Abstract The liquid fermentative conditions of Enterococcus sp. were studied through single factor and orthogonal design experiment. The best medium for L-α-Glycerophosphate oxidase(GPO) formation was composed of glucose 0.1%, proteose-peptone 1%, yeast extract 1%, K2HPO4 0.5%, glycerol 0.2%, Salt solution 0.25 ml , the initial pH was 6.8 .Furthermore, some research about the conditions of fermentation was done. When the germ grown in 250 ml flask containing 100 ml of the medium on the rotary shaker (200r/min) at 30℃ for 18h , GPO had maximal activity, which was 33.8 times than original production, During the course of scale-up culture, keeping other culture conditions, culture circle was shorter 6h than the one of flask fermentation, and the former enzyme activity increased 61% than the later, when the fermentation speed was 100r/min and dissolved oxygen was 6L/min. In addition, optimum temperature and pH of the enzyme action were 37℃ and 7.5~8, respectively. When GPO was treated with in the range of pH9.0～9.5 at 37℃ for 1h,the enzyme activity was kept only 33%.
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