Industrialization Of Alginate Lyase From Vibrio Sp. 510-64 And Their Applications

After u.v. and EMS mutagenizing on a marine bacterium strain Vibrio sp. 510, the alginate lyase secreted by the mutant strain Vibrio sp. 510-64 reached 289.1EU/ml and was higher than the original strain (136.4EU/ml). The optimum alginate lyase producing media are as follows: AlgNa 5‰, NaCl 20‰, CaCl2 0.1‰, KH2PO4 1‰, NH4Cl 3‰, initial pH 6. After 68 batches of continuous fermentations, the strain 510-64 demonstrated a good stability and was suitable to pilot study. Crude alginate lyase from the cell free supernatant of cultures was purified by ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sepharose and two steps of gel filtration on Superdex 75. The purified alginate lyase was detected as a single protein band upon SDS-PAGE, with a molecular weight of 34.6kDa. The N-terminal sequence of the alginate lyase is: AEILATDDAD using Edman degrading method. The alginate lyase showed optimum activity at a temperature of 35C and a pH 7.5 and showed stability below this temperature and at pH range 6.5-9. This alginate lyase was promoted by Mg2+, Ca2+, Li+, Na+, K+and inhibited by EDTA, Ba2+ Zn2+, Hg2+ Sn2+. After inactivated by EDTA, alginate lyase could be revived by the Ca2+,Mg2+ and Sn2+ to some extent.The optimum digestion conditions for alginate lyase releasing protoplasts from Undaria pinnatiflda were at the temperature of 28 C , digestion time 2h, 213.36EU/0.5g fresh leaf, NaCl 50%, rotary speed of 150 r/min. Under this conditions, the yield of protoplasts released from Undaria pinnatiflda reached 2.62 + 0.09x106cell/0.5g. The digestion course could be activated by Fe2+, Mn2+(0.05, 0.08, 0.10M) and totally inhibited by Ca2+.Two alginate-derived oligosaccharides (ADOs) of different molecular weightwere sulphated with the formamide-chlorosulphonic acid method. The antitumour activities of the two ADOs and their sulphated substitution derivatives that exhibited no direct cytotoxic effects on tsFT210 cells, were subsequently determined on Kunming mice. The best antitumour performer was Oligosaccharide A (molecular weight 3798 Da, sulphation degree 1.3), which exhibited 70.4 and 66.0% tumour inhibition against solid Sarcoma 180 at doses of 100 and 50 mg kg-1, respectively. For ADOs and their sulphated derivatives, an indirect antitumour effect via modulation of the host-mediated immune defences is postulated.The influence of alginate-derived oligosaccharide (molecular weight 1445 Da) was tested on the maize seed germination at different concentrations. Assays of a- and B-amylase and protease activities showed the highest response at 0.75 ‰. Compared with the control, root growth on days 3 and 7 showed increases of 34% and 18%, respectively; shoot growth on day 7 increases of 46%. In the case of protease activity, treatments with both 0.75‰ and 1.50‰ alginate-derived oligosaccharide gave higher activities than the control. These results indicate that the rate of seed germination was enhanced by increasing the activities of several enzymes beneficial for germination.Regarding to the industrialization production of alginate lyase secreted by Vibrio sp. 510-64, a pilot study was conducted and suggested a very good utilization future.