Characterization of a Novel Glutaminase-free L-asparaginase from Bifidobacterium Thermophilum

doi:10.21203/rs.3.rs-3113826/v1

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Characterization of a Novel Glutaminase-free L-asparaginase from Bifidobacterium Thermophilum

https://doi.org/10.21203/rs.3.rs-3113826/v1

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L-asparaginase (ASNase), as a pivotal amidohydrolase enzyme, has been used in removing acrylamide in food processing and treating acute lymphoblastic leukaemia in clinic. In this study, a novel ASNase from Bifidobacterium thermophilum (BtASNase) was successfully cloned and heterologously expressed in E. coli host. BtASNase was identified to share maximum 40% structural similarity with other ASNases in PDB database. The purified BtASNase with monomeric size about 35 kDa had the highest specific activity (554.82 IU/mg) at 55℃ and pH 8.0. Further investigation indicated that BtASNase showed great stability at wide pH range (6.0–11.0), and retained more than 85% of its activity for 50 min at 37℃. To be noted, BtASNase exhibited high L-asparaginase specificity and zero glutaminase activity. To our knowledge, this is the first time to explore ASNase from Bifidobacterium thermophilum, and explored BtASNase could be a potential candidate with desirable advantages for unraveling glutaminase activity, narrow pH range stability, and low thermostability restrict in industry applications of ASNase.

L-asparaginase

Bifidobacterium thermophilum

Glutaminase-free

pH stability

Thermal stability

L-asparaginase (EC3.5.1.1, ASNase) is a hydrolase that catalyzes L-asparagine (L-ASN, the non-essential amino acid) into aspartic acid (L-ASP) and ammonia(Eisele et al.2011), and converts less efficiently L-glutamine (L-Gln) leading to lymphoblastic cell death from nutritional deficiencies and protein synthesis inhibition( Lopes et al.2017). Hence, ASNases have attracted significant attentions in pharmaceutical industry as the potential anticancer compound (Asselin BL et al.1989). They have been applied as one kind of the main drugs for the therapy of acute myelomonocytic leukemia, acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia(Ghasemian et al.2019). In addition, it has hydrolysis potency of asparagine (major precursor of acrylamide) for efficiently producing acrylamide-free food products (Shabana et al.2021). Therefore, it has also been extensively used in various heat-processed foods for ensuring food safety. Considering its remarkable potential in food and clinic industry, ASNase with desired enzyme activity has attracted great interest.

ASNase widely exists in a variety of organisms including plants, animals, and microorganisms(Dumina et al.2021). To date, commercial ASNases has been used in the clinical treatment only from Erwinia chrysanthemi and Escherichia coli with low yield and glutaminase activities causing certain toxic side effects(Jia et al.2021; Castro et al.2021). Besides, only ASNases from Aspergillus oryzae and Aspergillus niger are applied for food processing with narrow range stability of pH 5.0–9.0 and pH 6.0–9.0(Hendriksen et al.2009; Pedreschi F et al.2008; Wang et al.2021). The shortcomings remarkably restrict ASNases from these organisms to be applied in industries. Therefore, it is essential to explore an alternative source of ASNase for facilitating its application.

Bifidobacterium as the well-known health-promoting and probiotic GARS microbe, belongs to actinobacteria phylum which can produce a variety of bioactive molecules and enzymes with better features than bacteria and fungus( Invernici et al.2018; Chua et al.2021; Ali et al.2021). However, there are rare report on ASNases derived from Bifidobacterium. Herein, our study successfully realized the expression of a novel ASNase from Bifidobacterium thermophilum in E. coli host. To our knowledge, expression and biochemical features of ASNase were firstly performed from Bifidobacterium thermophilum source.

2.1 Strains and reagents

B. thermophilum ASNase (Uniprot number A0A2N3QH30) gene was synthesized into the vector pET-28a by Sangon. Its expression was performed in E. coli BL21 (DE3) host. Chemicals for this study were all reagent grade from Aladdin Industrial Inc. (Shanghai, China) or Sangon Biotech (Shanghai, China).

2.2 Phylogenetic analysis and in silico sequence

The amino acid sequences of BtASNase were searched using NCBI BLAST and UniProt BLAST from database sequences. Identities among amino acid sequences were calculated using MEGA-X. The molecular masses and theoretical pI values were achieved through the ProtParam Expasy.

2.3 Structure analysis

The prediction of secondary structure for BtASNase was performed through the PSIPRED program. 3D structural modeling was predicted by online server (Swiss-Model). The sequence alignment of the B. thermophilum ASNase with homologous ASNase was performed through Clustal Omega and ESPript 3.0.

2.4 Expression and purification of BtASNase in E. coli

E. coli BL21 (DE3) containing pET-28a-BtASNase was cultured at 37°C in flask shaker (180 rpm). IPTG was added to the final concentration of 100 mg/mL when OD600 reached 0.6–0.8, then the flask was kept for the incubation at 16°C for 20 h. The cell pellets were then gathered by 5min centrifugation (5000 rpm), and suspended with buffer (20mM Tris-HCl, 300mM NaCl, pH8.0). Mixture were performed with a 15 min sonication at 300 W. After removing the debris by centrifugating at 15,000 rpm and 4°C for 30 min. Supernatant was sent onto Ni-NTA purification column. The recombinant BtASNase was validated by 4–20% SDS-PAGE.

2.5 The activity assay of BtASNase

Protein concentration was firstly performed through Bradford method( Bradford 1976). The activity of BtASNase was determined through the Nessler measurement with Nessler’s reagent following by Labrou and Muharram (2016). A mixture of 50 mM PBS buffer of pH 8.0 (700 µL), 200 mM L-asparagine (100 µL) and diluted enzyme (100 µL) were applied for 10 min incubation at 37℃ and abolished by supplying 25% trichloroacetic acid solution (100 µL). Then, reaction solution (40 µL) with additional Nessler’s reagent (100 mL) were gather together with deionized water (860 µL). The ammonia release quantity was analyzed through OD436 after 5 min.

2.6 Effects of temperature and pH on the activity and stability of BtASNase

The analysis for BtASNase optimum temperature was determined through testing ASNase activity at different temperatures (25°C-80°C). The highest activity of BtASNase was set as 100% for the relative activity calculation of other temperature in this temperature range. Temperature stability was determined through measuring the residual activity after 50 min incubation under different temperatures (4°C-50°C) followed by ice bath incubation (30 min). The activity of BtASNase at beginning was set as 100% activity (Jiao et al.2020).

The optimum reaction pH of BtASNase was analyzed by measure catalytic activity at pH 3.0 to pH 12.0 range. pH buffers for reactions were citrate-sodium buffer (pH 3.0–6.0), phosphate buffer (pH 6.0–9.0), glycine-sodium hydroxide buffer (pH 9.0–10.0), carbonate-sodium hydroxide buffer (pH 10.0–11.0), disodium hydrogen phosphate-sodium hydroxide buffer (pH 11.0–12.0)( Mukherjee J et al.2000; Chi H et al.2021). The maximum activity of ASNase was defined as 100% for the relative activity calculation. To determine pH stability, ASNase was firstly incubated in various pH buffers for 24 h, then back to optimum pH for determining activity. Enzyme activity at beginning was set as hundred percent activity to determine the residual activity according to its initial activity.

2.7 Effect of metal ions and inhibitors on BtASNase activity

The influences of metal ions and inhibitors on BtASNase activities were performed through adding 1 mM of various metal ions (NaCl, KCl, MgCl₂, CaCl₂, CuSO₄, ZnCl₂, FeSO₄, MnCl₂, FeCl₃, NiSO₄, and AlCl₃,) and inhibitors (SDS, EDTA). BtASNase activity without adding any compound was defined as hundred percent.

2.8 Substrate specificity assay of BtASNase

Different substrates at 20mM final concentration (L-asparagine (L-ASN), D-asparagine (D-ASN), L-glutamine (L-GLN), and L-glutamic acid (L-GLU)) were applied for substrate specificity determinations. The activity was performed through the same Nessler assay. BtASNase activity to L-asparagine was defined as hundred percent.

3.1 In silico sequence and phylogenetic analysis of B. thermophilum ASNase

The open reading frame consisting of 335 amino acids for BtASNase gene had the theoretical molecular mass of 35.370 kDa along with pI of 4.68. The amino acid sequences of BtASNase were aligned with the sequences of Escherichia coli ASNase, Erwinia carotovora ASNase, Helicobacter pylori ASNase, Erwinia chrysanthemi ASNase, Acinetobacter glutaminasificans ASNase, Wolinella succinogenes ASNase, Campylobacter jejuni ASNase, and Bifidobacterium boum ASNase. As the phylogenetic tree shown (Fig. 1), BtASNase showed identity of the amino acid sequence of ranging from 58 to 100% among the other ASNases.

3.2 Structure analysis of B. thermophilum ASNase

As shown in Fig. 2A, secondary structure of BtASNase was computated with a maximum of thirteen β-strands and nine α-helix with maximum hydrophilic molecules, along with a huge number of favorable coil regions in the deduced amino acid sequences. The three-dimensional structure model for BtASNase was simulated by the online server (Swiss-Model) to achieve its tertiary structure.

3.3 Expression and purification of B. thermophilum ASNase in heterologous E. coli

The gel band of purified BtASNase was around 35 kDa on SDS-PAGE (Fig. 3), which is consistent with its theoretical value (35.370 kDa). This result is concordant with those ASNases from B. altitudinis (35 kDa) and P. fluorescens (35 kDa)( Sindhu and Manonmani 2018; Prakash et al.2019). To further explore its characteristics, the purified and crude enzymes were applied for activity analysis. The specific activity of BtASNase was about 554.82 IU/mg (Table 1), which displayed 46.51 times higher compared with that of crude enzyme.

Table 1

Separation and purification process of *BtAsNase*
	Total Activity (IU)	Total Protein (mg)	Specific Activity (IU mg^− 1)	Purification Fold	Yield (%)
Crude enzyme	5464.50	441.97	11.93	1	100
Nickel affinity purified BtASNase	2,916.95	3.96	554.82	46.51	53.38

3.4 Effects of temperature and pH on the activity and stability of BtASNase

Generally, temperature is the critical parameter in biocatalysis, the activity of purified BtASNase was determined under different temperatures (25–80℃). As shown in Fig. 4A, 55℃ was the optimum reaction temperature for BtASNase. Further, thermal stability of BtASNase was analysis by determining its residual activity under different temperatures (4–50℃). As shown in Fig. 4B, the residual activity of BtASNase exhibited rare loss at 4℃ to 37℃ for 50 min incubation, indicating significant thermostability over this range. When the temperature raised to 40℃, its residual activity dropped to 13.7% after 50 min incubation. With the temperature reached 50℃, BtASNase was abolished with zero residual activity after 10 min. According to the reference, A. bacterium ASNase is stable under 4 to 10℃(Dash et al.2016), BtASNase exhibited high thermobility.

To explore the optimum reaction pH of BtASNase, its catalytic activity was measured under different pHs. As shown in Fig. 4C, BtASNase displayed the activity above 40% in pH range of 7.0–11.0, and highest at pH 8.0. Moreover, the residual activity of BtASNase was determined to explore its pH stability (Fig. 4D). BtASNase retained more than 40% in the range of pH 6.0–11.0, and over 80% in the range of pH 7.0–8.0. Compared with ASNases from A. niger stable at pH 5.0–9.0 and A. oryzae stable at pH 5.8–8.75(Hendriksen et al.2009).

3.5 Effect of metal ions and inhibitors on the activity of BtASNase

The effects of different metal ions as well as inhibitors on the activities of BtASNase were displayed in Table 2. Obviously, Mg²⁺ and Mn²⁺ could increase enzymatic activity for BtASNase by 42.97%, and 24.61%, respectively, which were concordant with those of ASNases from B. amyloliquefaciens and A. bacterium (Dash et al.2016; Hendriksen et al.2009;Yim and Kim 2019). However, Ca²⁺, Cu²⁺, and Ba²⁺ could lead to slight reduction of BtASNase activity, indicating they should be avoided during the fermentation process. Besides, EDTA also had slight inhibited effect on BtASNase activity, suggesting BtASNase not as a metalloprotein. To be noted, SDS could abolish the activity of BtASNase, which was similar to the reported ASNase from F. culmorum(Meghavarnam and Janakiraman S 2018).

Table 2

Effect of metal ions and compounds on *BtASNase* activity
Metal ions and inhibitors	Relative activity (%)
Control	100
K+	103.24 ± 2.12
Na+	106.08 ± 4.45
Ca2+	94.13 ± 3.67
Mg2+	142.97 ± 4.87
Cu2+	79.14 ± 2.08
Ni2+	108.51 ± 3.16
Zn2+	107.80 ± 2.95
Ba2+	92.56 ± 3.80
Mn2+	124.61 ± 4.47
Co2+	103.76 ± 2.89
EDTA	85.98 ± 1.26
SDS	0

3.6 Substrate specificity assay of BtASNase

As shown in Table 3, BtASNase only had enzymatic activity to D-asparagine (23.4%) and L-asparagine (100%), and exhibited no enzymatic activity towards the other two substrates, showing it had strict L-asparaginase activity without glutaminase activity.

Table 3

Substrate specificity of *BtASNase*.
Substrate	Relative activity (%)
L-ASN	100
D-ASN	23.40 ± 0.14
L-GLN	0
L-GLU	0

Bifidobacterium is a type of probiotic GARS microbe that can promote human health, which can produce a variety of bioactive molecules and enzymes with better features than bacteria and fungu. Our study successfully realized the expression of a novel ASNase from Bifidobacterium thermophilum in E. coli host, which rare report on ASNases derived from Bifidobacterium. As far as we know, expression and biochemical features of ASNase were firstly performed from Bifidobacterium thermophilum source. BtASNase showed identity of the amino acid sequence of ranging from 58 to 100% among the other ASNases Expressed as the phylogenetic tree. To be noted, BtASNase shared the maximum percentage of similarity with Bifidobacterium boum ASNase, which is also a kind of actinomycetes suggesting the divergence between the organisms.

Compared with its template from Yersinia pestis ASNase structure (PDB:3NTX), BtASNase monomer displayed two domains and the linker, which is consistant with the structure of most L-asparaginases, such as Helicobacter pylori ASNase (PDB: 2WLT), E. coli ASNase (6PA3), and Thermococcus kodakarensis ASNase (5OT0). Most importantly, BtASNase shared a maximum 40% similarity compared with the other ASNases in PDB, indicating BtASNase a potential novel L-asparaginase.

Although it was lower than ASNases from B. altitudinis (800 IU/mg) (Prakash et al.2019), B. sonorensis (4438.62 IU/mg) (Aly etal.2020), and P. furiosus (11,203.5 IU/mg)( Saeed etal.2020), it was much higher than those ASNases from many other species, such as A. terreus (339.34 IU/mg) ( Shabana et al.2021), S. brollosae (76.671 IU/mg)( Shabana et al.2021), B. licheniformis (36.08 IU/mg)( Alrumman et al.2019), and P. fluorescens (26 IU/mg) ( Sindhu and Manonmani 2018). Therefore, BtASNase in our study could be potentially applied for industrial applications for its high specific activity.

We sill have deeply studied the effects of temperature and pH on the activity and stability of BtASNase. Although most ASNases maintain their highest activity at 24–40℃, BtASNase displayed its maximum activity beyond this range, which was identical to those of ASNases from S. noursei (Selvakumar D2011) and P. furiosus(Saeed et al.2020). However, the optimum reaction temperature of BtASNase was still lower compared with that of ASNase from T.kodakarensis, whose maximum enzymatic activity was at 85℃(Chohan et al.2020). The residual activity of BtASNase exhibited more significant thermostability over the range of 4℃ to 37℃ compared with A. bacterium ASNase. Forthermore, BtASNase showed the wide pH range stability, which could meet pH demands for food processing application.

It is important to study the effect of metal ions and inhibitors on the activity of BtASNase. Mg²⁺ and Mn²⁺ can increase enzymatic activity for BtASNase. However, Ca²⁺, Cu²⁺, and Ba²⁺ should be avoided during the fermentation process due to their slight reduction of BtASNase activity. Otherwise, EDTA have slight inhibited effect on BtASNase activity,and SDS can abolish the activity of BtASNase.

L-glutaminase activity has been found in current commercial ASNases (E. coli ASNase with glutaminase activity of about 10% and E. chrysanthemi ASNase with glutaminase activity of approximately 1.5%)(Ollenschläger et al 1988;Eden et al 1990), causing many serious toxic side effects to ASNase treatment of patients(Derst et al.2000). Thus, different substrates were applied for investigating the substrate specificity of BtASNase to identify whether it had glutaminase activity for undesired L-glutamine production. BtASNase have less and most enzymatic activity to D-asparagine and L-asparagine respectively. However, it exhibited no enzymatic activity towards the other two substrates, showing it had strict L-asparaginase activity without glutaminase activity. Notably, glutaminase-free activity is the remarkably significant feature of ASNase for clinical treatment and food processing(Jia et al.2021; Vo et al.2020; Cecconello et al.2019), no glutaminase activity led BtASNase to have potential advantage in the pharmaceutical and food industry applications.

In all, a novel ASNase from Bifidobacterium thermophilum (BtASNase) was successfully expressed and characterized. BtASNase exhibited great features containing high asparaginase activity with zero glutaminase activity and a wide range pH stability. This explored BtASNase could be a future candidate agent of commercial ASNases for industry applications.

Author contribution ZZY conceived and designed the study. JJ and LQQ conducted the experiments. JJ wrote the manuscript. ZZY reviewed the manuscript. All authors read and approved the manuscript

Data availability Data will be made available on request.

Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest The authors declare no competing interests.

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Characterization of a Novel Glutaminase-free L-asparaginase from Bifidobacterium Thermophilum

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Strains and reagents

2.2 Phylogenetic analysis and in silico sequence

2.3 Structure analysis

2.4 Expression and purification of BtASNase in E. coli

2.5 The activity assay of BtASNase

2.6 Effects of temperature and pH on the activity and stability of BtASNase

2.7 Effect of metal ions and inhibitors on BtASNase activity

2.8 Substrate specificity assay of BtASNase

3. Results

3.1 In silico sequence and phylogenetic analysis of B. thermophilum ASNase

3.2 Structure analysis of B. thermophilum ASNase

3.3 Expression and purification of B. thermophilum ASNase in heterologous E. coli

3.4 Effects of temperature and pH on the activity and stability of BtASNase

3.5 Effect of metal ions and inhibitors on the activity of BtASNase

3.6 Substrate specificity assay of BtASNase

4. Discussion

Declarations

References

Status:

Version 1