[Purification and characterization of beta-N-acetyl hexosaminidase from Aspergillus tamarii].
A beta-N-acetylhexosaminidase from mycelium-free culture filtrate of Asp tamarii S215 was purified to PAGE homogenous by ammonium sulfate and polyethylene glycol fractionation precipitation followed by Sephadex G-50 desalt, DEAE-Sephadex A-25 chromatography, hydroxyapatite chromatography and DEAE-Sephadex A-25 rechromatography with 170-fold purification and 24.7% recovery. The ratio of the beta-GlcNAcase and beta-GalNAcase was 2.5 and remained constant throughout the purification. The Mr estimated with concentration gradient PAGE was 140,000 and subunit Mr determined with SDS-PAGE was 72,000, the number of subunit were 2. The pI was 4.2 determined by PAGIEF. The optimum pH was 5.5-6.5 and 5.0-6.0 for beta-GlcNAcase and beta-GalNAcase respectively with stable pH range 5.5-8.3 for both. The optimum temperature was 60 degrees C for beta-GlcNAcase and beta-GalNAcase. The residual activity of beta-GlcNAcase was 52.7% after treated at 50 degrees C for 8 h and it was 44.9% for beta-GalNAcase. The residual activities of both were down to 1% after treated at 62 degrees C for 10 min. The activity was slightly activated by Mn2+ or Fe2+, while strongly inhibited by Hg2+ and slightly by Ag+, Cu2+, Pb2+, Cd2+ or Zn2+. Analyses of amino acids composition showed that the beta-HexNAcase contained about 24.2% acidic amino acids and 14.9% basic amino acids and only 0.6% methionine.
Tao
Y
Y
Institute of Microbiology, Academic Sinica, Beijing.
Yan
Z
Z
Sun
J
J
Cheng
X
X
Zhang
S
S
D000118:Acetylglucosaminidase / Q000032:analysis; D000596:Amino Acids / Q000032:analysis; D001230:Aspergillus / Q000032:analysis*; D006596:Hexosaminidases / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D013696:Temperature; D048828:beta-N-Acetyl-Galactosaminidase; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification*
D000596:Amino Acids; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D001619:beta-N-Acetylhexosaminidases; D048828:beta-N-Acetyl-Galactosaminidase