Characterization, modeling, and anticancer activity of L.arginase production from marine Bacillus licheniformis OF2

Abstract

Background: L-arginase, is a powerful anticancer that hydrolyzes L-arginine to L-ornithine and urea. This enzyme is widely distributed and expressed in organisms like plants, fungi, however very scarce from bacteria. Our study is based on isolating, purifying, and screening the marine bacteria that can produce arginase.

Results: The highest arginase producing bacteria will be identified by using microbiological and molecular biology methods as Bacillus licheniformis OF2. Characterization of arginase is the objective of this study. The activity of enzyme was screened, and estimated beside partial sequencing of arginase gene was analyzed. In silico homology modeling was applied to generate the protein's 3D structure, and COACH and COFACTOR were applied to determine the protein's binding sites and biological annotations based on the I-TASSER structure prediction. The purified enzyme was undergone an in vitro anticancer test.

Conclusions: L-arginase demonstrated more strong anti-cancer cells with an IC50 of 21.4 ug/ml in a dose-dependent manner. L-arginase underwent another investigation for its impact on the caspase 7 and BCL2 family of proteins (BCL2, Bax, and Bax/Bcl2). Through cell arrest in the G1/S phase, L-arginase signals the apoptotic cascade, which is supported by a flow cytometry analysis of cell cycle phases.

Keywords: Anti-cancer; Arginase; Bacillus licheniformis OF2; Silico homology.

Fig. 1

Quantitative L-arginase production from the selected strains

Fig. 2

Phylogenetic tree of the Bacillus licheniformis OF2 partial 16S rRNA sequence in comparison to closely similar sequences in GenBank databases

Fig. 3

a The effect of the incubation time on the production of L-arginase. b Effect of the pH on the production of L-arginase from marine Bacillus licheniformis OF2. c Effect of incubation temperature on L-arginase production from marine Bacillus licheniformis OF2. d Effect of Carbon sources on L-arginase production from marine Bacillus licheniformis OF2. e Effect of different concentrations of maltose on L-arginase production from marine Bacillus licheniformis OF2. f Effect of different nitrogen sources on L-arginase production from marine Bacillus licheniformis OF2. g Effect of different concentrations of L-arginine on L-arginase production from marine Bacillus licheniformis OF2

Fig. 4

a Effect of temperature on the purified L-arginase from marine Bacillus licheniformis OF2. b Effect of pH on the purified L-arginase from marine Bacillus licheniformis OF2 (Sodium phosphate buffer (pH 6 and 7), Tris–HCl buffer (pH 8), glycine buffer (pH 9), and NaHCO₃–NaOH buffer (pH 10 and 11). c pH stability of L-arginase from marine Bacillus licheniformis OF2. d Effect of metal ions on L-arginase from marine Bacillus licheniformis OF2

Fig. 5

Lineweaver–Burk plot of the purified L-arginase with L-arginine

Fig. 6

a The 3D structures of Bacillus licheniformis OF2 arginase enzymes by a) I-Tasser and b) SWISS-MODEL. b Ramachandran’s plot calculations on the 3D models of arginase of Bacillus licheniformis OF2 computed by the SWISS-MODEL web-server to show the favored regions for backbone dihedral angles against amino acid residues in protein structure a) General (No Proline or Glycine) b) Glycine Only c) Pre-Proline Only d) Proline only. c predicted ligand binding sites

Fig. 7

a In vitro screening of the antiproliferative activities of L-arginase against human colorectal carcinoma (HCT-116 cell line), human breast carcinoma (MCF-7 cell line), human prostate cancer (PC3 cell line), human melanoma (Mel501 cell line), human pancreatic tumor cell line (Paca2), human lung carcinoma (A-549 cell line), human melanoma (A-375 cell line), human colon cancer (caco2 cell line), human liver carcinoma (HepG2). The preliminary concentration for screening was 100 μg/ml. Each result is a mean of 3 replicate samples and values are represented as % inhibition. b BCL2, BAX, BAX/BCL2 ratio and Casp 7 protein level in MCF-7 cells after treatment with L-arginase for 24 h. c Cellular mechanism of action of L-arginase (A, B) apoptotic induction of L-arginase. MCF-7 was treated with L-arginase for 24 h and analyzed by annexin V/PI staining. The percentage of apoptotic cells is the sum of early apoptotic (annexin V + /PI −) cell percentage and late apoptotic (annexin V + /PI +) cell percentage. (C, D) Cell cycle analysis of MCF-7 after incubation with compound L-arginase for 24 h. Untreated cells were used as a control