| Production of corn starch in our country commonly used traditional method of wet milling production, this method contains some steps as follows:first,the combustion of sulfur produces sulfur dioxide, second,sulfur dioxide introduces into the water in order to produce sulfurous acid, but the method has many disadvantages, such as the low productivity, long soaking time, energy consumption, much waste water emissions, the strong corrosion of sulfurous acid on the equipment and pipeline. Therefore, corn starch as a finished product and other downstream products deep processing, the high cost of raw materials can not adapt to the international market competition, It is urgent to innovate soaking process technology. Besides the traditional wet method process, Corn steeping also has enzymatic method. But the effect of the protease we can buy on the market can not meet the requirement of the effect of corn soaking, so in this paper we study the breeding of strains that can produce the protease that has high yied, From COFCO Biochemical Energy Limited (Gongzhuling) soaking solution of corn in the production workshop, we screened a protease producing strain, the morphology observing and molecular identification results of strain are as follows:1. using conventional dilution separation, from COFCO Biochemical Energy Limited (Gongzhuling) soaking solution of corn in the production workshop, we screened a protease producing strain, named CTD101, protease activity was measured to 80u/mL after fermentation, the growth curve showed that the strain entered the end of logarithmic growth after the 14h.Observation of morphological we found:The colony morphology on plates was round, smooth, the diameter is between lmm to 2mm,the edge of the colony is folded, low relief, colony color is white, opaque. According to CTD101 the 26SrDNA sequence results we performed BLAST analysis:Strains CTD101 had 99% of similarity with Pichia yeast srains and had closest relationship with Pichia Kudriavzevii.2. By UV mutagenesis, NTG mutagenesis, UV-NTG school compound mutation in order to improve the ability that the strains produce protease. UV mutagenesis results showedthat:the best broth dilution was 10-4, the best mutation time was at 20s, by the mutation protease activity increased from the 80u/mL that is before the mutation to 382u/mL. NTG mutagenesis results showed that:NTG concentration was 500ug/mL, the best broth dilution was 10-4, the best mutation time was at 40min, protease activity was 472 u/mL. After three rounds combined UV-NTG mutagenesis protease activity can reach 653 u/mL.Five times of genetic stability test of the strains were performed.3. CTD101 enzymatic properties of strains, the results showed that the substrate concentration of casein was 0.1% , the optimum pH that strain produced the protease was 6.5, the optimum temperature was 50℃, when pH was whin 6 to 7.5, the temperature was at the range of 40℃to 60℃, the protease activity was high.4. The results that Fermentation medium and fermentation conditions were optimized showed that:the best medium for fermentation contained that 2% of glucose,1% of yeast extract,C/Nis2,0.2% of KH2PO4,0.2% of(NH4)2SO4,0.05% of MgSO4·7H2O,0.02% of CaCl2,0.01% of FeSO4. Optimal fermentation conditions were that initial pH was 6.5, fermentation temperature was at 30℃,6% of inoculum, medium volume 50mL/250mL flask, speed 180rpm, using the best response surface method to optimize the best single factor,we found that:C/N is 2,the addition of MgSO4 was 0.05%,the addition of CaCl2 was 0.02%,6% inoculum, protease activity can reach 2504.8u/mL, it can increased by 4 times compared with the optimization of the former.5. The fermentation liquor of Strains on the effect of soaking maize:two-step immersion process, the soaking time was 3h, the quality of corn was100g, the ratio of corn to water was 2: 1 by adding fermentation broth that was 18% of the total volume, soaking temperature was 50℃, using response surface method optimization results were that the soaking time was 3h, soaked in pH value of 6.7, at this time starch yield was 67.3% .
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