PARTIAL-PURIFICATION AND CHARACTERIZATION OF 2 EXTRACELLULAR N-ACETYL-D-GLUCOSAMINIDASES PRODUCED BY THE ENTOMOPATHOGENIC FUNGUS BEAUVERIA-BASSIANA

Beauveria bassiana grown in a liquid medium containing N-acetyl-D-glucosamine and colloidal chitin produced two distinct N-acetyl-D-glucosaminidases, NAGase 1 and NAGase 2. NAGase 1 had a molecular weight of 97 000 and NAGase 2 was comprised of two subunits, of molecular weights 64 000 and 66 000. The optimal temperature and pH for NAGase 1 were 57-degrees-C and PH 5 and for NAGase 2 they were 37-degrees-C and pH 5. NAGase 1 was more thermostable than NAGase 2. The isoelectric points of NAGase 1 and 2 were ca. pH 9.5 and 5.5, respectively. NAGase 1 and 2 were unaffected by a 10 mM concentration of chloride salts of the ions Ca2+, Mg2+, or Zn2+, by 10 mM EDTA, and by 0.25-1 mM of short chain fatty acids. Dithiothreitol caused some inactivation of NAGase 2 while stimulating activity of NAGase 1. NAGase 1 had a K(m) of 0.38 mM and a K(cat)/K(m) of 3923.88 M-1.s-1. NAGase 2 had a K(m) of 2.095 mM and a K(cat)/K(m) of 411.88 M-1.s-1 when p-nitrophenyl-beta-N-acetylglucosminide was used as the substrate.