【作者】 郭继平；

【导师】 马莺；

【作者基本信息】 哈尔滨工业大学 ， 化学工程与技术， 2008， 博士

【摘要】 米曲霉(Aspergillus oryzae)在食品工业中应用极为广泛,在豆酱酿造业中的地位尤为重要。为了保证质量和提高生产效率,豆酱的工厂化生产已采用纯菌种发酵,目前广泛使用的菌种是米曲霉(A. oryzae)3.042。为了不断发掘米曲霉新菌种并通过基因工程加以改良以满足豆酱工业化生产的要求,本研究以分离自东北地区的野生米曲霉为研究对象,对其进行紫外诱变,最终筛选出蛋白酶活力高且遗传性能稳定的突变株米曲霉(A. oryzae)Y29,其中性蛋白酶活力是米曲霉(A. oryzae)3.042的1.2倍。与米曲霉(A. oryzae)3.042相比,米曲霉(A. oryzae)Y29菌株发酵豆酱的氨态氮含量达到0.74g/100mL,发酵周期提前2d结束,其它指标无显著差异。豆酱的发酵过程主要是蛋白酶的作用,通过添加蛋白酶抑制剂的方法对发酵过程中的蛋白酶活力进行了测定,结果证明米曲霉碱性蛋白酶在发酵中起关键作用。偏酸性的发酵环境不利于米曲霉碱性蛋白酶更大限度的发挥作用,有必要对其定向进化使其最适pH值下降,这需要高效异源表达系统的支持。本试验采用RT-PCR的方法获得了米曲霉(A. oryzae)Y29的碱性蛋白酶基因(alp),分别采用天然信号肽和α-信号肽将该基因在毕赤酵母(Pichia pastoris)GS115中进行异源表达,表达量分别为513mg/L和336mg/L。这两种信号肽均能被毕赤酵母识别、加工,并发现前导区对米曲霉alp基因在毕赤酵母中表达是必须的。为了研究表达后的重组米曲霉碱性蛋白酶的性质,采用硫酸铵沉淀、离子交换层析和凝胶过滤层析对该酶进行了纯化,其纯度已达到电泳纯。重组和天然米曲霉碱性蛋白酶均未被糖基化,最适温度和最适pH也均为40℃和9.0,两者具有相似的pH稳定性和对金属离子及蛋白酶抑制剂的敏感性,但重组酶的热稳定性稍低一些。对含天然信号肽的重组米曲霉碱性蛋白酶的诱导条件进行优化,当诱导温度为28.13℃、诱导pH为6.56、甲醇添加量为1.23%时,其表达量可达648mg/L。以毕赤酵母表达系统和纯化条件为基础,采用重叠PCR法对米曲霉碱性蛋白酶进行了定向进化。首先利用SWISS-MODEL分子模建服务系统同源模建了米曲霉碱性蛋白酶三维结构。再通过同源蛋白序列比对和三维结构结果,找出在活性中心附近且非常保守的氨基酸G78、A230和G236作为待突变位点。以重组米曲霉碱性蛋白酶野生型为对照,通过重叠PCR法进行定点突变并将米曲霉碱性蛋白酶突变酶基因在毕赤酵母中表达,表达产物经纯化后对其酶学性质进行比较,发现突变酶G78R(将78位氨基酸G突变为R)和A230E的最适pH值没有改变,突变酶G78D和G236D的最适pH值均下降了0.5个单位。突变酶G78D在pH值为5.5~8.5时的碱性蛋白酶的活力均高于野生型(平均高12%左右);在40℃、pH值9.0时,该突变酶的Km为野生型的115.27%,Vmax和Kcat分别是野生酶的85.89%和82.01%;但热稳定性与野生型差异不显著。而突变酶G236D的比酶活只有野生型的60%左右,所以只有突变酶G78D符合本试验的要求。为了将改造后的米曲霉碱性蛋白酶突变型G78D基因再回到米曲霉中发挥作用,首先构建了含米曲霉alp基因的同源臂和潮霉素B抗性(hygr)基因的置换型打靶载体pHC2,将其线性化后转化米曲霉(A. oryzae)Y29,得到了alp基因敲除的菌株米曲霉(A. oryzae)Y30。又构建了含米曲霉碱性蛋白酶突变型G78D基因的打靶载体pHC3,通过PEG介导转化米曲霉(A. oryzae)Y30,最终得到了米曲霉(A. oryzae)Y31工程菌株。与改造前相比,米曲霉(A. oryzae)Y31菌株的酸性蛋白酶活力和中性蛋白酶活力都相应增加了11.23%和13.36%。以米曲霉(A. oryzae)Y29和3.042为对照,用米曲霉(A. oryzae)Y31发酵豆酱,其氨态氮的含量达0.80g/100mL(Y29和3.042菌株分别为0.74g/100mL和0.70g/100mL),发酵周期为35d(Y29和3.042菌株分别为38d和40d),酱色稍深,鲜味更浓,其它指标无明显差异但均符合豆酱国家专业标准(SB/T 10309-1999)。更多还原

【Abstract】 Aspergillus oryzae has been broadly used in food industry, especially for producing soybean paste. Because of the demand for high quality and yiled, single strain of A. oryzae is adopted in soybean paste fermentation operation the manufacturing production of soybean paste. Nowdays A. oryzae 3.042 is the most popular strain. In this study, a wild A. oryzae originated from north-east region was induced by ultraviolet light, and a mutant Y29 with high genetic stability was finally separated from the induced strains. It was found that the amount of protease of Y29 is 1.2 times higher than A.3.042, and content of ammoniacal nitrogen is 0.74g/100ml, a little higher than A.3.042, the fermentation period is 2 days shorter than 3.042. Y29 and A.3.042 don’t show significant differences in other parameters. The fermentation of soybean paste is dominated by the enzym, in A. oryzae. The research also proved that alkaline protease plays a key role in fermentation.Because alkaline protease in A. oryzae can not effectively catalyze substrate in the acid environment, it is imerative to make protease efficiency work at lower pH. This is accomplished by the directed evolution system with effective heterogeneous expressing. The alp gene in Y29 was amplified by RT-PCR. The alp gene was expressed heterogenously in P. pastoris GS115 with native signal peptide and withα-signal peptide. Two types of signal peptide were both identified and processed by P. pastoris. The yield of recombinant alkaline protease was 513mg/L and 336mg/L respectively. It was found that pro-region part of alp gene was important for its expression in P. pastoris.Recombinant alkaline protease from A. oryzae was purified by ion exchange and gel filtration for further characteristics. Recombinant and native alkaline protease from A. oryzae were not methylated. Their optimal temperature and pH were idenfical: 40℃and 9.0, and both are similar in pH stability and sensitivity to metallic ions and protease inhibitors as well. However, thermal stability of recombinant alkaline protease was inferior to native alkaline protease. The optimum inducement condition for recombinant alkaline protease was found to be few preferable 28.13℃, pH 6.56, methanol concentration 1.23%, the yield of recombinant alkaline protease from A. oryzae could be as high as 648mg/L.The directed evolution of alkaline protease from A. oryzae was carried out by over-lap PCR method. Three dimensional structure of alkaline protease was constructed homologously with the SWISS-MODEL system. Then based on dimensional structure and the results of three conservative amino acid sites of G78, A230 and G236 which are all near the active center, were chosen as mutant sites. The mutant genes were expressed in P. pastoris. The expressed mutant alkaline protease was purified for compare their characters. The results showed that the optimum pH of mutant enzyme G48R and A230E remain unchanged, whereas pH of mutant enzyme G78D and A230D reduced by 0.5 unit. Enzyme activity of G78D was 12% higher than wild alkaline protease enzyme activity of G236D was 40% lower than wild alkaline protease. At 40℃and pH 9.0, Km of G78D was 115.27% than wild enzyme, and Vmax and Kcat was 85.89% and 82.01% than wild enzyme respectively. Therefore, G78D was selected for further studies.To make functions in A. oryzae, two replacing target vectors were constructed. One vector, which includes two homologous arms and hygromycin B, was transformed into Y29 to get the mutant A. oryzae without alp gene Y30. The other vector, which includes mutant alp gene of G78D, was transformed into Y30 to get the mutant A. oryzae with alp gene (G78D) Y31. Compared with Y29, enzyme activity of acid and alkaline protease in Y31 was 14% and 20% respectively. Soybean paste was fermented with Y29, Y31 and A. oryzae 3.042. The ammoniacal nitrogen content was 0.74g/100mL, 0.80g/100mL and 0.70g/100mL, and fermentation period was 38d, 35d and 40d respectively. Soybean paste fermented with Y31 showed color darker. All testing parameters for Y31 met the national standard of soybean paste (SB/T 10309-1999).更多还原