Molecular characterization and computational analysis of a highly specific L-glutaminase from a marine bacterium Bacillus australimaris NIOT30

Abstract

An alkaline active L-glutaminase (BALG) producing bacterium was screened and identified from seamount sediment samples of the Arabian Sea. The isolate was confirmed to be Bacillus australimaris NIOT30 based on morphological characteristics and 16 S rRNA gene sequencing. The glutaminase gene, balg was PCR amplified, cloned and expressed in E. coli BL21 (DE3) host. The molecular weight of purified BALG was estimated to be 36 kDa and the enzyme showed a specific activity of 507 ± 27 Umg^-1 against L-glutamine under optimal assay conditions of pH 7.0 and temperature at 37 °C for 15 min. The enzyme showed maximum activity at pH 7 and retained 95% activity at pH 10. BALG retained a relative activity of about 82% and 45% at 45 °C and 60 °C respectively. The kinetic parameters of BALG, Km and Kcat/Km were determined to be of 210 ± 11 mM and 4.4 × 10² M s^-1 respectively. Homology modeling and substrate ligand interaction studies revealed the stability of the enzyme-substrate complex. The present study highlights the characterization of a highly active L-glutaminase from B. australimaris NIOT30. Further, mutational analyses of ligand binding residues would show insights into the affinity of L-Glutaminase.

Keywords: Bacillus australimaris; Characterization; L-Glutaminase; Sediment; Specificity.

Fig. 1

Production of L-Glutaminase from selected positive strains. The strains were grown in minimal medium containing 5% L-Glutamine at 30 °C, 150 rpm for 48 h. The lysed cell pellets were assayed for LG activity.

Fig. 2

Characterization of NIOT30 strain. (A) Scanning electron micrograph of B. australimaris NIOT30. (B) Phylogenetic tree showing the relatedness of B. australimaris NIOT30 to other bacterial species, using neighbor-joining method. The numbers at the nodes show the bootstrap values from 1000 resampling analyses.

Fig. 3

Phylogenetic tree showing the relatedness of B. australimaris NIOT30 BALG to glutaminases form other bacterial species.

Fig. 4

Cloning, expression and purification of L-glutaminase. (A) L-glutaminase encoding gene of 1 kb was PCR amplified and cloned into pET28a. Lane 1: Double digested pET28a carrying balg gene. (B) SDS-PAGE analysis of purified BALG. Lane P: His-tagBALG purified using Ni-NTA affinity chromatography. Lane M: Precision plus prestained protein molecular weight marker (BioRad). Lane 1,2&3 - Purified BALG with an apparent molecular mass of 36 kDa on SDS-PAGE.

Fig. 5

Characterization of the functional properties of purified BALG. (A) Effect of pH on the activity and (B) Stability of purified BALG. (C) Effect of temperature on the activity and (D) Stability of purified BALG.

Fig. 6

(A) Effect of substrate concentration on the activity of BALG. (B) Line-Weaver Burk plot to determine Km and Vmax of purified BALG.

Fig. 7

Modeling, docking and model validation by ProSA (A) Homology model of BALG (B) The Z score is represented by a large dot.

Fig. 8

ConSurf results depicting the conserved functional regions of BALG.

Fig. 9

Binding patterns of BALG and L-Gln docked complex. (A) The orange cartoon represents the docked complex before 100 ns MD. (B) The blue cartoon represents the docked complex after 100 ns MD. The yellow dash line represents the hydrogen bond. (C) MD simulation of the BALG – L-Gln docked complex. The graph shows the RMSD of the backbone C atoms of the BALG protein with respect to the first snapshot during the 100 ns MD simulation.