Thermoactive B-N-Acetylhexosaminidase Production by a Soil Isolate of Penicillium Monoverticillium CFR 2 under Solid State Fermentation: Parameter Optimization and Application For N-Acetyl Chitooligosaccharides Preparation from Chitin

Two fungal strains were evaluated for
b-N-acetylhexosaminidase production by solid state fermentation
using different agro-industrial residues such as
commercial wheat bran (CWB) and shrimp shell chitin
waste (SSCW), of which Penicillium monoverticillium
CFR 2 a local soil isolate showed significantly (P B 0.001)
higher b-N-acetylhexosaminidase activity on CWB medium
as compared with the activity of Fusarium oxysporum
CFR 8. Fermentation parameters such as incubation temperature,
incubation time, initial moisture content and
inoculum concentration were optimized by statistically
designed experiments, using 3**(4–1) fractional factorial
design of Response Surface Methodology. The high R2
(0.9512) observed during validation experiment showed the
usefulness of the model. Highest level of enzyme activity
(311.84 U/g IDS) was predicted at 75% (w/w) initial
moisture content, 26 C incubation temperature, 168 h
incubation time and initial inoculum, at the highest concentration
tested (2.95 ml spore suspension/5 g substrate).
Statistical optimization yielded a 4.5 fold increase in b-Nacetylhexosaminidase
activity. The crude b-N-acetylhexosaminidase
showed optimum temperature of 57 ± 1 C
and pH of 3.6 and retained 50% activity after 1 h of incubation
at 57 ± 1 C. SDS–PAGE zymogram revealed
crude enzyme was a monomer with an apparent molecular
weight *110 kDa. The crude enzyme formed 6.81 ±
0.03 mM/l of N-acetyl chitooligosaccharides from colloidal
chitin in 24 h of incubation. HPLC analysis revealed
hydrolysate contained 37.57% N-acetyl chitotriose and
62.43% N-acetyl chitohexose, indicating its potential for
specific N-acetyl chitooligosaccharides production.