activity (Narta et al. 2007; Mahajan et al. 2014; Huang et al. 2014; Labrou and Muharram 2016). The l-GLNase activity in l-ASNase II is considered a drawback for its applications in therapeutics. However, the presence of l-GLNase activity in Ps44-ASNase II is inevitable. Therefore, in the present study, we report whole-genome analysis, heterologous expression, biochemical, and in-silico characterization of the Ps44-ASNase II isolated from high-altitude niches of the Indian Himalayan region. Further, in-silico and comparative docking analysis of Ps44-ASNase II and E. coli l-ASNase (Ec-ASNase II) with l-asparagine and l-glutamine were carried out to understand the molecular basis for higher l-GLNase activity in Ps44-ASNase II.

Materials and methods

Materials and bacterial strains

NEB Taq DNA Polymerase, T4 DNA Ligase, FastDigest SacII and XhoI endonuclease, 1.0 kb DNA ladder, HisPur Cobalt resin superflow were purchased from Thermo Fisher Scientific, USA. The expression vector pET-47b(+) and E. coli BL-21(DE3) were purchased from Novagen, USA. The amino acids (l-asparagine, l-glutamine), Nessler’s reagent, inducer (IPTG, isopropy l-β-d-1 thiogalactopyranoside), and antibiotics such as kanamycin were obtained from Sigma-Aldrich, USA. All the media chemicals were obtained from HiMedia, India.

Bacterial isolate PCH44

The bacterial isolate PCH44 was previously isolated and identified in our laboratory (Thakur et al. 2018). It was screened qualitatively for l-ASNase activity on a modified M9 medium (components in g/L; 6.0 g Na2HPO4.2H2O, 3.0 g KH2PO4, 0.5 g NaCl, 2.0 mM MgSO4.7H2O, and 0.1 mM CaCl2·2H2O) supplemented with l-asparagine (0.5%, w/v) as a nitrogen source and glucose (0.2%, w/v) as a carbon source and phenol red (0.003%) as an indicator dye (Kumar et al. 2019). The plate was incubated at 28 °C up to 120 h, and a qualitative color change from yellow to pink was observed. PCH44 was cultured in a modified M9 medium, and quantitative l-ASNase activity was estimated from 12 to 35 h of incubation. The enzyme activity was checked after every 3 h interval using Nessler’s method. The 16S rDNA-based molecular identification of strain PCH44 was carried out (3130 xl genetic analyzer, Applied Biosystems, USA). The EzTaxon server was used to examine the sequence obtained following 16S rDNA sequencing (http://www.eztaxon.org/), and a partial 16S rDNA sequence was submitted to GenBank database (Thakur et al. 2018; Kumar et al. 2019). The phylogenetic tree of 16S rDNA sequences was constructed using MEGA X (Kumar et al. 2018).

Whole-genome analysis of bacterial isolate PCH44

The genomic DNA of bacterial isolate PCH44 was isolated; DNA library preparation, and whole-genome sequencing were performed as described earlier (Kumar et al. 2020). Briefly, the quality and quantity of genomic DNA were estimated using a Dropsense96 (Trinean, Gentbrugge, Belgium) and Qubit 2.0 Fluorometer (Invitrogen, USA) following the manufacturer’s instructions. A PacBio RSII (Pacific Biosciences, Menlo Park, USA) was used to sequence the whole-genome. The sequenced data were assembled using Canu 2.1 (Koren et al. 2017). The genome sequence of Pseudomonas sp. PCH44 was deposited in GenBank under reference GCF_018304925.1 and the best matching genome was determined to be Pseudomonas vlassakiae (GCA_014269035.2) with an ANI similarity value of 96.28. The genome was annotated with the Prokaryotic Genome Annotation Pipeline (PGAP) (Tatusova et al. 2016) and the Rapid Annotation Subsystems Technology (RAST) (Aziz et al. 2008).

Bioinformatics analysis and cloning of the gene for l-ASNase

Gene encoding l-ASNase II was identified from the annotated genome and retrieved the sequence. The nucleotide sequence was analyzed and translated using the ExPASy translate tool (Swiss Institute of Bioinformatics). The molecular mass and theoretical isoelectric point (pI) of l-ASNase II were calculated using the ProtParam tool [https://web.expasy.org/protparam/]. SignalP 5.0 was used to ascertain the presence of signal peptide (Armenteros et al. 2019). The protein sequence was further analyzed in UniProt [https://www.uniprot.org/blast/] against the database sequence of l-ASNase. Multiple sequence alignment was performed using ClustalW [https://www.genome.jp/toolsbin/clustalw] and ESPript 3.0 (Robert and Gouet 2014).

The complete nucleotide sequence of Ps44-asn II (NCBI Protein ID: JIQ88_05230) was retrieved from the whole-genome of Pseudomonas sp. PCH44 (BioProject ID PRJNA689707). The forward (5′-TCCCCGCGGTTATGAAAGAAGCCGAAACCCAGCAG-3′) and reverse (5′-CCGCTCGAGTCAGTACTCCCAGAAAATCCGCTGCA-3′) primers for the Ps44-asn II gene flanked by SacII and XhoI restriction sites (underlined), respectively, were designed excluding the signal peptide nucleotide sequences (75 bp). The properties (Tm, cross dimer, ∆G, and self-dimer) of designed primers were analyzed using NetPrimer (Premier Biosoft, USA). The genomic DNA (30–50 ng) of PCH44 was used to amplify Ps44-asn II. The PCR amplification was performed using initial denaturation for 3 min at 95 °C and 35 cycles of each of 30 s at 94 °C, 40 s at 53 °C, 1 min at 72 °C, and a final extension of 7 min at 72 °C. The amplified PCR product and the pET-47b(+) vector were double digested with SacII and XhoI. The double-digested PCR product and plasmid were ligated using T4 DNA ligase. The standard heat-shock method was used to transform of the ligated product into the E. coli BL-21(DE3) (Bergmans et al. 1981) and further incubated overnight at 37 °C on LB plates containing antibiotic kanamycin (25.0 µg/mL). The positive clones carrying pET-47b-Ps44-asn II recombinant plasmid were confirmed by colony PCR and sequencing. The recombinant plasmid was extracted using a plasmid purification kit (Favorgen, Taiwan) and sequenced using a vector-specific primer (T7 P and T7 T). FinchTV 1.4 was used to analyze and get the consensus sequence.

Expression of Ps44-ASNase II in E. coli BL-21(DE3)

E. coli BL-21(DE3) culture harboruring recombinant pET-47b-Ps44-asn II plasmid was inoculated in 100.0 mL modified M9 medium supplemented with yeast extract (1.0%) and kanamycin (25.0 μg/mL), and kept for growth at 37 °C × 200 rpm. When the cell OD (600 nm) reached 0.8, the bacterial culture was induced by adding 0.5 mM IPTG to express Ps44-ASNase II. Bacterial culture was incubated for 16 h (overnight) at 37 °C. The cell pellet was recovered by centrifugation at 8000 g for 20 min, washed thoroughly with buffer (50.0 mM Tris–HCl, pH 8.5) to remove residual medium traces, and suspended in basic buffer (50.0 mM Tris–HCl: pH 8.5, 300.0 mM sodium chloride, and 5.0 mM imidazole). The cell suspension was sonicated (8 s pulse on; 10 s pulse off, and 30% amplitude) for 40 min on ice. The cell lysate was centrifuged at 8000 g for 25 min to obtain cell-free extract (CFE). The specific activity and total protein content of the CFE were also measured.

Purification of recombinant Ps44-ASNase II

Ps44-ASNase II was purified using affinity-based chromatography with HisPur Cobalt Superflow. The CFE (0.45 µM filtered) was loaded onto the HisPur Cobalt Superflow chromatography column pre-equilibrated with a basic buffer. The matrix was washed with 10 column volumes (CV) of basic buffer to remove non-specific proteins. The bound Ps44-ASNase II was eluted using elution buffer (50.0 mM Tris–HCl pH 8.5, 300.0 mM NaCl, and 150.0 mM imidazole) in different fractions. The amount of protein in each fraction was estimated by Bradford assay (Bradford 1976) using bovine serum albumin as standard. The purity of different fractions was analyzed by 10% SDS-PAGE (Laemmli 1970). The purified fractions were pooled and dialyzed using 50.0 mM Tris–HCl (pH 8.5). The native molecular weight of purified Ps44-ASNase II was determined by gel exclusion chromatography (Superdex 200, GE Healthcare, USA) using different molecular weight markers (Merck-Sigma Aldrich, USA).

Ps44-ASNase II assay

The activity of Ps44-ASNase II was estimated spectrophotometrically (Synergy H1, BioTek, Agilent, USA) at 480 nm using Nessler’s reagent, which measures the amount of ammonia released in the reaction mixture (Imada et al. 1973). Briefly, the enzyme assay was performed in a 1.0 mL volume of a reaction containing 50.0 mM Tris–HCl, 5.0 mM l-asparagine, and 2.0 µg (enzyme suspension in reaction buffer) of purified Ps44-ASNase II. All reaction components were pre-incubated at 37 °C for 30 min. The reaction was carried out at 37 °C for 15 min and terminated by adding 250.0 µL (1.5 M) of TCA (Trichloroacetic acid). The control and blank were prepared simultaneously. The reaction was diluted as per necessity before adding Nessler’s reagent, and the optical density was measured at 480 nm. A standard curve was also prepared using ammonium chloride. The specific activity of purified Ps44-ASNase II was expressed in U/mg protein (µmoles min−1 mg−1). One unit (IU) of Ps44-ASNase II is defined as the amount of enzyme liberating 1.0 µmol of ammonia per minute under standard reaction conditions.

Biochemical characterization and kinetic evaluation of Ps44-ASNase II

The biochemical parameters for maximum Ps44-ASNase II activity were assessed. Ps44-ASNase II activity was measured in 50.0 mM buffer of sodium citrate (pH 3.0–5.0), potassium phosphate (pH 6.0–7.0), Tris–HCl (pH 8.0–10.0), sodium carbonate-bicarbonate (pH 9.0–10.0), sodium carbonate-NaOH (pH 10.0–11.0), and potassium chloride-NaOH (pH 11.0–13.0). The buffer system with maximum activity was selected for further experiments. The ambient temperature of Ps44-ASNase II was optimized by assaying the enzyme activity at different temperatures range (10–80 °C). The thermal stability of purified Ps44-ASNase II was investigated. The enzyme was incubated at 28, 37, 45, 50, and 60 °C in 50.0 mM Tris–HCl buffer (pH 8.5), and residual enzyme activity was measured at fixed time intervals. The half-life of Ps44-ASNase II was calculated from the thermal stability graph. Similarly, the specificity of Ps44-ASNase II for l-asparagine and l-glutamine was measured at different concentrations (0.05–5.0 mM). The Km and Vmax were calculated using the Michaelis–Menten equation and plotting 1/s and 1/v values in the Lineweaver–Burk plot. The kcat of the Ps44-ASNase II was deduced by using the equation kcat = Vmax/[E0], where [E0] is the initial enzyme concentration in the reaction and Vmax (µmol/min) is the maximum reaction rate.

Effect of metal ions and protein modifying agents

The effect of various metal ions and protein modifying agents on purified Ps44-ASNase II activity was examined. The Ps44-ASNase II was pre-incubated at 37 °C for 60 min with 1.0 mM CuSO4, CoCl2, and 2.0 mM KCl2, NaCl, CaCl2, and ZnSO4 in separate reactions. Similarly, the effect of protein-modifying agents using 1.0 mM dithiothreitol (DTT), sodium dodecyl sulfate (SDS), and 2.0 mM dimethyl sulfoxide (DMSO), ethylenediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and 2-mercaptoethanol (β-ME) were also measured.

Homology modeling and structural validation of Ps44-ASNase II

A template search for homology modeling of Ps44-ASNase II was performed using BLASTp (http://blast.ncbi.nlm.nih.gov/) against the Protein Data Bank (PDB). The homology model of Ps44-ASNase II was generated using SWISS-MODEL v.4.0 (https://swissmodel.expasy.org/) based on predicted PDB structure with maximum sequence identity and percent coverage (Arnold et al. 2006). The quality of the model was assessed by analyzing the Ramachandran plot, PROCHECK, and structural analysis and verification server (SAVES v5.0) (https://saves.mbi.ucla.edu/). The secondary structure was predicted using SOPMA (Geourjon and Deleage 1995).

Docking analysis of Ps44-ASNase II and Ec-ASNase II

Three-dimensional structures of l-asparagine and l-glutamine were used as ligands for docking and were downloaded from the library of 3D Molecular Structures (https://pubchem.ncbi.nlm.nih.gov/compound/). The ligands were docked with the catalytic site of Ps44-ASNase II (Thr21 and Thr101) and Ec-ASNase II (Thr12 and Thr89) using Auto Dock Vina software (Trott and Olson 2010). The docked complexes of Ps44-ASNase II were evaluated for the amino acid-ligand interactions in close proximity (≤3 Å) of a catalytic site using Molegro Molecular viewer and LIGPLOT+ (Laskowski and Swindells 2011). Further, a comparative analysis of the interactions of l-glutamine with catalytic sites of Ps44-ASNase II is performed with that of Ec-ASNase II to address the molecular basis of high l-GLNase activity in Ps44-ASNase II.

Statistical analysis

All the experiments were performed in triplicates. The data were represented in the form of mean ± standard deviation (±SD).

Results and discussion

Screening of PCH44 for l-ASNase activity

In our earlier studies, the bacterial isolate PCH44 was qualitatively screened for l-ASNase activity on a modified M9 medium (Kumar et al. 2019). The formation of pink color was observed after 48 h for every 24 h interval, suggesting l-ASNase producing ability (Fig. S1). Quantitatively, l-ASNase activity was measured after 12 h and subsequent readings after every 3 h intervals. The maximum enzymatic activity (0.58 U/mL crude) was obtained after 15 h of incubation at 28 °C and 180 rpm. Interestingly, the isolate PCH44 produces high l-ASNase-associated l-GLNase activity in a short time (15 h) compared to other potential isolates studied earlier (Kumar et al. 2019). Therefore, the PCH44 was selected for whole-genome sequencing to reveal genomic insights. The phylogenetic tree of 16S rDNA sequencing of isolate PCH44 (GenBank accession number KY628862) showed the best match with Pseudomonas alloputida Kh7(T) (Fig. S2) (the earlier best match was with Pseudomonas hunanensis) (Thakur et al. 2018).

Whole-genome analysis and in-silico characterization

The PacBio RSII platform was used to sequence the whole-genome of Pseudomonas sp. PCH44. The raw sequences were assembled using Canu 2.1 and generated seven contigs with a total genome size of 6.41 Mb. The genome was submitted to the NCBI database under the BioProject ID PRJNA689707 and BioSample ID SAMN17215112. The RAST server displayed 536 subsystems, 6092 coding sequences, and 62.2% GC content (Fig. S3 and Table S1). The two l-ASNase protein sequences (NCBI Protein ID: JIQ88_05230 and JIQ88_19890) were identified through PGAP and RAST annotation server. SignalP 5.0 server revealed the presence of signal peptide in JIQ88_05230 only, which corresponds to periplasmic localization. The pI of periplasmic Ps44-ASNase II was calculated at 6.33 using the ProtParam tool (Table S2). The periplasmic l-ASNase protein-encoding gene with signal peptide was selected for heterologous expression.

Cloning, expression, and purification of Ps44-ASNase II

The L-asn II gene (1.0 kb) was amplified using gene-specific primers (Fig. S4). The l-asn II was cloned and expressed in the host E. coli BL-21(DE3) under the control of T7 promoter in pET-47b(+) vector. The positive clones were grown in a modified M9 media and induced with 0.5 mM IPTG to express a heterologous protein. Maximum expression of Ps44-ASNase II was obtained at 37 °C after 20 h incubation. After expression, cell pellet was lysed, and Ps44-ASNase II was obtained as soluble and active CFE. The CFE was subjected to purification using HisPur Cobalt Superflow Agarose. The purified Ps44-ASNase II was obtained from CFE in a single chromatographic step. A final yield of 55.88% with 28-fold purification was achieved (Table 1). Ps44-ASNase II was obtained as a single distinct band of 37.0 kDa on SDS-PAGE analysis (Fig. 1A). The native molecular weight was estimated at 148.0 kDa, revealing a tetrameric form of Ps44-ASNase II, consistent with earlier reported native protein size (Fig. 1B). It was similar in size to l-ASNase of E. coli, used to treat ALL (Jackson and Handschumacher 1970). Also, l-ASNase from E. carotovora and Vibrio cholerae (V. cholerae) has a molecular weight of 36.6 kDa (Warangkar and Khobragade 2010; Radha et al. 2018), suggesting the expected size of Ps44-ASNase II.

Table 1 Protein purification profile of recombinant Ps44-ASNase II expressed in E. coli BL-21(DE3) by HisPur Cobalt Superflow affinity chromatography
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Fig. 1
figure 1

Polyacrylamide gel electrophoresis and molecular weight estimation of Ps44-ASNase II. A SDS-PAGE (10%) analysis of recombinant Ps44-ASNase II. Lane 1 is total cell lysate; lane 2 is flow-through; lane 3–6 eluted protein; lane 7 is a wash, and M is protein molecular weight marker. B Determination of the molecular mass of the native Ps44-ASNase II. Gel filtration chromatography was performed using Superdex 200 (10/300 GL) column. Arrow indicates the log MW of the Ps44-ASNase II. Ferritin, aldolase, conalbumin, and ovalbumin were used as protein molecular weight standards for standard curves. Ve and Vo indicate for elution volume of each protein and void volume, respectively

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Effect of pH on Ps44-ASNase II activity

The buffering environment of the reaction is a key deciding factor for optimal enzyme activity. In the present study, maximum Ps44-ASNase II activity (34.48 ± 3.79 U/mg) was observed in 50.0 mM Tris–HCl buffer (pH 8.5). The comparable activity (26.83 ± 2.39 U/mg) was also reported in potassium phosphate buffer (pH 7.5). However, Tris–HCl provides optimum buffering conditions with enhanced stability (Fig. S5). Ps44-ASNase II was found active in a broad pH range from 4.5 to 11.0 (Fig. 2A). In a similar study, l-ASNase from Bacillus sp. was reported to be active over a pH range between 4.5 and 10.0 (Safary et al. 2019). Also, the optimum pH of 8.5 for l-ASNase in the current study was similar to earlier reported pH from Yersinia pseudotuberculosis (Y. pseudotuberculosis, pH 8.0), Staphylococcus sp. OJ82 (pH 8.0–9.0), and Halomonas elongate (H. elongate, pH 8.0) (Pokrovskaya et al. 2012; Han et al. 2014; Ghasemi et al. 2017). The optimum enzyme activity of pH 8.0 is critical for therapeutic application since blood pH ranges from 7.35 to 7.45.

Fig. 2
figure 2

Optimization of different reaction parameters for Ps44-ASNase II. A Effect of pH (3–13) on Ps44-ASNase II at 37 °C. B Effect of incubation temperature (10–80 °C) on Ps44-ASNase II at pH 8.5. C Thermostability profile of Ps44-ASNase II was studied by incubating at 28−50 °C for 200 min in Tris–HCl buffer (50.0 mM and 8.5 pH), and the residual Ps44-ASNase II activity was calculated. D Effect of metal ions and protein modifier agents on Ps44-ASNase II incubating at 37 °C for 60 min

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Effect of temperature on Ps44-ASNase II activity

The purified Ps44-ASNase II exhibited activity at a temperature range from 10 to 75 °C with optimum activity at 45 °C (40.84 ± 0.9 U/mg). Above 50 °C, a sharp decrease in specific activity was observed (Fig. 2B). In literature, purified l-ASNase from E. chrysanthemi NCPPB1125 and Paenibacillus barengoltzii (P. barengoltzii) has optimum activity at 45 °C (Nguyen et al. 2016a, b; Shi et al. 2017). l-ASNase of Pyrococcus furiosus (P. furiosus) showed optimum activity at 50 °C (Saeed et al. 2020). The thermostability of Ps44-ASNase II was evaluated by incubating enzyme at different temperatures. Ps44-ASNase II retained 76.53% of enzyme activity at 45 °C after 120 min of incubation (Fig. 2C). The thermal inactivation of Ps44-ASNase II follows the theoretical curve of a first-order reaction. The half-life (t1/2) of Ps44-ASNase II was calculated using linear regression of data acquired from thermal stability. The t1/2 of Ps44-ASNase II at 45 °C in Tris–HCl (pH 8.5) was 600 min with a dissociation constant (Kd) of 1.10 × 10–3 min−1 (Table S3). l-ASNase from P. furiosus showed 72% residual activity after 60 min at 45 °C (Saeed et al. 2020). The t1/2 (min) values for V. cholerae at 40 and 45 °C were 751 ± 21.5 and 13.6 ± 0.38, and the Kd (min−1) values were 0.92 × 10–3 and 51.2 × 10–3 at 40 and 45 °C, respectively (Radha et al. 2018).

Effect of metal ions and protein modifier agents

Enzyme was assayed in the presence of different metal ions and protein modifying agents. It was found that none of the metal ions have a positive effect on Ps44-ASNase II activity (Fig. 2D). The presence of Ca2+, K+, Na+, Zn2+, Co2+, and Cu2+ ions in reaction decreases the enzymatic activity. The high inhibitory effect of Zn2+ (52%) and EDTA (36%) on Ps44-ASNase II activity was reported. Inhibition of enzyme activity by using divalent ions might be due to the chelation of sulfhydryl groups of enzyme with metal ions. The sulfhydryl groups are critically important for enzyme activity to produce catalysis (Sokolov 1976; Radha et al. 2018). It showed that the Ps44-ASNase II does not essentially require monovalent and divalent ions for its activity. Besides DTT, protein modifiers like PMSF, β-ME, SDS, and DMSO had negatively affected the enzyme activity (Fig. 2D). The inhibition of l-ASNase activity by metal ions such as Zn2+ in V. cholerae (Radha et al. 2018), Cu2+, Ca2+, and Zn2+ in P. furiosus (Saeed et al. 2020) has also been reported. The Cu2+ showed inhibition in Streptomyces brollosae NEAE-115 (S. brollosae) l-ASNase (El-Naggar et al. 2018) and a complete loss of enzyme activity in V. cholerae l-ASNase (Radha et al. 2018) was observed. Similarly, protein-modifying agents such as SDS, EDTA, and β-ME also had an inhibitory effect on l-ASNase activity of R. miehei (Huang et al. 2014). DTT is widely used for protein structure maintenance by establishing a reducing environment for the SH group of cysteine-containing proteins. For instance, 1.0 mM DTT enhanced the activity of E. coli l-ASNase by 46% (Nguyen et al. 2016a, b). However, cysteine residue was not found in Ps44-ASNase II in this investigation. As a result, the addition of DTT did not affect enzyme activity.

Substrate specificity and kinetic parameters of Ps44-ASNase II

The Lineweaver–Burk plot was drawn to estimate the kinetic parameters by taking a different concentration of l-asparagine and l-glutamine (Fig. 3A). The Km values of purified Ps44-ASNase II for l-asparagine and l-glutamine were found to be 0.559 ± 0.12 and 0.728 ± 0.086 mM, respectively. Similarly, Vmax values of 29.41 ± 4.20 and 50.12 ± 3.54 U/mg for l-asparagine and l-glutamine were obtained, respectively (Table 2, Fig. 3B). The result shows that enzyme has more affinity for l-asparagine than l-glutamine. However, the turnover number (kcat) for l-glutamine is more (30.91 ± 2.18 s−1) than for l-asparagine (18.06 ± 2.59 s−1) (Table 2). The Km values of l-ASNase for B. subtilis B11-06 (Jia et al. 2013) and Pectobacterium carotovorum MTCC 1428 (Kumar et al. 2011) were 0.43 and 0.657 mM, respectively. The Vmax of Ps44-ASNase II is considerably higher for l-glutamine than l-asparagine. In contrast, the Km value is lower for l-ASNase, indicating that the enzyme acts on l-asparagine at lower concentrations than l-glutamine. Such a feature can be a favorable attribute where l-GLNase activity is not desired, like treating ALL. The high l-GLNase activity of l-ASNase has been reported as an undesirable feature for a therapeutic application. Therefore, the structural studies of Ps44-ASNase II were performed to know the possible molecular basis of a higher affinity for one substrate (l-asparagine) and a high turnover number for another (l-glutamine).

Fig. 3
figure 3

Kinetic study of Ps44-ASNase II. A Effect of varying l-asparagine and l-glutamine concentration (0.25–5.0 mM) on Ps44-ASNase II activity. B Lineweaver–Burk plot for determining a kinetic parameter of Ps44-ASNase II for l-asparagine and l-glutamine

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Table 2 Steady-state kinetic parameters for recombinant Ps44-ASNase II by using l-asparagine and l-glutamine (0.05–5.0 mM) and incubating the reaction at 37 °C for 15 min
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In-silico analysis of Ps44-ASNase II

BLASTp analysis of Ps44-ASNase II using the UniProtKB/Swiss-Prot database showed 95% similarity with l-ASNase of P. putida KT2440 (Q88K39.1). Further, the percent identity of Ps44-ASNase II was 85.64 with Pseudomonas fluorescens bv. A (P. fluorescens) (O68897.1), 61.89 with Acinetobacter glutaminasificans (A. glutaminasificans) (P10172.1), 48 with E. coli K-12 (P00805.2), and 47.26 with E. chrysanthemi (P06608.1) (Table S4). The Ps44-ASNase II showed two recognized structural domains, IlATGGTIA (residue 15–23) and GiVitHGTDTL (residue 94–104) when compared with selected l-ASNase sequences from most similarity index values (Fig. 4). The predicted active site for Ps44-ASNase II was Thr21 in the first domain and Thr101 in the second domain. The secondary structure analysis indicated random coil protein (40.83%), α-helices (34.91%), extended strands (19.82%), and β-turn (4.9%). Similarly, secondary structure analysis for l-ASNase of P. putida KT2440, Pseudomonas 7A, E. coli, and E. chrysanthemi has a similar trend as Ps44-ASNase II (Table S5).

Fig. 4
figure 4

Multiple sequence alignment of Ps44-ASNase II sequences using UniProt identifiers. Alignment of l-ASNase was performed by using ClustalW and ESPript 3.0. ‘α’ represents α-helix; ‘β’ represents β-sheet. The secondary structure was observed from the 3D structure (PDB ID: 3PGA) and reported at the top

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Homology modeling of Ps44-ASNase II

BLASTp search in PDB database showed l-ASNase II of Pseudomonas sp. 7A (PDB ID: 3PGA) as the best template with 95.3% identity and 100% coverage. The crystal structure of l-ASNase from Pseudomonas sp. 7A was characterized by 2.0 Å resolution and studied for its catalytic residues and enzyme activity (Lubkowski et al. 1994). The crystal structure of l-ASNase of Pseudomonas sp. 7A was used as a template for homology modeling due to high sequence similarity, high query coverage, and phylogenetically closeness to Ps44-ASNase II. The modeled structure of Ps44-ASNase II was homotetrameric, and the topology of each chain was identical to the template (Fig. S6). The Ramachandran plot of the model showed 90.9% of amino acid residues in the most favored region, and 7.6% in the additional allowed region suggested for a good model (Fig. S7). Furthermore, verify3D score showed that 96.21 residues are arranged in secondary structure, and the ERRAT score was 96.68, which signifies the model’s accuracy (Fig. S8). The overall structural analysis and validation suggest that the model structure of Ps44-ASNase II was suitable for further analysis.

Docking of l-asparagine and l-glutamine with Ps44-ASNase II and Ec-ASNase II provides a molecular basis for high l-GLNase activity in Ps44-ASNase II

The substrates l-asparagine and l-glutamine were docked with a homology model of Ps44-ASNase II and reference structure of l-ASNase (PDB ID: 1NNS) from E. coli (Ec-ASNase II) (Sanches et al. 2003). The catalytic role of threonine (Thr) for l-ASNase II activity is well studied using crystallography and mutational analysis (Kozak et al. 2000; Nomme et al. 2012; Brumano et al. 2019; Saeed et al. 2020). In short, l-ASNase mechanism of action involves the hydroxyl group of Thr, which initiates the nucleophilic attack on amine group of l-asparagine and forms an intermediate complex. It is followed by adding water molecules, resulting in aspartic acid and ammonium ions formation (Nguyen et al. 2016a, b; Lubkowski et al. 2020). Since the molecular structure of l-glutamine is similar to l-asparagine except for an additional methyl group, the mechanism of action is anticipated to be the same for both the substrates (Lubkowski et al. 2020). Based on the literature and current findings, Thr12 and Thr89 in Ec-ASNase II and Thr21 and Thr101 in Ps44-ASNase II were selected for docking grid. The docking analysis of Ps44-ASNase II and Ec-ASNase II showed average binding energy of −3.11, −4.33 for l-glutamine and −2.59, −4.05 for l-asparagine, respectively. The binding energy of Ec-ASNase II for l-glutamine was lesser than Ps44-ASNase II, which showed a lower catalysis rate (Reddy et al. 2016; Lubkowski et al. 2020). Further, the distance from nucleophilic residue Thr101 and Thr21 to carbonyl NH2 group of l-glutamine were 2.65 and 4.07 Å, whereas, for l-asparagine, the distance was observed at 2.95 and 3.62 Å, respectively (Fig. 5). The low binding energy of l-glutamine and closer proximity to catalytic Thr101 of Ps44-ASNase II correlate with kinetic data having a high turnover number for l-glutamine (30.91 s−1) as compared to l-asparagine (18.06 s−1).

Fig. 5
figure 5

Docking of l-ASNase II with l-asparagine and l-glutamine. A, B interaction of an amino group of l-asparagine and l-glutamine with the hydroxyl group of Thr21, Thr101 of Ps44-ASNase II. C, D Interaction of amino group of l-asparagine and l-glutamine with the hydroxyl group of Thr12, Thr89 of Ec-ASNase II. Distance between the NH2 group of l-asparagine and l-glutamine with OH group of Thr21, Thr101 is 3.62, 2.95, and 4.07, 2.65, respectively, and in Ec-ASNase II Thr12 and Thr89 is 3.16, 2.60 and 3.15, 2.63 for l-asparagine and l-glutamine, respectively

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The docked complexes of Ps44-ASNase II showed differences in the amino acids close (≤3.0 Å) with l-asparagine and l-glutamine ligands. For l-asparagine, the residues Thr101, Ser68, Glu69, and Asp102 are within ≤ 3.0 Å, whereas Thr101, Thr21, Ser68, Glu69, and Asp102 are close to l-glutamine (Fig. 6A, B). The closeness of an additional nucleophile Thr21 to the carbonyl group of l-glutamine might be responsible for higher l-GLNase activity. In Ec-ASNase II, nucleophile Thr12 and Thr 89 are close to l-asparagine and l-glutamine. However, Thr12 is away from the carbonyl group of l-glutamine (Fig. 6C, D). The active sites of Ps44-ASNase II and Ec-ASNase II showed both conserved and diverged residues. The diverged residue within ≤3 Å of docked l-glutamine was Gln69 in Ps44-ASNase II and Ala114 for Ec-ASNase II. The variation of amino acids within 3.0 to 5.0 Å of the active site in Ps44-ASNase II and Ec-ASNase II were Ala37, Ala38, Ala67, Ser70, Ser126, and Glu 69 in place of Val, Gly, Gly, Asp, Ala, and Gln at the respective positions. These variations of amino acids might be responsible for higher l-GLNase activity of Ps44-ASNase II. Thus, comparing the docked complex of l-asparagine and l-glutamine provides a molecular basis for high l-GLNase activity in Ps44-ASNase II. The identified amino acid residues can be targeted in future studies for enzyme improvement through mutagenesis.

Fig. 6
figure 6

LigPlot of interacting atoms of l-ASNase II. A Ps44-ASNase II with l-asparagine. B Ps44-ASNase II with l-glutamine. C Ec-ASNase II with l-asparagine. D Ec-ASNase II with l-glutamine

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Conclusions

In the present study, the l-ASNase producing Pseudomonas sp. PCH44 was isolated from a high-altitude niche. The whole-genome sequencing and analysis of Pseudomonas sp. PCH44 revealed two l-asn genes belonging to Type I and II, where l-asn II is produced extracellularly. The Ps44-asn II gene was successfully cloned and expressed in a bacterial host E. coli. The Ps44-ASNase II was purified, and a native molecular weight of 148.0 kDa size in homotetrameric conformation was estimated, which is consistent with other reported molecular weights in the literature. However, Ps44-ASNase II has unique activity features of a wide pH range, high thermal stability, and half-life. In addition, the enzyme activity was not influenced positively by the metal ions and protein modifying agents. The in-silico studies revealed structural similarity of Ps44-ASNase II with l-ASNase of P. putida KT2440, Pseudomonas sp. 7A, E. coli, and E. chrysanthemi. The experimental data have shown that Ps44-ASNase II has a higher affinity for l-asparagine unlike a higher turnover number for l-glutamine. The contrasting observation was validated through the in-silico analysis of Ps44-ASNase II, where the possible amino acid residues were found for higher l-GLNase co-activity. In the future, critical amino acid residue of Ps44-ASNase II identified in present study can be targeted for mutagenesis to evaluate the enzyme for therapeutic applications.