Purification and characterization of glutamine synthetase from alkalophilic Bacillus No. 170.

Abstract

Glutamine synthetase from an alkalophilic Bacillus No. 170 was purified to homogeneity from cells grown in synthetic medium at pH 9.0. The purified enzyme had a molecular weight of 600,000 and a subunit size of 50,000, similar to the enzymes from Bacillus subtilis and Bacillus cereus. Either Mg2+ or Mn2+ activated the enzyme; however, the highest activity was obtained when 20 mM Mg2+ with 0.2 mM Mn2+ were added to the assay mixture. The peculiar divalent cation dependency of the glutamine synthetase was similar to that of other Bacillus enzymes but differed from that of the enteric bacteria enzymes. The kinetic properties of the enzyme, such as optimum pH, Km for each substrate, Vmax values, and inhibition by glutamine, were similar to those in the case of other Bacillus enzymes. The enzyme from this alkalophilic Bacillus was modified by iodoacetoamide or the ATP analog, 5' p-fluorosulfonyl benzoyladenosine (FSBA). Only Mg2+-dependent activity was decreased by iodoacetamide, whereas each of the Mn2+-, Mg2+-, and Mg2+ plus Mn2tdependent activities was decreased with FSBA. The sequence of the NH2-terminal 20 amino acids of alkalophilic Bacillus glutamine synthetase was similar to those of other Bacillus enzymes.Glutamine synthetase [L-glutamate: ammonia ligase (ADP-forming) EC 6.3.1.2] is an important enzyme in nitrogen metabolism and ammonia assimilation in many prokaryotes, eukaryotes, and archaebacteria. Consequently, the enzyme is subject to a variety of control mechanisms, and its regulation has been extensively studied in Escherichia coli and some enteric bacteria (11, 21, 23) . In these bacteria,

Adaptive Laboratory Evolution of Bacillus subtilis 168 for Efficient Production of Surfactin Using NH4Cl as a Nitrogen Source

“…Iron homeostasis in bacteria is maintained by various iron uptake systems, which are indispensable for many key enzymes of the cell, such as ribonucleotide reductases, superoxide dismutases, and various energy-generating and regulatory proteins of heme or iron-sulfur cluster cofactors [23]. Mn 2+ and Mg 2+ enhance nitrogen assimilation by increasing glutamine synthetase (GS) and glutamate synthetase (GOGAT) activities [21,24,25].…”