Liquid Fermentation Of Fucoidanase From Aspergillus Wentii PZ322

Fucoidan and low molecular weight fucoidan (LMWF) had some biological activities, LMWF could obviously decrease side effects and antigenicity caused by fucoidan. Fucoidanase could hydrolyze fucoidan to produce sulfated LMWF without removal of its side substitute groups because of its substrate hydrolysis property. So it could additionally be used as a tool for elucidating the structure of fucoidan. Our study focused on investigating the screening, identification and liquid fermentation of fucoidanase from PZ322.In this study, many kinds of samples, including seawater, sea sand, sea mud, sea invertebrata, sea moss and decayed wood, were collected from Xiamen sea region. 129 strains marine-fungi were isolated from the above samples. With fucoidan as sole carbon source, 28 fucoidanase producing strains were obtained by primary and secondary screening, accounting for 21.7% of total fungi. It was found that the rate of marine-fungi producing fucoidanse isolated from seawater mud and decayed wood to the total of fungi isolated from different samples was high, 33.3%. PZ322 was definited as original strain for its topmost enzyme yield of 3.83 IU/ml and stable fermentation.Then morphological analysis of the strain initially identified as the genus Aspergillus member. Strain PZ322 ITS sequence was compared with the GENBANK sequence. And the significant sequence is sequence GU183175.1 (Aspergillus fumigatus isolate HF11) and AY373884.1 (Aspergillus wentii strain ATCC 1023), and Sequence similarity was 100%. Aspergillus fumigatus conidia production structure is single-layer, while the strains PZ322 morphological identification of the result was double-layer. So we identified this strain as Aspergillus wentii PZ322. Aspergillus wentii fucoidanase has not yet been reported on the current literature.In order to improve fucoidanase production, the compositions of medium and cultivation conditions were optimized. After single factor test, the optimum liquid fermentation medium of PZ322 was consisted of wheat bran 5%, kelp powder 2%, (NH₄)₂SO₄ 0.4%, The initial pH of medium was 6.5. The inoculation was 5%. The volume was 80 mL in 250 mL flask. After shaken at 180rpm and cultured in the above medium for 48h at 28℃, the fucoidanase activity reached 6.90U/mL. To further enhance the yield of fucoidanase, we have done three low energy (15 Kev) mutations to PZ322 induced by nitrogen ion implantation. The results showed that a lower injection dose (30 units), the production of beneficial mutants. After two rounds of mutagenesis, the strain 2041 was screened with high yield. The fucoidanase activity after 48h fermentation liquid is 8.89IU/ml.Finally, we tried to purify Aspergillus wentti PZ322 fucoidanase. Crude enzyme solution was classificated to five fractions by pretreatment and ultrafiltering by 100 000Da, 50 000Da, 30 000Da, 10 000 Da membrane. And the fucoidanase activity test showed that fucoidanase is mainly in below 30 000Da fraction and 50 000-100 000 Da fraction. Then fraction 50 000-100 000 Da was purified by Sephadex G-100 column chromatography and Superdex 200 column chromatography respectively, but PAGE identification results are not a single band, need for further purification.