Study Of Characteristics Of Rhizopus Oryzae Glucoamylase And Its Effects On L-lactic Acid Fermentation

Glucoamylase is a kind of extracellular enzyme of Rhizopus oryzae induced in amylum culture medium. Its activity has a great impact on L-lactic acid yield and L-lactic acid fermentation time. Purification and characteristics of glucoamylase in Rhizopus oryzae As3.819 with high L-lactic acid yield were carried out in this paper and the L-lactic acid conversion and glucoamylase activity were also studied during fermentation in different culture conditions. The results were as follows:The glucoamylase was purified by ammonium sulphate precipitation, dialysis and Sephadex G-100 gel filtration and its specific activity increased by 23.58 times. The SDS-PAGE showed that the purified enzyme was a homogeneous protein with molecular weight around 75.5 kD. The optimum reaction pH and temperature were about 4.0-6.0 and 40-50℃, respectively. It was stable under 50℃. The glucoamylase was activated by Ca²+ and Mg²+ but inhibited by Pb²+ and Fe²+.The results analyzed by SAS 8.0 indicated that the effects of ZnSO₄ and KH₂PO₄ on glucoamylase activity were notable among the four factors(the concentration of ZnSO₄, KH₂PO₄, MgSO₄ and the sampling tine) and that the effects of the interaction of temperature and broth volume was also notable among the three factors(temperature, rotation rate and broth volume). The outputs of L-lactic acid and glucoamylase were highest when compositions of fermentation medium(%) were sweet potato starch 10, (NH4)₂SO₄0.40, KH₂PO₄0.043, ZnSO₄·7H₂O0.024, MgSO₄·7H₂O0.024 and the optimal culture temperature, rotation rate and broth volume were 30. ℃, 198r/min and 49mL/250mL respectively. The addition of 1% bran and using maltose as the carbon substrate of culture could induce glucoamylase activity.