Thermoactive β-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.

Abstract

Two fungal strains were evaluated for β-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≤0.001) higher β-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 R(2) (0.9512) observed during validation experiment showed the usefulness of the model. Highest level of enzyme activity (311.84U/g IDS) was predicted at 75% (w/w) initial moisture content, 26°C incubation temperature, 168h incubation time and initial inoculum, at the highest concentration tested (2.95ml spore suspension/5g substrate). Statistical optimization yielded a 4.5fold increase in β-N-acetylhexosaminidase activity. The crude β-N-acetylhexosaminidase showed optimum temperature of 57±1°C and pH of 3.6 and retained 50% activity after 1h of incubation at 57±1°C. SDS-PAGE zymogram revealed crude enzyme was a monomer with an apparent molecular weight ~110kDa. The crude enzyme formed 6.81±0.03mM/l of N-acetyl chitooligosaccharides from colloidal chitin in 24h 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.