| Aspergillus oryzae(A. oryzae) is a filamentous fungus listed as a “Generally Recognized as Safe(GRAS)” organism by the US Food and Drug Administration. It has a long history of use in the food industry in the production of traditional fermented foods, due to its high proteolytic activity. Neutral protease from A. oryzae which could be used for soy sauce production and other relevant applications because it can hydrolyze the proteins of the raw materials thoroughly and reduce the bitterness. Soy sauce mash is unique due to its low pH. The protease activity and stability decreased sharply when koji has low pH. Consequently, a A. oryzae with high catalytic efficiency at acidic pH is important in order to improve the quality of the soy sauce, to shorten the time for the maturation process and to improve the efficiency of raw materials utilization. In this study, the effect of glycosylation on rNP? activity have been analyzed. furthermore construct the produce high neutral protease activity engineering fungus based on food-grade. The main study contents are as follows:1 Three-dimensional structure of the neutral protease has been simulated, and the glycosylation sites are on the surface of the protease. Plasmid pPIC9K-NP?-P56Y-6His and pPIC9K-NP?-N41AP56Y-6His were constructed by site-specific mutagenesis and eletrotransformed into Pichia Pastoris Gs115 to express. After 60 hour' expression induced by methanol in BMMY, the rNP?-P56 Y supernatant was collected and the neutral protease' s activity reached 42 U/mL, that of rN41AP56 Y is 72 h and 60 U/m L, respectively. The crude enzyme of rNP?-P56 Y and rNP?-N41AP56 Y were treated with solid(NH4)2SO4 at 70% saturation and purified by nickel-affinity chromatography column, and the molecular weight of rNP? were about 70 kDa and 50 kDa, respectively. The purified rNP?' specific activity is 217 U/mg, those of rNP?-P56 Y and rNP?-N41AP56 Y are 242 U/mg and 147 U/mg, respectively. The thermal stability of the rNP?-N41AP56 Y was from 40 oC to 65 oC with 60% of the residual enzyme activity, which was no obvious change compared with rNP?. rNP?-P56 Y can be stable from 40 oC to 70 oC.2 In previous work, the uridine auxotroph mutant strain was obtained and named as A. oryzae AS11. Formation and regeneration conditions of protolasts from A. oryzae AS11 was optimized. 0.8 M NaCl was used as osmotic stabilizer, under this condition, the protolasts concentration of A. oryzae AS11 have reached 8.5×106/m L and the regeneration rate was 22%. Plasmid pNP-NP?-6His, pNP-NP?Y122FK246ID382V-6His and pNP-NP?Y122FK246ID382VY186S-6His, were constructed by site-specific mutagenesis and transformated into A. oryzae AS11 protolasts to express. Three different transformants were obtained and named as A. oryzae D(pNP-NP?-6His), A. oryzae E(pNP-NP?-Y122FK246ID382V-6His) and A. oryzae F(pNP-NP?-6His). The crude enzyme of A. oryzae AS11, A. oryzae B, A. oryzae D, A. oryzae E and A. oryzae F were 3100.36 U/g, 2290.81 U/g, 3978.41 U/g, 4435.62 U/g and 3540.27 U/g, respectively. |
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