ABSTRACT
Abstract L-glutaminase was produced by Streptomyces canarius FR (KC460654) with an apparent molecular mass of 44 kDa. It has 17.9 purification fold with a final specific activity 132.2 U/mg proteins and 28% yield recovery. The purified L-glutaminase showed a maximal activity against L-glutamine when incubated at pH 8.0 at 40 °C for 30 min. It maintained its stability at wide range of pH from 5.0 11.0 and thermal stable up to 60 °C with Tm value 57.5 °C. It has high affinity and catalytic activity for L-glutamine (Km 0.129 mM, Vmax 2.02 U/mg/min), followed by L-asparagine and L-aspartic acid. In vivo, L-glutaminase showed no observed changes in liver; kidney functions; hematological parameters and slight effect on RBCs and level of platelets after 10 days of rabbit's injection. The anticancer activity of L-glutaminase was also tested against five types of human cancer cell lines using MTT assay in vitro. L-glutaminase has a significant efficiency against Hep-G2 cell (IC50, 6.8 μg/mL) and HeLa cells (IC50, 8.3 μg/mL), while the growth of MCF-7 cells was not affected. L-glutaminase has a moderate cytotoxic effect against HCT-116 cell (IC50, 64.7 μg/mL) and RAW 264.7 cell (IC50, 59.3 μg/mL).
Subject(s)
Animals/chemistry , Animals/drug effects , Animals/enzymology , Animals/metabolism , Animals/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/drug effects , Antineoplastic Agents/enzymology , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Biocatalysis/chemistry , Biocatalysis/drug effects , Biocatalysis/enzymology , Biocatalysis/metabolism , Biocatalysis/pharmacology , Cell Proliferation/chemistry , Cell Proliferation/drug effects , Cell Proliferation/enzymology , Cell Proliferation/metabolism , Cell Proliferation/pharmacology , Enzyme Stability/chemistry , Enzyme Stability/drug effects , Enzyme Stability/enzymology , Enzyme Stability/metabolism , Enzyme Stability/pharmacology , Glutaminase/chemistry , Glutaminase/drug effects , Glutaminase/enzymology , Glutaminase/metabolism , Glutaminase/pharmacology , Glutamine/chemistry , Glutamine/drug effects , Glutamine/enzymology , Glutamine/metabolism , Glutamine/pharmacology , HeLa Cells/chemistry , HeLa Cells/drug effects , HeLa Cells/enzymology , HeLa Cells/metabolism , HeLa Cells/pharmacology , /chemistry , /drug effects , /enzymology , /metabolism , /pharmacology , Humans/chemistry , Humans/drug effects , Humans/enzymology , Humans/metabolism , Humans/pharmacology , Kinetics/chemistry , Kinetics/drug effects , Kinetics/enzymology , Kinetics/metabolism , Kinetics/pharmacology , Streptomyces/chemistry , Streptomyces/drug effects , Streptomyces/enzymology , Streptomyces/metabolism , Streptomyces/pharmacology , Substrate Specificity/chemistry , Substrate Specificity/drug effects , Substrate Specificity/enzymology , Substrate Specificity/metabolism , Substrate Specificity/pharmacologyABSTRACT
The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min-1 mg-1, using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+. The substrate inhibition observed with SSA was uncompetitive with respect to NAD+. While product inhibition by succinate was not observed, NADH inhibited the enzyme competitively with respect to NAD+ and noncompetitively with respect to SSA. Dead-end inhibition by AMP and p-hydroxybenzaldehyde (pHB) was analyzed. The pHB inhibition was competitive with SSA and uncompetitive with NAD+; AMP competed with NAD+. Consistent with the kinetic data, a sequential, ordered Bi Bi mechanism is proposed for this enzyme.