Fermentation Optimization And Catalytic Properties Of Acetyl Xylan Esterase

In this paper, we studied on fermentation conditions for acetyl xylan esterase production by yeast strains KM71H (Muts) and its catalytic properties. The experimental results were as follows:To further increase the production of AXE, the factors which affected acetyl xylan esterase fermentation in baffled flask were screened out by single factor analysis. The result showed that three prominent factors were methanol concentration and the amount of casein added, initial pH in the medium. Then, Box- Behnken design and Response Surface Methodology was employed for optimizing the fermentation condition. The obtained optimal fermentation conditions were methanol 2.8%, casein 0.63%, initial pH 8.0, inoculum OD600=30, temperature 25℃.Under these conditions, the yield of AXE increased to 463IU/ mL.In a 7.5 L fermenter, we found that the best condition for AXE production was inoculating amount 10%, casein 0.7%, pH of induction phase 6.5, temperature 25℃. Methanol concentra-tion 0.35%-0.5%, and the activity of enzyme reached 1385IU/mL after 84 hours'fermentation.AXE was purified by affinity chromatography using Ni-NTA agrose. SDS-PAGE analysis revealed that the purified AXE migrated as two bands with molecular masses of 48 KDa and 66KDa, Both turned out to be 45KDa after being treated with EndoH. In the assay of testing the effects of metal ions on AXE and deglycosylated AXE, Zn2+ and Cu2+ showed highly inhibition on them, while Mn2+ and Ca2+ could slightly activate them, however, no dramatic effects were found in Mg2+ or Co2+. Also the catalytic activity of deglycosated AXE on three substrates de-clined, the activity of the enzyme reduced to 44%-90% of the AXE activity. The proteolytic stability of deglycosylated AXE decreased somewhat, it become 87% of the acetyl xylan es-terase. The thermal stability of AXE dropped as well after being deglycosylated. PMSF strongly inhibited the enzyme. The remaining enzymatic activity was only 2% by incubation in 0.1mM PMSF.The purified enzyme showed a mild but significant synergistic effect in combination with xylanase from Volvariella volvacea, on the degradation of oat-spelt xylan, birchwood xylan, and DSWB with a 5%,11%,1% increase in the amount of reducing sugar released, compared to the expected amount of individual enzymes alone.