米曲霉碱性蛋白酶的异源表达和定向进化以及遗传改造
|
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)。
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).
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)。
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).
引文
1刘丽萍,刘丽华.米曲霉研究进展与应用.中国调味品. 2008, 4: 28～32
2 Brian J.B. Wood主编,徐岩译.发酵食品微生物学(第二版).北京,中国轻工业出版社, 2001, 294～298
3 Y. Nakagawa. Alkaline Proteinases from Aspergillus. Methods in Enzymology. 1970, 19: 581～589
4 H. Tatsumi, Y. Ogawa, S. Murakami, et al. A Full Length cDNA Clone for the Alkaline Protease from Aspergillus oryzae: Structural Analysis and Expression in Saccharomyces cerevisiae. Molecular & General Genetics. 1989, 219: 33～38
5 S. Cheevadhanarak, D. V. Renno, G. Saunders, et al. Cloning and Selective Overexpression of an Alkaline Protease-encoding Gene from Aspergillus oryzae. Genes. 1991, 108: 151～153
6 K. Abe, K. Gomi, F. Hasegawa. Impact of Aspergillus oryzae Genomics on Industrial Production of Metabolites. Mycopathologia. 2006, 162: 143～153
7朱国萍,罗丹,蔡云飞等. Q20L及G247D定点突变对葡萄糖异构酶酶活和最适pH的改善.生物工程学报. 2000, 16(4): 469～473
8 E. J. Mullaney, C. B. Daly, T. Kim, et al. Site-directed Mutagenesis of Aspergillus niger NRRL 3135 Phytase at Residue 300 to Enhance Catalysis at pH 4.0. Biochemical and Biophysical Research Communications. 2002, 297: 1016～1020
9 J. He, J. Deng, Y. Zheng, et al. A Synergistic Effect on the Production of S-adenosyl-l-methionine in Pichia pastoris by Knocking in of S-adenosyl-l-methionine Synthase and Knocking out of Cystathionine-βSynthase. Journal of Biotechnology. 2006, 126: 519～527
10 C. Ruckert, A. Puhler, J. Kalinowski. Genome-wide Analysis of the L-methionine Biosynthetic Pathway in Corynebacterium glutamicum by Targeted Gene Deletion and Homologous Complementation. Journal of Biotechnology. 2003, 104: 213～228
11方继功.酱类制品生产技术.北京,中国轻工业出版社, 1997, 36～40
12赵龙飞,徐亚军.米曲霉的应用研究进展.中国酿造. 2006, 3: 8～10
13张彦茹,肖霄,吴瑞华等.不同菌种对豆酱品质的影响.中国调味品. 2008, 2: 81～83
14上海酿造科学研究所.发酵调味品生产技术.上海,中国轻工业出版社, 1999, 82～84
15朱史齐.试论当前我国酱油生产的技术状况.中国调味品. 1999, 11: 2～5
16付祖姣,陈宇,莫湘涛等.复合菌株发酵猪血粉的条件研究.微生物学通报. 2003, 30(5): 28～32
17张炳文,郝征红,黎荣静.对中国大豆食品产业走技术创新之路的探讨.中国调味品. 2000, 7: 6～9
18赵丰丽,卫红春.快速法酿造豆酱.中国酿造. 2002, 3: 16～18
19高秀芝,王小芬,李献梅等.传统发酵豆酱发酵过程中养分动态及细菌多样性.微生物学通报. 2008, 35(5): 748～753
20牛天娇,马莺.中国传统黄豆酱中微生物的发掘与利用.食品科学. 2004, 25(增刊): 213～216
21鲁迎新,高进秀,刘志成.酱油曲霉优良菌株的评选.中国调味品. 1992, 4: 7～9
22叶树山.日本豆酱的生产工艺.江苏调味副食品. 2001, 70: 23～24
23曲大亮.日本酱油生产的技术进展及其存在问题.食品科学. 1981, 2: 10～14
24张树政.酶制剂工业(下).北京,科学出版社, 1998, 154～159
25 A. Manavalan, A. Kalaichelvan, V. Kalyanasundaram, et al. Purification and Partial Characterization of Serine Protease from Thermostable Alkalophilic Bacillus laterosporus -AK1. World Journal of Microbiology and Biotechnology. 2007, 23: 475～481
26 M. Ueda, T. Kubo, K. Miyatake, et al. Purification and Characterization of Fibrinolytic Alkaline Protease from Fusarium sp. BLB. Applied Microbiology and Biotechnology. 2007, 74: 331～338
27 M. B. Rao, A. M. Tanksale, M. S. Ghatge, et al. Molecular and Biotechnological Aspects of Microbial Proteases. Microbiology and Molecular Biology Reviews. 1998, 62: 597～635
28 B. B. Samal, B. Karan, T. C. Boone, et al. Isolation and Characterization of the Gene Encoding a Novel, Thermostable Serine Proteinase from the Mould Tritirachium album Limber. Molecular Microbiology. 1990, 4(10):1789～1792
29 M. M. Taylor, D. G. Bailey, S. H. Feairheller. A Review of the Use of Enzymes in the Tannery. Journal of the American Leather Chemists Association. 1987, 82: 153～165
30 A. Dayanandan, J. Kanagaraj, L. Sounderraj, et al. Application of an Alkaline Protease in Leather Processing: an Ecofriendly Approach. Journal of Cleaner Production. 2003, 11(5): 533～536
31 S. George, B. Sivasankar, K. Jayaraman, et al. Production and Separation of the Methionine-rich Fraction from Chick Pea Protein Hydrolysate Generated by Proteases of Bacillus amyloliguefaciens. Process Biochemistry. 1997, 32(5): 401～404
32 A. Impoolsup, A. Bhumiratana, T. W. Flegel. Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. Columnaris, a Soy Sauce Koji Mold. Applied and Environmental Microbiology. 1981, 42(4), 619～628
33 Q. H. Chen, G. Q. He, Y. C. Jiao, et al. Effects of Elastase from a Bacillus Strain on the Tenderization of Beef Meat. Food Chemistry. 2006, 98(4): 624～629
34 H. Takagi, I. Ohtsu, S. Nakamori. Construction of Novel Subtilisin E with High Specificity, Activity and Productivity Through Multiple Amino Acid Substitutions. Protein Engineering. 1997, 10(9): 985～989
35 C. M. Yeh, H. K. Chang, H. M. Hsieh, et al. Improved Translational Efficiency of Subtilisin YaB Gene with Different Initiation Codons in Bacillus subtilis and Alkalophilic Bacillus YaB. Journal of Applied Microbiology. 1997, 83(6): 758～763
36毕汝昌,储乃明.枯草杆菌蛋白酶与蛋白质工程.生物化学与生物物理进展. 1991, 18(5): 329～335
37 H. Takagi, H. Matsuzawa, T. Ohta, et al. Studies on the Structure and Function of Subtilisin E by Protein Engineering. Annals of the New York Academy of Sciences. 1992, 672: 52～59
38 E. J. Mullaney, C. B. Daly, T. Kim, et al. Site-directed Mutagenesis of Aspergillus niger NRRL 3135 Phytase at Residue 300 to Enhance Catalysis at pH 4.0. Biochemical and Biophysical Research Communications. 2002, 297: 1016～1020
39 D. W. Goddette, T. Christianson, B. F. Ladin et al. Strategy and Implementation of a System for Protein Engineering. Journal of Biotechnology, 1993, 28(1): 41～54
40 S. Davail, G. Feller, E. Narinx, et al. Cold Adaptation of Proteins. Purification, Characterization, and Sequence of the Heat-labile Subtilisin from the Antarctic Psychrophile Bacillus TA41. Journal of Biological Chemistry. 1994, 269(26): 17448～17453
41 S. Taguchi, A. Ozaki, H. Momose. Engineering of a Cold-adapted Protease by Sequential Random Mutagenesis and a Screening System. Applied and Environmental Microbiology. 1998, 64(2): 492～495
42 J. M. Cregg, J. F. Tschopp, C. Stillman, et al. High-level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast Pichia pastoris. Bio/Technology, 1987, 5(4): 479～485
43 J. L. Cereghino, J. M. Cregg. Heterologous Protein Expression in the Methylotrophic Yeast Pichia pastoris. FEMS Microbiology Reviews. 2000, 24(1) 45～66
44 C. A. Scorer, J. J. Clare, W. R. McCombie, et al. Rapid Selection Using G418 of High Copy Number Transformants of Pichia pastoris for High-level Foreign Gene Expression. Bio/Technology, 1994, 12(2): 181～184
45 A. J. Brake. Secretion of Heterologous Proteins Directed by the Yeastα-factor Leader. In: P. J. Barr, A. J. Brake, P. Valenzuela. Yeast Genetic Engineering Butterworths, Boston, 1989, 269～280
46 J. M. Cregg, K. J. Barringer, A. Y. Hessler, et al. Pichia pastoris as a Host System for Transformations. Molecular and Cellular Biology. 1985, 5(12): 3376～3385
47马兴元,谭建华,朱平等.巴斯德毕赤酵母(Pichia pastoris)表达系统及其在外源蛋白生产中的优势与应用前景.中国兽医学报. 2003, 23(1): 98～101
48 K. Sreekrishna, K. A. Barr, A. Hoards, et al. Expression of Human Serum Albumin in Pichia pastoris. Yeast, 1990, 6: 447～450
49 M. Yamada, T. Azuma, T. Matsuba, et al. Secretion of Human Intracellular Aspartic Proteinase Cathepsin E Expressed in the Methylotrophic Yeast, Pichia pastoris and Characterization of Produced Recombinant Cathepsin E.Biochim Biophys Acta. 1994, 1206: 279～285
50 C. A. Scorer, R. G. Buckholz, J. J. Clare, et al. The Intracellular Production and Secretion of HIV-1 Envelope Protein in the Methylotrophic Yeast Pichia pastoris. Gene. 1993, 136(1): 111～119
51 J. J. Clare, F. B. Rayment, S. P. Ballantine, et al. High-level Expression of Tetanus Toxin Fragment C in Pichia pastoris Strains Containing Multiple Tandem Integration of the Gene. Bio/Technology. 1991a, 9: 455～460
52赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析.生物工程学报. 2000, 16(3): 308～311
53姚斌,张春义,王建华等.高效表达具有生物学活性的植酸酶的毕赤酵母.中国科学(C辑). 1998b, 28(3): 237～243
54 K. Sreekrishna, L. Nelles, R. Potenz, et al. High-level Expression, Purification, and Characterization of Recombinant Human Tumor Necrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris. Biochemistry. 1989, 28: 4117～4125
55 M. A. Romanos, J. J. Clare, K. M. Beesley, et al. Recombinant Bordetella Pertussis (P69) from the Yeast Pichia pastoris: High-level Production and Immunological Properties.Vaccine. 1991, 9: 901～906
56 G. P. Thill, G. R. Davis, C. Stillman, et al. Positive and Negative Effects of Multi-copy Integrated Expression Vectors on Protein Expression in Pichia pastoris. Paris: Societe Franaise de Microbiologie. 1990, 2: 477～490
57 S. H. Ayman, R. C. Matilde, M. S. Angela, et al. Cloning, Expression, and Properties of a Noneuronal Secreted Acetylcholinesterase from the Parasitic Nematode Nippostrongylus brasiliensis. Journal of Biological Chemistry. 1999, 274: 9312～9316
58 L. Holm, J. Park. Dalilite Workbench for Protein Structure Comparison. Bioinofrmatics. 2000, 16(6): 566～567
59 A. Zemla. A Method for Finding 3D Similarities in Protein Structures. Nucleic Acids Research. 2003, 31(13): 211～215
60 M. S. Madhusudhan, M. A. Marti-Renom, U. Pieper, et al. Comparative Protein Structure Modeling. 2005, 831～860
61 M. A. Marti-Renom, A. C. Stuart, A. Fiser, et al. Comparative Protein Structure Modeling of Genes and Genomes. Annual Review of Biophysicsand Biomolecular Structure. 2000, 29: 291～325
62 A. V. Finkelstein, O. B. Ptitsyn. Why Do Globular Proteins Fit the Limited Set of Folding Patterns?. Progress in Biophysics & Molecular Biology. 1987, 50(3): 171～190
63 C. Chothia. Proteins. One Thousand Families for the Molecular Biologist. Nature. 1992, 357 (6379): 543～544
64 Z. X. Wang. A Re-estimation for the Total Numbers of Protein Folds and Superfamilies. Protein Engineering. 1998, 11(8): 621～626
65 C. B. Anfinsen. Principles That Govern the Folding of Protein Chains. Science. 1973, 181(96): 223～230
66 J. B. Heale, J. E. Isaac, D. Chandler. Prospects for Strain Improvement in Entomopathogenic Fungi. Pesticide Science. 1989, 26: 79～92
67裴炎,方卫国,张永军.昆虫病原真菌致病寄主的机制和基因工程改良.农业生物技术学报. 2003, 11: 221～222
68 A. D. Laird, D. K. Morrison, D. Shalloway. Characterization of Raf-1 Activation in Mitosis. Journal of Biological Chemistry. 1999, 274(7): 4430～4439
69 J. L. Dai, R. K. Bansal, S. E. Kern. G1 Cell Cycle Arrest and Apoptosis Induction by Nuclear Smad4/Dpc4: Phenotypes Reversed by a Tumorigenic Mutation. Proceedings-National Academy of Sciences USA, 1999, 96(4): 1427～1432
70周丽萍.聚合酶链式反应介导的定点突变方法探索.江苏大学学报(医学版). 2003, 13(4): 33～36
71 B. A. Larder, P. Kellan, S. D. Kemp. Zidovudine Resistance Predicted by Direct Detection of Mutations in DNA from HIV-infected Lymphocytes. Acquired Immune Deficiency Syndrome, 1991, 5: 137～144
72 W. P. Stemmer. DNA Shuffling by Random Fragmentation and Reassembly: in Vitro Recombination for Molecular Evolution. Proceedings-National Academy of Sciences USA. 1994, 91:10747～10751
73 M. Kikuchi, K. Ohnishi, S. Harayama. An Effective Family Shuffling Method Using Single-stranded DNA. Gene. 2000, 243: 133～137
74 J. T. Chern, Y. P. Chao. Chitin-binding Domain Based Immobilization of D-hydantoinase. Journal of Biotechnology. 2005, 117: 267～275
75 M. R. Capecchi. Altering the Genome by Homologous Recombination. Science. 1989, 24: 1288～1292
76谭晓红,程首,杨晓.微生物基因打靶系统的研究进展.生物技术通讯. 2004, 15(3): 263～266
77 W. Chen, H. Z. Zeng, H. R. Tan. Cloning, Function, and Expression of Sans: a Gene Essential for Nikkomycin Biosynthesis of Strepeomyces ansochromogenes. Current Microbio1ogy. 2000, 41: 312～316
78 W. L. Li, G. Liu, H. R. Tan. Disruption of SabR Affects Nikkomycin Biosynthesis and Morphogenesis in Streptomyces ansochromogenes. Biotechnology. 2003, 25: 1491～1497
79 G. Liu, Y. Q. Tian, H. H. Yang. A Pathway-specific Transcriptional Regulatory Gene for Nikkomycin Biosynthesis in Streptomyces ansochromogenes That also Influences Colony Development. Molecular Microbiology. 2005, 55(6): 1855～1866
80 M. K. Chaveroche, J. M. Ghigo. A Rapid Method for Efficient Gene Replacement in the Filamentous Fungus Aspergillus nidulans. Nucleic Acids Research. 2006, 28(22): 97～103
81 J. H. Yu, Z. Hamari, K. H. Han, et al. Double Joint PCR: a PCR Based Molecular Tool for Gene Manipulations in Filamentous Fungi. Fungal Genetics and Biology. 2004, 41(11): 973～981
82 J. W. Gordon, G. A. Scangos, D. J. Plotkin, et al. Genetic Transformation of Mouse Embryos by Microinjection of Purified DNA. Proceedings of the National Academy of Sciences of the United States of America. 1980, 77(12): 7380～7384
83 G. Brem, B. Brenig, H. M. Goodman, et al. Production of Transgenic Mice Rabbits and Pigs by Microinjection. Theriogenology. 1986, 25(1): 143～146
84 J. P. Selgrath, M. A. Memon, T. E. Smith, et al. Collection and Transfer of Microinjectable Embryos from Dairy Goats. Theriogenology. 1990, 34(6): 1195～1205
85罗刚,魏泓.一种转化率较高的CaCl2转化法.中国现代实用医学杂志. 2004, 3(4): 24～25
86王友如. CaCl2浓度对感受态细胞转化效率的影响.湖北师范学院学报:自然科学版. 2006, 26(3): 30～32
87 E. Neumann, M. Schaefer-Ridder, Y. Wang, et al . Genetransfer into Mouse Lyoma Cells by Electroporation in High Electric Fields. The EMBO Journal. 1982, (1): 841～845
88 M. Ward, K. H. Kodama, L. J. Wilson. Transformation of Aspergillus awamori and A. niger by Electroporation. Experimental Mycology. 1989, 13(3): 289～293
89 A. Hinnen, J. B. Hicks, G. R. Fink. Transformation of Yeast. Proceedings of the National Academy of Sciences of the United States of America. 1978, 75 (4): 1929～1933
90 M. E. Case, M. Schweizer, S. R. Kushner, et al. Efficient Transformation of Neurospora crassa by Utilizing Hybrid Plasmid DNA. Proceedings of the National Academy of Sciences of the United States of America. 1979, 76(10): 5259～5263
91高兴喜,杨谦. PEG-CaCl2介导苏云金杆菌CryⅠA(b)基因转化哈茨木霉.哈尔滨工业大学学报. 2005, 37(10): 1333～1336
92 S. Demaneche, E. Kay, F. Gourbiere, et al. Natural Transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in Soil. Applied and Environmental Microbiology. 2001, 67(6): 2617～2621
93 P. Bundock, A. den Dulk-Ras, A. Beijersbergen, et al. Trans-kindom T- DNA Transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. The EMBO Journal. 1995, 14: 3206～3214
94 E. M. Lai, O. Chesnokova, L. M. Banta, et al. Genetic and Environmental Factors Affecting T-Pilin Export and T-Pilus Biogenesis in Relation to Flagellation of Agrobacterium tumefaciens. The Journal of Bacteriology, 2000, 182(13): 3705～3716
95 T. Kunik, T. Tzfira, Y. Kapulnik, et al. Genetic Transformation of HeLa Cells by Agrobacterium. Proceedings of the National Academy of Sciences of the United States of America. 2004, 98(4): 1871～1876
96卢萍,王宝兰.基因枪法转基因技术的研究综述.内蒙古师范大学学报(自然科学汉文版). 2006, 35(1): 106～109
97 F. D. Smith, D. M. Gadoury, P. R. Harpending, et al. Transformation of a Powdery Mildew, Uncinula necator, by Microprojectile Bombardment. Phytopathology. 1992, 82:247
98 A. M. Barley, G. L. Mena, L. Herrera-Estrella. Transformation of Four Pathogenic Phytophthora spp. by Microprojectile Bombardment on Intact Mycelia. Current Genetics. 1993, 23: 42～46
99 K. Lakrod, C. Chaisrisook, D. Z. Skinner. Transformation of Monascus Purpureus to Hygromycin b Resistance with Cosmid Pmocosx Reduces Fertility. Electronic Journal of Biotechnology. 2003, 6(2): 143～147
100 H. Gu, J. D. Marth, P. C. Orban, et al. Deletion of a DNA Polymerase Beta Gene Segment in T Cells Using Cell Type-specific Gene Targeting. Science. 1994, 265(5168): 103～106
101 A. Zimmer, P. Gruss. Production of Chimaeric Mice Containing Embryonic Stem (ES) Cells Carrying a Homoeobox Hox 1.1 Allele Mutated by Homologous Recombination. Nature. 1989, 338(6211): 150～153
102葛菁萍,宋刚,孙宗祥等.乙醇脱氢酶I基因敲除的酿酒酵母重组菌构建的初步研究.食品科学. 2008, 29(2): 210～212
103应明,班睿.枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响.微生物学报. 2006, 46(1): 23～27
104 S. L. Mansour,K. R. Thomas, M. R. Capecchi. Disruption of the Proto-oncogene int-2 in Mouse Embryo-derived Stem Cells: a General Strategy for Targeting Mutations to Non-selectable Genes. Nature, 1988, 336: 345～352
105 P. Hasty, R. Ramirez-Solis, R. Krumlauf, et al. Introduction of a Subtle Mutation into the Hox-2.6 Locus in Embryonic Stem Cells. Nature. 1991, 350: 243～246
106 P. P. Lakhlani, L. B. Macmillan, T. Z. Guo, et al. Substitution of a Mutantα2a-adrenergic Receptor Via“hit and run”Gene Targeting Reveals the Role of This Subtype in Sedative, Analgesic, and Anesthetic-sparing Responses in vivo. Proceedings of the National Academy of Sciences of the United States of America. 1997, 94: 9950～9955
107 G. R. Askew, T. Doetschman, J. B. Lingrel. Site-directed Point Mutations in Embryonic Stem Cells: a Gene Targeting Tag-and-exchange Strategy. Molecular and Cellular Biology. 1993, 13: 4115～4124
108 L. M. Whyatt, P. D. Rathjen. Introduction of Precise Alterations into the Mouse Genome with High Efficiency by Stable Tag-exchange Gene Targeting: Implications for Gene Targeting in ES Cells. Nucleic AcidsResearch. 1997, 25: 2381～2388
109 R. Kuhn, F. Schwenk, M. Aguet, et al. Inducible Gene Targeting in Mice.Science. 1995, 269(5229): 1427～1429
110单晓映,李蓓,张举仁.利用FLP/frt重组系统产生无选择标记的转基因烟草植株.生物工程学报. 2006, 22(5): 744～750
111 S. Tateishi, H. Niwa, J-I. Miyazaki, et al. Enhanced Genomic Instability and Defective Postreplication Repair in RAD18 Knockout Mouse Embryonic Stem Cells. Molecular and Cellular Biology. 2003, 23: 474～481
112 T. Doetschman, R. G. Gregg, N. Maeda, et al. Targetted Correction of a Mutant HPRT Gene in Mouse Embryonic Stem Cells. Nature. 1987, 330: 576～578
113许杨,涂追.丝状真菌基因敲除技术研究进展.食品与生物技术学报. 2007, 26(1): 120～126
114李瑞国,安晓荣,苟克勉等.基因打靶技术及其应用前景.生物技术通报. 2002, 5: 6～9
115方中达.植病研究法.北京,中国农业出版社, 1998, 456～459
116汪家政,范明.蛋白质技术手册.北京,科学出版社, 2000, 77～90
117 S. Jitendra, S. Avtar, K. Rejesh, et al. Partial Purification of an Alkaline Protease from a New Strain of Aspergillus oryzae AWT 20 and Its Enhanced Stabilization in Entrapped Ca-alginate Beads. Internet Journal of Microbiology. 2006, 2 (2)
118 D. C. Montgomery著,汪仁官、陈荣昭译.实验设计与分析.北京,中国统计出版社, 1998, 159～165
119 G. E. P. Box, W. G. Hunter, J. S. Hunter. Statistics for Experiments: an Introduction to Design, Data Analysis and Model Building. New Nork: Wiley, 1990
120李铁,臧威,刘井权等.米曲霉沪酿3.042原生质体制备和再生的研究.中国酿造. 2007, 2: 19～22
121邱雁临,曾莹,王伟平等.两种曲霉产蛋白酶特性.湖北工学院学报. 1996, 11: 39～43
122次素琴,陈珊,刘东波等.紫外线诱变选育高产PHB解聚酶的菌株.微生物学通报. 2005, 32(1): 38～43
123陈睦,王选一,张月琴.维吉霉素产生菌株X-435的诱变育种.微生物学通报. 2007, 34(6): 1090～1092
124赵琼,杨谦.细菌纤维素高产菌株的紫外诱变育种研究.食品与发酵工业. 2007, 33(7): 26～28
125乔殿华,唐国敏,钟丽蝉等.黑曲霉糖化酶基因表达调控的研究Ⅰ.高产及低产菌株糖化酶表达的总体分析和比较.微生物学报. 1997, 37(5): 349～354
126范晓春,仇润祥,刘丽等.黑曲霉诱变株Aspergillus niger T21糖化酶产量提高的分子基础.中国科学(C辑). 2001, 31(3): 202～207
127贡汉坤,姚清海.传统酱类自然发酵的微生物学分析.中国调味品. 2003, 10: 9～12
128 H. Ikemura, H. Takagi, M. Inouye. Requirement of Pro-sequence for the Production of Active Subtilisin E in Escherichia coli. Journal of Biological Chemistry. 1987, 262: 7859～7864
129 J. L. Silen, D. A. Agard. The Alpha-lytic Protease Pro-region Does Not Require a Physical Linkage to Activate the Protease Domain in Vivo. Nature. 1989, 341(6241): 462～464
130 J. R. Winther, P. Sorensen. Propeptide of Carboxypeptidase Y Provides a Chaperone-like Function as Well as Inhibition of the Enzymatic Activity. Proc Natl Acad Sci USA. 1991, 88: 9330～9334
131 L. X. Song, L. D. Fricker. The Pro Region is Not Required for the Expression or Intracellular Routeing of Carboxypeptidase E. Biochemical Journal. 1997, 323(1): 265～271
132 X. H. Mei, Y. L. Hao, H. L. Zhu, et al. Cloning of Pectin Methylesterase Inhibitor from Kiwi Fruit and its High Expression in Pichia pastoris. Enzyme and Microbial Technology. 2007, 40: 1001～1005
133 J. M. Cregg, J. F. Tschopp, C. Stilman, et al. High-level Expression and Efficient Assembly of Hepatitis B Surface Antigen in Methylotrophic Yeast, Pichia pastoris. Bio/Technology. 1987, 5: 479～485
134 K. Kunishige, T. Kazuhiro, S. Yoko, et al. High-level Expression of Myrothecium verrucaria Bilirubin Oxidase in Pichia pastoris, and Its Facile Purification and Characterization. Protein Expression and Purification. 2005, 41: 77～83
135 D. Destoumieux, P. Bulet, J. M. Strub, et al. Recombinant Expression andRange of Activity of Penaedins, Antimicrobial Peptides from Penaeid Shrimp. European Journal of Biochemistry. 1999, 266: 335～346
136 S. J. Ding, W. Ge, J. A. Buswell, Secretion, Purification and Characterisation of a Recombinant Volvariella volvacea Endoglucanase Expressed in the Yeast Pichia pastoris. Enzyme and Microbial Technology. 2002, 31: 621～626
137 C. J. Beall, S. M. Breckenridge, L. Chakravarty, et al. Expression of Human Monocyte Chemoattractant Protein-1 in the Yeast Pichia pastoris. Protein Expression and Purification. 1998, 12: 145～150
138 W. Liu, Y. Chao, S. Liu, et al. Molecular Cloning and Characterization of a Laccase Gene from the Basidiomycete Fome lignosus and Expression in Pichia pastoris. Applied Microbiology and Biotechnology. 2003, 63: 174～181
139 U. P. Shinde, M. Inouye. Folding Pathway Mediated by an Intramolecular Chaperone: Characterization of the Structural Changes in Pro-subtilisin E Coincident with Autoprocessing. Journal of Molecular Biology. 1995, 252(1): 25～30
140 P. C. Sijmons, B. M. Dekker, B. Schrammeijer, et al. Production of Correctly Processed Human Serum Albumin in Transgenic Plants. Bio/Technology, 1990, 8(3): 217～221
141 S. J. Kalil, F. Maugeri, M. I. Rodrigues. Response Surface Analysis and Simulation as a Tool for Bioprocess Design and Optimization. Process Biochemistry, 2000, 35: 539～550
142顾园,诸欣平,王少华.毕赤酵母表达蛋白质的糖基化.生命的化学. 2004, 24(4): 353～355
143 H. T. Song, Z. B. Jiang, L. X. Ma. Expression and Purification of Two Lipases from Yarrowia lipolytica AS 2.1216. Protein Expression and PuriWcation. 2006, 47: 393～397
144 C. S. Wright, R. A. Alden, J. Kraut. Structure of Subtilisin BPN' at 2.5 ? Resolution. Nature. 1969, 221: 235～242
145 S. j. Ding, W. Ge, J. A. Buswell. Secretion, Purification and Characterisation of a Recombinant Volvariella volvacea Endoglucanase Expressed in the Yeast Pichia pastoris. Enzyme and Microbial Technology. 2004, 31:621～626
146 R. Sareen, U. T. Bornscheuer, P. Mishra. Cloning, Functional Expression and Characterization of an Alkaline Protease from Bacillus licheniformis. Biotechnology Letters. 2005, 27: 1901～1907
147 D. Anandan, W. N. Marmer, R. L. Dudley. Isolation, Characterization and Optimization of Culture Parameters for Production of an Alkaline Protease Isolated from Aspergillus tamari. Journal of Industrial Microbiology & Biotechnology. 2007, 34: 339～347
148 D. Kazan, A. A. Denizci, M. N. K. Omer, et al. Purification and Characterization of a Serine Alkaline Protease from Bacillus clausi GMBAE 42. Journal of Industrial Microbiology & Biotechnology. 2005, 32: 335～344
149张丞斌,傅正伟.影响外源蛋白在巴斯德毕赤酵母中表达和分泌的研究进展.现代生物医学进展. 2008, 8(7): 1382～1384
150 S. K. Ahuja, G. M. Ferreira, A. R. Moreira. Application of Plackett-Burman Design and Response Surface Methodology to Achieve Exponential Growth for Aggregated Shipworm Bacterium. Biotechnology and Bioengineering. 2004, 85: 666～675
151肖亚中,伍传金,尤凡等.用蛋白质工程方法改变葡萄糖异构酶最适pH和最适温度.生物化学与生物物理学报. 1995, 27(5): 469～476
152 R. Helland, A. N. Larsen, A. O. Smalas, et al. The 1.8 ? Crystal Structure of a Proteinase K-like Enzyme from a Psychrotroph Serratia Species. FEBS Journal. 2006, 273(1): 61～71
153 C. Betzel, S. Gourinath, P. Kumar, et al. Structure of a Serine Protease Proteinase K from Tritirachium album limber at 0.98 ? Resolution. Biochemistry. 2005, 40(10): 3080～3088
154 A. Tomschy, R. Brugger, M. Lehmann, et al. Engineering of Phytase for Improved Activity at Low pH. Applied and Environmental Microbiology. 2002, 68: 1907～1913
155 S. H. Bhosale, M. B. Rao and V. V. Deshpande. Molecular and Industrial Aspects of Glucose Isomerase. Microbiology and Molecular Biology Reviews. 1996, 60(2): 280～300
156施家琦,夏家辉.真核生物中基因打靶的策略.生命科学研究. 1999, 3(2): 97～100
157 M. J. Shulman, L. Nissen, C. Collins. Homologous Recombination in Hybridoma Cells: Dependence on Time and Fragment Length. Molecular and Cellular Biology. 1990, 10: 4466～5591
158 P. Hasty, P. J. Reuer, A. Bradley. The Length Required for Gene Targeting in Embryonicstem Ells. Molecular and Cellular Biology. 1991, 11: 5586～5593
159 Y. Fujitani, K. Yamatnoto, I. Kobayashi. Dependence of Frequency of Homologous Recombination on the Homology Length. Genetics. 1995, 140:797～809
160 C. A. M. J. J. van den Hondel and P. J. Punt. Gene Transfer Systems and Vector Development for Filamentous Fungi. Applied molecular genetics of fungi. Cambridge, Cambridge Univ Press, 1991, 1～28
161王昌禄,杜连祥,顾晓波等.抗酸酵母遗传特性的初步研究.微生物学通报. 1998, 25(4): 213～217
162马先勇,姚开泰.同源重组技术研究进展.生物工程进展. 1996, 16(3): 16～23
163 K. Tatebayashi, J. Kato, H. Ikeda. Structural Analyses of DNA Fragments Integrated by Illegitimate Recombination in Schizosaccharomyces pombe. Molecular and General Genetics. 1994, 244: 111～119
164周礼红,李国琴,王正祥.红曲霉原生质体的制备、再生及其遗传转化系统.遗传. 2005, 27(3): 423～428
165廖勇,张增艳,徐惠君等.基因枪法介导转人工合成Rs-AFP2基因小麦的获得和检测.麦类作物学报. 2006, 26(4): 15～19
166 R. V. Merrihew, K. Marburger, S. L. Pennington, et al. High-frequency Illegitimate Integration of Transfected DNA at Preintegrated Target Sites in a Mammalian Genome. Molecular and Cellular Biology. 1996, 16: 10～18
167孙晶,李景鹏,王敖全等.黑曲霉基因pepB缺失菌株的构建及其功能分析.微生物学报. 2004, 44(6): 766～770
168廖军,徐冲,杨永辉等.变性双链法实现葡萄糖异构酶基因敲除的研究.遗传学报. 2000, 27: 449～454
169唐雪明,邵蔚蓝,王正祥等.整合型碱性蛋白酶基因工程菌中抗性基因的敲除.微生物学通报. 2003, 30(3): 1～5
170胡少新,韩翠萍,江连洲等.豆酱的加工现状与安全性分析.大豆通报. 2007, 4: 26～29
171 M. T. Boylan, M. J. Holland and W. E. Timberlake. Saccharomyces cerevisiae Centromere CEN11 Does Not Induce Chromosome Instability when Integrated into the Aspergillus nidulans Genome. Molecular and Cellular Biology. 1986, 6(11): 3621～3625
172 B. Chen, G. H. Ghoi, D. L. Nuss. Mitotic Stability and Nuclear Inheritance of Integrated Viral cDNA in Engineered Hypovirulent Strains of the Chestnut Blight Fungus. The EMBO Journal, 1993, 12(8): 2991～2998
173 A. Upshall, T. Gilbert, G. Saari, et al. Molecular Analysis of the argB Gene of Aspergillus nidulans. Molecular Genetics and Genomics. 1986, 204: 349～354
2 Brian J.B. Wood主编,徐岩译.发酵食品微生物学(第二版).北京,中国轻工业出版社, 2001, 294～298
3 Y. Nakagawa. Alkaline Proteinases from Aspergillus. Methods in Enzymology. 1970, 19: 581～589
4 H. Tatsumi, Y. Ogawa, S. Murakami, et al. A Full Length cDNA Clone for the Alkaline Protease from Aspergillus oryzae: Structural Analysis and Expression in Saccharomyces cerevisiae. Molecular & General Genetics. 1989, 219: 33～38
5 S. Cheevadhanarak, D. V. Renno, G. Saunders, et al. Cloning and Selective Overexpression of an Alkaline Protease-encoding Gene from Aspergillus oryzae. Genes. 1991, 108: 151～153
6 K. Abe, K. Gomi, F. Hasegawa. Impact of Aspergillus oryzae Genomics on Industrial Production of Metabolites. Mycopathologia. 2006, 162: 143～153
7朱国萍,罗丹,蔡云飞等. Q20L及G247D定点突变对葡萄糖异构酶酶活和最适pH的改善.生物工程学报. 2000, 16(4): 469～473
8 E. J. Mullaney, C. B. Daly, T. Kim, et al. Site-directed Mutagenesis of Aspergillus niger NRRL 3135 Phytase at Residue 300 to Enhance Catalysis at pH 4.0. Biochemical and Biophysical Research Communications. 2002, 297: 1016～1020
9 J. He, J. Deng, Y. Zheng, et al. A Synergistic Effect on the Production of S-adenosyl-l-methionine in Pichia pastoris by Knocking in of S-adenosyl-l-methionine Synthase and Knocking out of Cystathionine-βSynthase. Journal of Biotechnology. 2006, 126: 519～527
10 C. Ruckert, A. Puhler, J. Kalinowski. Genome-wide Analysis of the L-methionine Biosynthetic Pathway in Corynebacterium glutamicum by Targeted Gene Deletion and Homologous Complementation. Journal of Biotechnology. 2003, 104: 213～228
11方继功.酱类制品生产技术.北京,中国轻工业出版社, 1997, 36～40
12赵龙飞,徐亚军.米曲霉的应用研究进展.中国酿造. 2006, 3: 8～10
13张彦茹,肖霄,吴瑞华等.不同菌种对豆酱品质的影响.中国调味品. 2008, 2: 81～83
14上海酿造科学研究所.发酵调味品生产技术.上海,中国轻工业出版社, 1999, 82～84
15朱史齐.试论当前我国酱油生产的技术状况.中国调味品. 1999, 11: 2～5
16付祖姣,陈宇,莫湘涛等.复合菌株发酵猪血粉的条件研究.微生物学通报. 2003, 30(5): 28～32
17张炳文,郝征红,黎荣静.对中国大豆食品产业走技术创新之路的探讨.中国调味品. 2000, 7: 6～9
18赵丰丽,卫红春.快速法酿造豆酱.中国酿造. 2002, 3: 16～18
19高秀芝,王小芬,李献梅等.传统发酵豆酱发酵过程中养分动态及细菌多样性.微生物学通报. 2008, 35(5): 748～753
20牛天娇,马莺.中国传统黄豆酱中微生物的发掘与利用.食品科学. 2004, 25(增刊): 213～216
21鲁迎新,高进秀,刘志成.酱油曲霉优良菌株的评选.中国调味品. 1992, 4: 7～9
22叶树山.日本豆酱的生产工艺.江苏调味副食品. 2001, 70: 23～24
23曲大亮.日本酱油生产的技术进展及其存在问题.食品科学. 1981, 2: 10～14
24张树政.酶制剂工业(下).北京,科学出版社, 1998, 154～159
25 A. Manavalan, A. Kalaichelvan, V. Kalyanasundaram, et al. Purification and Partial Characterization of Serine Protease from Thermostable Alkalophilic Bacillus laterosporus -AK1. World Journal of Microbiology and Biotechnology. 2007, 23: 475～481
26 M. Ueda, T. Kubo, K. Miyatake, et al. Purification and Characterization of Fibrinolytic Alkaline Protease from Fusarium sp. BLB. Applied Microbiology and Biotechnology. 2007, 74: 331～338
27 M. B. Rao, A. M. Tanksale, M. S. Ghatge, et al. Molecular and Biotechnological Aspects of Microbial Proteases. Microbiology and Molecular Biology Reviews. 1998, 62: 597～635
28 B. B. Samal, B. Karan, T. C. Boone, et al. Isolation and Characterization of the Gene Encoding a Novel, Thermostable Serine Proteinase from the Mould Tritirachium album Limber. Molecular Microbiology. 1990, 4(10):1789～1792
29 M. M. Taylor, D. G. Bailey, S. H. Feairheller. A Review of the Use of Enzymes in the Tannery. Journal of the American Leather Chemists Association. 1987, 82: 153～165
30 A. Dayanandan, J. Kanagaraj, L. Sounderraj, et al. Application of an Alkaline Protease in Leather Processing: an Ecofriendly Approach. Journal of Cleaner Production. 2003, 11(5): 533～536
31 S. George, B. Sivasankar, K. Jayaraman, et al. Production and Separation of the Methionine-rich Fraction from Chick Pea Protein Hydrolysate Generated by Proteases of Bacillus amyloliguefaciens. Process Biochemistry. 1997, 32(5): 401～404
32 A. Impoolsup, A. Bhumiratana, T. W. Flegel. Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. Columnaris, a Soy Sauce Koji Mold. Applied and Environmental Microbiology. 1981, 42(4), 619～628
33 Q. H. Chen, G. Q. He, Y. C. Jiao, et al. Effects of Elastase from a Bacillus Strain on the Tenderization of Beef Meat. Food Chemistry. 2006, 98(4): 624～629
34 H. Takagi, I. Ohtsu, S. Nakamori. Construction of Novel Subtilisin E with High Specificity, Activity and Productivity Through Multiple Amino Acid Substitutions. Protein Engineering. 1997, 10(9): 985～989
35 C. M. Yeh, H. K. Chang, H. M. Hsieh, et al. Improved Translational Efficiency of Subtilisin YaB Gene with Different Initiation Codons in Bacillus subtilis and Alkalophilic Bacillus YaB. Journal of Applied Microbiology. 1997, 83(6): 758～763
36毕汝昌,储乃明.枯草杆菌蛋白酶与蛋白质工程.生物化学与生物物理进展. 1991, 18(5): 329～335
37 H. Takagi, H. Matsuzawa, T. Ohta, et al. Studies on the Structure and Function of Subtilisin E by Protein Engineering. Annals of the New York Academy of Sciences. 1992, 672: 52～59
38 E. J. Mullaney, C. B. Daly, T. Kim, et al. Site-directed Mutagenesis of Aspergillus niger NRRL 3135 Phytase at Residue 300 to Enhance Catalysis at pH 4.0. Biochemical and Biophysical Research Communications. 2002, 297: 1016～1020
39 D. W. Goddette, T. Christianson, B. F. Ladin et al. Strategy and Implementation of a System for Protein Engineering. Journal of Biotechnology, 1993, 28(1): 41～54
40 S. Davail, G. Feller, E. Narinx, et al. Cold Adaptation of Proteins. Purification, Characterization, and Sequence of the Heat-labile Subtilisin from the Antarctic Psychrophile Bacillus TA41. Journal of Biological Chemistry. 1994, 269(26): 17448～17453
41 S. Taguchi, A. Ozaki, H. Momose. Engineering of a Cold-adapted Protease by Sequential Random Mutagenesis and a Screening System. Applied and Environmental Microbiology. 1998, 64(2): 492～495
42 J. M. Cregg, J. F. Tschopp, C. Stillman, et al. High-level Expression and Efficient Assembly of Hepatitis B Surface Antigen in the Methylotrophic Yeast Pichia pastoris. Bio/Technology, 1987, 5(4): 479～485
43 J. L. Cereghino, J. M. Cregg. Heterologous Protein Expression in the Methylotrophic Yeast Pichia pastoris. FEMS Microbiology Reviews. 2000, 24(1) 45～66
44 C. A. Scorer, J. J. Clare, W. R. McCombie, et al. Rapid Selection Using G418 of High Copy Number Transformants of Pichia pastoris for High-level Foreign Gene Expression. Bio/Technology, 1994, 12(2): 181～184
45 A. J. Brake. Secretion of Heterologous Proteins Directed by the Yeastα-factor Leader. In: P. J. Barr, A. J. Brake, P. Valenzuela. Yeast Genetic Engineering Butterworths, Boston, 1989, 269～280
46 J. M. Cregg, K. J. Barringer, A. Y. Hessler, et al. Pichia pastoris as a Host System for Transformations. Molecular and Cellular Biology. 1985, 5(12): 3376～3385
47马兴元,谭建华,朱平等.巴斯德毕赤酵母(Pichia pastoris)表达系统及其在外源蛋白生产中的优势与应用前景.中国兽医学报. 2003, 23(1): 98～101
48 K. Sreekrishna, K. A. Barr, A. Hoards, et al. Expression of Human Serum Albumin in Pichia pastoris. Yeast, 1990, 6: 447～450
49 M. Yamada, T. Azuma, T. Matsuba, et al. Secretion of Human Intracellular Aspartic Proteinase Cathepsin E Expressed in the Methylotrophic Yeast, Pichia pastoris and Characterization of Produced Recombinant Cathepsin E.Biochim Biophys Acta. 1994, 1206: 279～285
50 C. A. Scorer, R. G. Buckholz, J. J. Clare, et al. The Intracellular Production and Secretion of HIV-1 Envelope Protein in the Methylotrophic Yeast Pichia pastoris. Gene. 1993, 136(1): 111～119
51 J. J. Clare, F. B. Rayment, S. P. Ballantine, et al. High-level Expression of Tetanus Toxin Fragment C in Pichia pastoris Strains Containing Multiple Tandem Integration of the Gene. Bio/Technology. 1991a, 9: 455～460
52赵翔,霍克克,李育阳.毕赤酵母的密码子用法分析.生物工程学报. 2000, 16(3): 308～311
53姚斌,张春义,王建华等.高效表达具有生物学活性的植酸酶的毕赤酵母.中国科学(C辑). 1998b, 28(3): 237～243
54 K. Sreekrishna, L. Nelles, R. Potenz, et al. High-level Expression, Purification, and Characterization of Recombinant Human Tumor Necrosis Factor Synthesized in the Methylotrophic Yeast Pichia pastoris. Biochemistry. 1989, 28: 4117～4125
55 M. A. Romanos, J. J. Clare, K. M. Beesley, et al. Recombinant Bordetella Pertussis (P69) from the Yeast Pichia pastoris: High-level Production and Immunological Properties.Vaccine. 1991, 9: 901～906
56 G. P. Thill, G. R. Davis, C. Stillman, et al. Positive and Negative Effects of Multi-copy Integrated Expression Vectors on Protein Expression in Pichia pastoris. Paris: Societe Franaise de Microbiologie. 1990, 2: 477～490
57 S. H. Ayman, R. C. Matilde, M. S. Angela, et al. Cloning, Expression, and Properties of a Noneuronal Secreted Acetylcholinesterase from the Parasitic Nematode Nippostrongylus brasiliensis. Journal of Biological Chemistry. 1999, 274: 9312～9316
58 L. Holm, J. Park. Dalilite Workbench for Protein Structure Comparison. Bioinofrmatics. 2000, 16(6): 566～567
59 A. Zemla. A Method for Finding 3D Similarities in Protein Structures. Nucleic Acids Research. 2003, 31(13): 211～215
60 M. S. Madhusudhan, M. A. Marti-Renom, U. Pieper, et al. Comparative Protein Structure Modeling. 2005, 831～860
61 M. A. Marti-Renom, A. C. Stuart, A. Fiser, et al. Comparative Protein Structure Modeling of Genes and Genomes. Annual Review of Biophysicsand Biomolecular Structure. 2000, 29: 291～325
62 A. V. Finkelstein, O. B. Ptitsyn. Why Do Globular Proteins Fit the Limited Set of Folding Patterns?. Progress in Biophysics & Molecular Biology. 1987, 50(3): 171～190
63 C. Chothia. Proteins. One Thousand Families for the Molecular Biologist. Nature. 1992, 357 (6379): 543～544
64 Z. X. Wang. A Re-estimation for the Total Numbers of Protein Folds and Superfamilies. Protein Engineering. 1998, 11(8): 621～626
65 C. B. Anfinsen. Principles That Govern the Folding of Protein Chains. Science. 1973, 181(96): 223～230
66 J. B. Heale, J. E. Isaac, D. Chandler. Prospects for Strain Improvement in Entomopathogenic Fungi. Pesticide Science. 1989, 26: 79～92
67裴炎,方卫国,张永军.昆虫病原真菌致病寄主的机制和基因工程改良.农业生物技术学报. 2003, 11: 221～222
68 A. D. Laird, D. K. Morrison, D. Shalloway. Characterization of Raf-1 Activation in Mitosis. Journal of Biological Chemistry. 1999, 274(7): 4430～4439
69 J. L. Dai, R. K. Bansal, S. E. Kern. G1 Cell Cycle Arrest and Apoptosis Induction by Nuclear Smad4/Dpc4: Phenotypes Reversed by a Tumorigenic Mutation. Proceedings-National Academy of Sciences USA, 1999, 96(4): 1427～1432
70周丽萍.聚合酶链式反应介导的定点突变方法探索.江苏大学学报(医学版). 2003, 13(4): 33～36
71 B. A. Larder, P. Kellan, S. D. Kemp. Zidovudine Resistance Predicted by Direct Detection of Mutations in DNA from HIV-infected Lymphocytes. Acquired Immune Deficiency Syndrome, 1991, 5: 137～144
72 W. P. Stemmer. DNA Shuffling by Random Fragmentation and Reassembly: in Vitro Recombination for Molecular Evolution. Proceedings-National Academy of Sciences USA. 1994, 91:10747～10751
73 M. Kikuchi, K. Ohnishi, S. Harayama. An Effective Family Shuffling Method Using Single-stranded DNA. Gene. 2000, 243: 133～137
74 J. T. Chern, Y. P. Chao. Chitin-binding Domain Based Immobilization of D-hydantoinase. Journal of Biotechnology. 2005, 117: 267～275
75 M. R. Capecchi. Altering the Genome by Homologous Recombination. Science. 1989, 24: 1288～1292
76谭晓红,程首,杨晓.微生物基因打靶系统的研究进展.生物技术通讯. 2004, 15(3): 263～266
77 W. Chen, H. Z. Zeng, H. R. Tan. Cloning, Function, and Expression of Sans: a Gene Essential for Nikkomycin Biosynthesis of Strepeomyces ansochromogenes. Current Microbio1ogy. 2000, 41: 312～316
78 W. L. Li, G. Liu, H. R. Tan. Disruption of SabR Affects Nikkomycin Biosynthesis and Morphogenesis in Streptomyces ansochromogenes. Biotechnology. 2003, 25: 1491～1497
79 G. Liu, Y. Q. Tian, H. H. Yang. A Pathway-specific Transcriptional Regulatory Gene for Nikkomycin Biosynthesis in Streptomyces ansochromogenes That also Influences Colony Development. Molecular Microbiology. 2005, 55(6): 1855～1866
80 M. K. Chaveroche, J. M. Ghigo. A Rapid Method for Efficient Gene Replacement in the Filamentous Fungus Aspergillus nidulans. Nucleic Acids Research. 2006, 28(22): 97～103
81 J. H. Yu, Z. Hamari, K. H. Han, et al. Double Joint PCR: a PCR Based Molecular Tool for Gene Manipulations in Filamentous Fungi. Fungal Genetics and Biology. 2004, 41(11): 973～981
82 J. W. Gordon, G. A. Scangos, D. J. Plotkin, et al. Genetic Transformation of Mouse Embryos by Microinjection of Purified DNA. Proceedings of the National Academy of Sciences of the United States of America. 1980, 77(12): 7380～7384
83 G. Brem, B. Brenig, H. M. Goodman, et al. Production of Transgenic Mice Rabbits and Pigs by Microinjection. Theriogenology. 1986, 25(1): 143～146
84 J. P. Selgrath, M. A. Memon, T. E. Smith, et al. Collection and Transfer of Microinjectable Embryos from Dairy Goats. Theriogenology. 1990, 34(6): 1195～1205
85罗刚,魏泓.一种转化率较高的CaCl2转化法.中国现代实用医学杂志. 2004, 3(4): 24～25
86王友如. CaCl2浓度对感受态细胞转化效率的影响.湖北师范学院学报:自然科学版. 2006, 26(3): 30～32
87 E. Neumann, M. Schaefer-Ridder, Y. Wang, et al . Genetransfer into Mouse Lyoma Cells by Electroporation in High Electric Fields. The EMBO Journal. 1982, (1): 841～845
88 M. Ward, K. H. Kodama, L. J. Wilson. Transformation of Aspergillus awamori and A. niger by Electroporation. Experimental Mycology. 1989, 13(3): 289～293
89 A. Hinnen, J. B. Hicks, G. R. Fink. Transformation of Yeast. Proceedings of the National Academy of Sciences of the United States of America. 1978, 75 (4): 1929～1933
90 M. E. Case, M. Schweizer, S. R. Kushner, et al. Efficient Transformation of Neurospora crassa by Utilizing Hybrid Plasmid DNA. Proceedings of the National Academy of Sciences of the United States of America. 1979, 76(10): 5259～5263
91高兴喜,杨谦. PEG-CaCl2介导苏云金杆菌CryⅠA(b)基因转化哈茨木霉.哈尔滨工业大学学报. 2005, 37(10): 1333～1336
92 S. Demaneche, E. Kay, F. Gourbiere, et al. Natural Transformation of Pseudomonas fluorescens and Agrobacterium tumefaciens in Soil. Applied and Environmental Microbiology. 2001, 67(6): 2617～2621
93 P. Bundock, A. den Dulk-Ras, A. Beijersbergen, et al. Trans-kindom T- DNA Transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. The EMBO Journal. 1995, 14: 3206～3214
94 E. M. Lai, O. Chesnokova, L. M. Banta, et al. Genetic and Environmental Factors Affecting T-Pilin Export and T-Pilus Biogenesis in Relation to Flagellation of Agrobacterium tumefaciens. The Journal of Bacteriology, 2000, 182(13): 3705～3716
95 T. Kunik, T. Tzfira, Y. Kapulnik, et al. Genetic Transformation of HeLa Cells by Agrobacterium. Proceedings of the National Academy of Sciences of the United States of America. 2004, 98(4): 1871～1876
96卢萍,王宝兰.基因枪法转基因技术的研究综述.内蒙古师范大学学报(自然科学汉文版). 2006, 35(1): 106～109
97 F. D. Smith, D. M. Gadoury, P. R. Harpending, et al. Transformation of a Powdery Mildew, Uncinula necator, by Microprojectile Bombardment. Phytopathology. 1992, 82:247
98 A. M. Barley, G. L. Mena, L. Herrera-Estrella. Transformation of Four Pathogenic Phytophthora spp. by Microprojectile Bombardment on Intact Mycelia. Current Genetics. 1993, 23: 42～46
99 K. Lakrod, C. Chaisrisook, D. Z. Skinner. Transformation of Monascus Purpureus to Hygromycin b Resistance with Cosmid Pmocosx Reduces Fertility. Electronic Journal of Biotechnology. 2003, 6(2): 143～147
100 H. Gu, J. D. Marth, P. C. Orban, et al. Deletion of a DNA Polymerase Beta Gene Segment in T Cells Using Cell Type-specific Gene Targeting. Science. 1994, 265(5168): 103～106
101 A. Zimmer, P. Gruss. Production of Chimaeric Mice Containing Embryonic Stem (ES) Cells Carrying a Homoeobox Hox 1.1 Allele Mutated by Homologous Recombination. Nature. 1989, 338(6211): 150～153
102葛菁萍,宋刚,孙宗祥等.乙醇脱氢酶I基因敲除的酿酒酵母重组菌构建的初步研究.食品科学. 2008, 29(2): 210～212
103应明,班睿.枯草芽孢杆菌ccpA基因敲除及对其核黄素产量的影响.微生物学报. 2006, 46(1): 23～27
104 S. L. Mansour,K. R. Thomas, M. R. Capecchi. Disruption of the Proto-oncogene int-2 in Mouse Embryo-derived Stem Cells: a General Strategy for Targeting Mutations to Non-selectable Genes. Nature, 1988, 336: 345～352
105 P. Hasty, R. Ramirez-Solis, R. Krumlauf, et al. Introduction of a Subtle Mutation into the Hox-2.6 Locus in Embryonic Stem Cells. Nature. 1991, 350: 243～246
106 P. P. Lakhlani, L. B. Macmillan, T. Z. Guo, et al. Substitution of a Mutantα2a-adrenergic Receptor Via“hit and run”Gene Targeting Reveals the Role of This Subtype in Sedative, Analgesic, and Anesthetic-sparing Responses in vivo. Proceedings of the National Academy of Sciences of the United States of America. 1997, 94: 9950～9955
107 G. R. Askew, T. Doetschman, J. B. Lingrel. Site-directed Point Mutations in Embryonic Stem Cells: a Gene Targeting Tag-and-exchange Strategy. Molecular and Cellular Biology. 1993, 13: 4115～4124
108 L. M. Whyatt, P. D. Rathjen. Introduction of Precise Alterations into the Mouse Genome with High Efficiency by Stable Tag-exchange Gene Targeting: Implications for Gene Targeting in ES Cells. Nucleic AcidsResearch. 1997, 25: 2381～2388
109 R. Kuhn, F. Schwenk, M. Aguet, et al. Inducible Gene Targeting in Mice.Science. 1995, 269(5229): 1427～1429
110单晓映,李蓓,张举仁.利用FLP/frt重组系统产生无选择标记的转基因烟草植株.生物工程学报. 2006, 22(5): 744～750
111 S. Tateishi, H. Niwa, J-I. Miyazaki, et al. Enhanced Genomic Instability and Defective Postreplication Repair in RAD18 Knockout Mouse Embryonic Stem Cells. Molecular and Cellular Biology. 2003, 23: 474～481
112 T. Doetschman, R. G. Gregg, N. Maeda, et al. Targetted Correction of a Mutant HPRT Gene in Mouse Embryonic Stem Cells. Nature. 1987, 330: 576～578
113许杨,涂追.丝状真菌基因敲除技术研究进展.食品与生物技术学报. 2007, 26(1): 120～126
114李瑞国,安晓荣,苟克勉等.基因打靶技术及其应用前景.生物技术通报. 2002, 5: 6～9
115方中达.植病研究法.北京,中国农业出版社, 1998, 456～459
116汪家政,范明.蛋白质技术手册.北京,科学出版社, 2000, 77～90
117 S. Jitendra, S. Avtar, K. Rejesh, et al. Partial Purification of an Alkaline Protease from a New Strain of Aspergillus oryzae AWT 20 and Its Enhanced Stabilization in Entrapped Ca-alginate Beads. Internet Journal of Microbiology. 2006, 2 (2)
118 D. C. Montgomery著,汪仁官、陈荣昭译.实验设计与分析.北京,中国统计出版社, 1998, 159～165
119 G. E. P. Box, W. G. Hunter, J. S. Hunter. Statistics for Experiments: an Introduction to Design, Data Analysis and Model Building. New Nork: Wiley, 1990
120李铁,臧威,刘井权等.米曲霉沪酿3.042原生质体制备和再生的研究.中国酿造. 2007, 2: 19～22
121邱雁临,曾莹,王伟平等.两种曲霉产蛋白酶特性.湖北工学院学报. 1996, 11: 39～43
122次素琴,陈珊,刘东波等.紫外线诱变选育高产PHB解聚酶的菌株.微生物学通报. 2005, 32(1): 38～43
123陈睦,王选一,张月琴.维吉霉素产生菌株X-435的诱变育种.微生物学通报. 2007, 34(6): 1090～1092
124赵琼,杨谦.细菌纤维素高产菌株的紫外诱变育种研究.食品与发酵工业. 2007, 33(7): 26～28
125乔殿华,唐国敏,钟丽蝉等.黑曲霉糖化酶基因表达调控的研究Ⅰ.高产及低产菌株糖化酶表达的总体分析和比较.微生物学报. 1997, 37(5): 349～354
126范晓春,仇润祥,刘丽等.黑曲霉诱变株Aspergillus niger T21糖化酶产量提高的分子基础.中国科学(C辑). 2001, 31(3): 202～207
127贡汉坤,姚清海.传统酱类自然发酵的微生物学分析.中国调味品. 2003, 10: 9～12
128 H. Ikemura, H. Takagi, M. Inouye. Requirement of Pro-sequence for the Production of Active Subtilisin E in Escherichia coli. Journal of Biological Chemistry. 1987, 262: 7859～7864
129 J. L. Silen, D. A. Agard. The Alpha-lytic Protease Pro-region Does Not Require a Physical Linkage to Activate the Protease Domain in Vivo. Nature. 1989, 341(6241): 462～464
130 J. R. Winther, P. Sorensen. Propeptide of Carboxypeptidase Y Provides a Chaperone-like Function as Well as Inhibition of the Enzymatic Activity. Proc Natl Acad Sci USA. 1991, 88: 9330～9334
131 L. X. Song, L. D. Fricker. The Pro Region is Not Required for the Expression or Intracellular Routeing of Carboxypeptidase E. Biochemical Journal. 1997, 323(1): 265～271
132 X. H. Mei, Y. L. Hao, H. L. Zhu, et al. Cloning of Pectin Methylesterase Inhibitor from Kiwi Fruit and its High Expression in Pichia pastoris. Enzyme and Microbial Technology. 2007, 40: 1001～1005
133 J. M. Cregg, J. F. Tschopp, C. Stilman, et al. High-level Expression and Efficient Assembly of Hepatitis B Surface Antigen in Methylotrophic Yeast, Pichia pastoris. Bio/Technology. 1987, 5: 479～485
134 K. Kunishige, T. Kazuhiro, S. Yoko, et al. High-level Expression of Myrothecium verrucaria Bilirubin Oxidase in Pichia pastoris, and Its Facile Purification and Characterization. Protein Expression and Purification. 2005, 41: 77～83
135 D. Destoumieux, P. Bulet, J. M. Strub, et al. Recombinant Expression andRange of Activity of Penaedins, Antimicrobial Peptides from Penaeid Shrimp. European Journal of Biochemistry. 1999, 266: 335～346
136 S. J. Ding, W. Ge, J. A. Buswell, Secretion, Purification and Characterisation of a Recombinant Volvariella volvacea Endoglucanase Expressed in the Yeast Pichia pastoris. Enzyme and Microbial Technology. 2002, 31: 621～626
137 C. J. Beall, S. M. Breckenridge, L. Chakravarty, et al. Expression of Human Monocyte Chemoattractant Protein-1 in the Yeast Pichia pastoris. Protein Expression and Purification. 1998, 12: 145～150
138 W. Liu, Y. Chao, S. Liu, et al. Molecular Cloning and Characterization of a Laccase Gene from the Basidiomycete Fome lignosus and Expression in Pichia pastoris. Applied Microbiology and Biotechnology. 2003, 63: 174～181
139 U. P. Shinde, M. Inouye. Folding Pathway Mediated by an Intramolecular Chaperone: Characterization of the Structural Changes in Pro-subtilisin E Coincident with Autoprocessing. Journal of Molecular Biology. 1995, 252(1): 25～30
140 P. C. Sijmons, B. M. Dekker, B. Schrammeijer, et al. Production of Correctly Processed Human Serum Albumin in Transgenic Plants. Bio/Technology, 1990, 8(3): 217～221
141 S. J. Kalil, F. Maugeri, M. I. Rodrigues. Response Surface Analysis and Simulation as a Tool for Bioprocess Design and Optimization. Process Biochemistry, 2000, 35: 539～550
142顾园,诸欣平,王少华.毕赤酵母表达蛋白质的糖基化.生命的化学. 2004, 24(4): 353～355
143 H. T. Song, Z. B. Jiang, L. X. Ma. Expression and Purification of Two Lipases from Yarrowia lipolytica AS 2.1216. Protein Expression and PuriWcation. 2006, 47: 393～397
144 C. S. Wright, R. A. Alden, J. Kraut. Structure of Subtilisin BPN' at 2.5 ? Resolution. Nature. 1969, 221: 235～242
145 S. j. Ding, W. Ge, J. A. Buswell. Secretion, Purification and Characterisation of a Recombinant Volvariella volvacea Endoglucanase Expressed in the Yeast Pichia pastoris. Enzyme and Microbial Technology. 2004, 31:621～626
146 R. Sareen, U. T. Bornscheuer, P. Mishra. Cloning, Functional Expression and Characterization of an Alkaline Protease from Bacillus licheniformis. Biotechnology Letters. 2005, 27: 1901～1907
147 D. Anandan, W. N. Marmer, R. L. Dudley. Isolation, Characterization and Optimization of Culture Parameters for Production of an Alkaline Protease Isolated from Aspergillus tamari. Journal of Industrial Microbiology & Biotechnology. 2007, 34: 339～347
148 D. Kazan, A. A. Denizci, M. N. K. Omer, et al. Purification and Characterization of a Serine Alkaline Protease from Bacillus clausi GMBAE 42. Journal of Industrial Microbiology & Biotechnology. 2005, 32: 335～344
149张丞斌,傅正伟.影响外源蛋白在巴斯德毕赤酵母中表达和分泌的研究进展.现代生物医学进展. 2008, 8(7): 1382～1384
150 S. K. Ahuja, G. M. Ferreira, A. R. Moreira. Application of Plackett-Burman Design and Response Surface Methodology to Achieve Exponential Growth for Aggregated Shipworm Bacterium. Biotechnology and Bioengineering. 2004, 85: 666～675
151肖亚中,伍传金,尤凡等.用蛋白质工程方法改变葡萄糖异构酶最适pH和最适温度.生物化学与生物物理学报. 1995, 27(5): 469～476
152 R. Helland, A. N. Larsen, A. O. Smalas, et al. The 1.8 ? Crystal Structure of a Proteinase K-like Enzyme from a Psychrotroph Serratia Species. FEBS Journal. 2006, 273(1): 61～71
153 C. Betzel, S. Gourinath, P. Kumar, et al. Structure of a Serine Protease Proteinase K from Tritirachium album limber at 0.98 ? Resolution. Biochemistry. 2005, 40(10): 3080～3088
154 A. Tomschy, R. Brugger, M. Lehmann, et al. Engineering of Phytase for Improved Activity at Low pH. Applied and Environmental Microbiology. 2002, 68: 1907～1913
155 S. H. Bhosale, M. B. Rao and V. V. Deshpande. Molecular and Industrial Aspects of Glucose Isomerase. Microbiology and Molecular Biology Reviews. 1996, 60(2): 280～300
156施家琦,夏家辉.真核生物中基因打靶的策略.生命科学研究. 1999, 3(2): 97～100
157 M. J. Shulman, L. Nissen, C. Collins. Homologous Recombination in Hybridoma Cells: Dependence on Time and Fragment Length. Molecular and Cellular Biology. 1990, 10: 4466～5591
158 P. Hasty, P. J. Reuer, A. Bradley. The Length Required for Gene Targeting in Embryonicstem Ells. Molecular and Cellular Biology. 1991, 11: 5586～5593
159 Y. Fujitani, K. Yamatnoto, I. Kobayashi. Dependence of Frequency of Homologous Recombination on the Homology Length. Genetics. 1995, 140:797～809
160 C. A. M. J. J. van den Hondel and P. J. Punt. Gene Transfer Systems and Vector Development for Filamentous Fungi. Applied molecular genetics of fungi. Cambridge, Cambridge Univ Press, 1991, 1～28
161王昌禄,杜连祥,顾晓波等.抗酸酵母遗传特性的初步研究.微生物学通报. 1998, 25(4): 213～217
162马先勇,姚开泰.同源重组技术研究进展.生物工程进展. 1996, 16(3): 16～23
163 K. Tatebayashi, J. Kato, H. Ikeda. Structural Analyses of DNA Fragments Integrated by Illegitimate Recombination in Schizosaccharomyces pombe. Molecular and General Genetics. 1994, 244: 111～119
164周礼红,李国琴,王正祥.红曲霉原生质体的制备、再生及其遗传转化系统.遗传. 2005, 27(3): 423～428
165廖勇,张增艳,徐惠君等.基因枪法介导转人工合成Rs-AFP2基因小麦的获得和检测.麦类作物学报. 2006, 26(4): 15～19
166 R. V. Merrihew, K. Marburger, S. L. Pennington, et al. High-frequency Illegitimate Integration of Transfected DNA at Preintegrated Target Sites in a Mammalian Genome. Molecular and Cellular Biology. 1996, 16: 10～18
167孙晶,李景鹏,王敖全等.黑曲霉基因pepB缺失菌株的构建及其功能分析.微生物学报. 2004, 44(6): 766～770
168廖军,徐冲,杨永辉等.变性双链法实现葡萄糖异构酶基因敲除的研究.遗传学报. 2000, 27: 449～454
169唐雪明,邵蔚蓝,王正祥等.整合型碱性蛋白酶基因工程菌中抗性基因的敲除.微生物学通报. 2003, 30(3): 1～5
170胡少新,韩翠萍,江连洲等.豆酱的加工现状与安全性分析.大豆通报. 2007, 4: 26～29
171 M. T. Boylan, M. J. Holland and W. E. Timberlake. Saccharomyces cerevisiae Centromere CEN11 Does Not Induce Chromosome Instability when Integrated into the Aspergillus nidulans Genome. Molecular and Cellular Biology. 1986, 6(11): 3621～3625
172 B. Chen, G. H. Ghoi, D. L. Nuss. Mitotic Stability and Nuclear Inheritance of Integrated Viral cDNA in Engineered Hypovirulent Strains of the Chestnut Blight Fungus. The EMBO Journal, 1993, 12(8): 2991～2998
173 A. Upshall, T. Gilbert, G. Saari, et al. Molecular Analysis of the argB Gene of Aspergillus nidulans. Molecular Genetics and Genomics. 1986, 204: 349～354