Characterization of a Novel Recombinant β-Lactamase from Bacillus subtilis R5

1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Abdul Qadir Jillani Road, Lahore, Pakistan. 2Riphah International University, Raiwind Road Campus, Lahore 3Department of Biotechnology, Virtual University of Pakistan 4Department of Chemistry, University of Agriculture Faisalabad, Faisalabad Article Information Received 18 July 2021 Revised 14 August 2021 Accepted 21 August 2021 Available online 07 December 2021 (early access)


INTRODUCTION
β -lactamases (EC 3.5.2.6) hydrolyze the amide bond between the beta carbon atom and the nitrogen atom of the four corner beta lactam ring which is core part of all beta lactam based antibiotics. β-lactamases render the antibiotics ineffective by breaking the beta lactam ring causing development of resistance in bacteria (Gaude and Hattiholli, 2013). These enzymes have a wide range of applications in pharmaceutics, waste water treatment, diagnostics, food analysis, contamination control and in cancer chemotherapy (Giang et al., 2014).
β-lactamases are produced only by bacterial strains (Therrien and Levesque, 2000). Four classes of β-lactamases, namely A, B, C and D have been recognized based on their amino acid sequences and molecular function. While the activities of A, C and D classes of β-lactamases are governed by the serine based mechanism, the metallo-β-lactamases or class B lactamases require zinc for their activity (Bush et al., 1995). Uptil now more than 890 β-lactamases have been reported from different bacterial strains (Bush and Jacoby, 2010). Bacteria have developed resistance against various antibiotics due to the presence of genes responsible for the production of β-lactamases, involved in catabolism of antibiotics (MacGowan and Macnaughton, 2017).
Bacteria are a diverse group of microbes, distributed in all environments and are involved in the production of enzymes. Genus Bacillus is being utilized for the fulfillment of industrial demand of enzymes worldwide (Salcido et al., 2013;Pasvolsky et al., 2014;Liu et al., 2014). Extracellular enzymes in bulk are produced industrially from the selected strains of bacteria thus it is thought that they occupy the most prominent place regarding industrial production of enzymes. Food and Drug Administration (FDA) has listed some Bacillus species to be safe for their usage in the food and drugs (Sorokulova et al., 2008).
Bacillus subtilis R5 are Gram positive, small rod shaped, highly motile cells that show optimal growth O n l i n e F i r s t A r t i c l e at 30°C (Jalal et al., 2009). The present study describes characterization of recombinant β-lactamase from Bacillus subtilis R5 and its utilization as a positive control for antibiotic resistance detection in diagnostic laboratories.

MATERIALS AND METHODS
All chemicals utilized in the current study were of purified grade and procured from Sigma Aldrich (USA). The DNA extraction kit, Ins T/A cloning kit and restriction endonucleases were procured from Thermo-Fischer Scientific and Invitrogen, Life Sciences, USA.

Culture maintenance and isolation of genomic DNA
The pure culture of Bacillus subtilis R5, a gift from School of Biological Sciences, University of the Punjab, Lahore, was maintained on LB medium and used for isolation of genomic DNA (Sambrook and Russell, 2001).

Cloning of β-lactamase gene
The β-lactamase gene was amplified by PCR using BL-3N: CATATGACAACTGAAGATG and BL-3C: TCACCTCTTCATTGTTTTAA as forward and reverse primers and the genomic DNA of B. subtilis R5 as template. β-lactamase gene sequence of B. subtilis 168 (NZ_CP010052), the closet homolog of B. subtilis R5 was utilized for designing the primers. The forward primer contained the unique NdeI restriction site. Ligation of purified PCR product was performed in pTZ57R/T using T4 DNA ligase (Thermo-Fisher, Life Sciences, USA) at 20°C for overnight. E. coli DH5α competent cells were transformed using the ligated mixture and blue white screening method was followed for the screening of positive clones (Sambrook and Russell, 2001). The white colonies from the plate were utilized for isolation of plasmid DNA (Jalal et al., 2009). The presence of insert in the recombinant vector was examined by single and double digestion using HindIII and NdeI endonucleases. The plasmid DNA after confirmation by restriction digestion, was utilized for sequencing of β-lactamase gene using M13 forward and reverse primers. The obtained nucleotide sequence was utilized for deduction of amino acid sequence and for homology analysis (Naas et al., 2003;Sabir et al., 2017).

Expression analysis of β-lactamase gene
The β-lactamase gene was transferred from recombinant pTZ57R/T to pET21a already restricted with same endonucleases and this recombinant pET21a having β-lactamase gene was used for the transformation of BL21 Codon Plus (DE3) cells. The expression of β-lactamase was analyzed by growing the recombinant cells in the presence of 100 µg/mL ampicillin. The overnight grown cells were diluted to 1% with fresh LB broth and were incubated at 37 °C until the optical density at 660 nm reached to 0.4. The cells were induced with 0.6 mM IPTG followed by further incubation of 5h at the same temperature under shaking conditions. The cell pellet after centrifugation (6000xg for 10 min) was resuspended in 10 mL sodium phosphate buffer (pH 7) and the cells were lysed by sonication. The production of recombinant β-lactamase was analyzed by 12% SDS-PAGE.

Optimal production of β-lactamase
The production of β-lactamase was examined by varying the IPTG concentration from 0.1 to 1mM and with post induction time from 1 to 6h. The samples were withdrawn after every hour and were utilized for the estimation of β-lactamase activity.

Purification of recombinant β-lactamase
The soluble part after sonication process was applied on pre-equilibrated DEAE Sephadex A-50 column. The equilibration and washing of unbound proteins in column was done with 50 mM sodium phosphate buffer (pH 7). The bound proteins in the column were eluted by NaCl gradient prepared in the same buffer. The fractions containing β-lactamase activity were pooled and was applied to pre-equilibrated Sephadex G-50 gel filtration column. The purity of the fractions with β-lactamase activity was analyzed on SDS-PAGE. The purified recombinant β-lactamase was called as LAC BS .
Enzyme activity assay β-lactamase activity was examined by following the method of Sawai and Yamagishi (1978). The assay mixture comprised of 0.1 mL enzyme solution, 2.4 mL 50 mM sodium phosphate buffer (pH 7) and 0.5 mL benzylpenicillin. The mixture was incubated at 37 °C for 10 min in a water bath and was allowed to stay at room temperature for 10 min followed by addition of 3 mL iodine solution with rapid mixing and absorbance was recorded at 540 nm. Two blanks were utilized for performing the activity assay. Blank A was comprised of 2.9 mL sodium phosphate buffer and 0.1 mL enzyme solution while Blank B was having 2.5 mL sodium phosphate buffer and 0.5 mL benzylpenicillin. The blanks were treated in the same way as described above. One β-lactamase unit was the amount of enzyme which hydrolyzed 1 µmol of substrate per min under the assay conditions.

Characterization of β-lactamase
Effect of temperature The enzyme activity was recorded at various temperatures ranging from 15 to 45°C. Regarding the activity assay, the assay mixture was incubated at specific temperature in water bath and the absorbance was recorded as 540 nm.

Effect of pH
The impact of pH on enzyme activity was recorded by examining the β-lactamase activity in the presence of 50 mM of each of sodium acetate buffer (pH 3-5), sodium phosphate buffer (pH 5-7), Tris HCl buffer (pH 7-9) and Glycine NaOH (pH 9-10).

Effect of metal cations and detergents
The enzyme activity was recorded in the presence of 1mM of each of metal cations including Ca 2+ , Mg 2+ , Fe 2+ , Zn 2+ , Cd 2+ , CO 2+ , Cu 2+ Ni 2+ and Mn 2+ as well as in the presence of 1mM EDTA. The activity was also recorded in the presence of 1% non-ionic (Triton X-100, Tween 20 and Tween 80) and ionic (SDS) detergents.

Kinetic studies of β-lactamase
β-lactamase activity was examined by changing the substrate concentration from 0.1 to 1%. The attained data was used for the construction of Lineweaver Burk/ double reciprocal plot and for the calculation of kinetic parameters.

Antibiotics degradative ability of recombinant β-lactamase
The β-lactamase activity was examined against various β-lactam ring containing antibiotics using LB agar plates and commonly available bacterial strains.

RESULTS
The PCR resulted in the amplification of 1 kb β-lactamase gene. Single digestion of recombinant pET21a with Nde1 resulted in the linearization of the vector and double digestion with HindIII and Nde1 resulted in the liberation of insert from the recombinant vector (Fig. 1). The DNA sequencing confirmed the presence of β-lactamase gene. The homology analysis based on deduced amino acid sequence showed the sequence identity of 100% with the non-characterized members of genus Bacillus whereas this lactamase did not show significant homology among the characterized counterparts.
Maximal production of β-lactamase was observed at 37°C when BL21 CodonPlus (DEL) cells having recombinant pET21a were induced with 0.6 mM IPTG (Fig. 2) with a post induction period of 5h. SDS-PAGE analysis of the purified protein after DEAE Sephadex A-50 column confirmed the molecular weight of β-LAC BS as 34 kDa (Fig. 3).  LAC BS activity was linearly increased with the increase in temperature from 15 to 37°C. Further increase in temperature beyond 37°C resulted in the decreased enzymatic activity (Fig. 4). LAC BS activity was found pH dependent and was able to perform its activity between pH ranges from 3 to 10 with an optimal activity at neutral pH (Fig. 5). The presence of 1mM EDTA in the assay mixture reduced the LAC BS activity to 91%. However, the presence

O n l i n e F i r s t A r t i c l e
of Zn 2+ , Co 2+ and Mn +2 in the activity assay enhanced the enzymatic activity to 144, 121 and 108% whereas, Mg 2+ , Ca 2+ and Fe 2+ reduced the activity to 90, 82 and 50% when used at a final concentration of 1mM. The presence of nonionic (Triton X-100, Tween 20, Tween 80) and ionic (SDS) detergents could reduce the LAC BS activity to 97, 94, 73 and 71% respectively when used at final concentration of 1% (Table I). Kinetic studies revealed the K m and V max values of 2.27 mM and 45.45 µmoles/min, respectively (Fig. 6). LAC BS has degraded the β-lactam ring containing antibiotics and due to the degradation of antibiotics the bacterial growth was observed in the LB plates as compared to the controls.    5. Effect of pH on LAC BS activity. X-axis shows the pH while Y-axis shows the LAC BS activity (%). Enzyme activity was examined in 50 mM of each of sodium acetate buffer (pH 3-5), Sodium phosphate buffer (5-7), Tris-HCl buffer (7-9) and Glycine/NaOH buffer (9-10) using benzylpenicillin (5μM) as a substrate. Fig. 6. Lineweaver-Burk plot for the estimation of kinetic parameters. X-axis shows 1/substrate concentration whereas Y-axis shows 1/enzyme velocity.
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O n l i n e F i r s t A r t i c l e
Characterization of a Novel Recombinant β-Lactamase Metal chlorides were used in β-LAC BS activity assay.

DISCUSSION
β-lactamases are enzymes that play important role as positive control in antimicrobial susceptibility testing in clinical laboratories. These are responsible for the hydrolysis of amide bond in the β-lactam ring to make the antibiotic ineffective. β-lactamases have been classified into various classes including A, B, C and D. Classes A, C and D have serine as active site amino acid and follow the development of acyl enzyme intermediate during the hydrolysis of β-lactam ring. Class B lactamases are metal dependent enzymes which require Zn 2+ for their activity (Fisher et al., 2005;Massova and Mobashery, 1998;Wilke et al., 2005).
Interestingly, β-lactamase from current study is unique as it didn't share the basic conserved domains required for the activity of class A, C and D of β-lactamases and this lactamase shows its activity even in the absence of Zn 2+ which is the unique character of class B β-lactamases. The lactamase from current study have ability of showing 90% of its activity even in the presence of 1mM EDTA; however LAC BS activity is increased in the presence of Zn 2+ . These characteristics make this enzyme novel.
Metallo β-lactamases require Zn 2+ for performing their activity and these enzymes can't work in the absence of cofactor (Paton et al., 1994;Concha et al., 1996;Salahuddin et al., 2018;Meini et al., 2015). LAC BS retains 90% activity in the presence of EDTA indicating the ability of enzyme to work even in the absence of Zn 2+ . At the same time its activity is being enhanced with the availability of Zn 2+ . This might be due the stabilizing impact of Zn 2+ on the structure of LAC BS . The results demonstrated that LAC BS requires 0.8 mM Zn 2+ for performing maximal activity. In contrast to this, β-lactamase VIM-4 from Pseudomonas aeruginosa possessed maximal activity at 0.4 mM concentration of Zn 2+ (Lassaux et al., 2011).
The presence of ionic and non-ionic detergents showed slight to moderate inhibitory impact on the LAC BS activity. Among the nonionic detergents, Tween 80 reduced the enzymatic activity to 73% as compared to Tween 20 or Triton X-100 which exhibited negligible impact and reduced the enzymatic activity to 94 or 97%, respectively.

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SDS reduced the LAC BS activity to 70%.

CONCLUSION
β-lactamase from current study is unique as it does not show significant homology with the previously characterized lactamases. Secondly, this enzyme shows its activity similar to class A, C and D of lactamases in a metal independent way, however the activity is being increased in the presence of Zn 2+ that is unique characteristic of class B of lactamases. The antibiotic hydrolyzing ability of this enzyme makes it suitable for its use in diagnostic laboratory. The domestic production of β-lactamase will result in cost effective availability of this enzyme and will save huge foreign exchange for the import of this enzyme.