硫色曲霉α-半乳糖苷酶基因的异源表达及其水解大豆寡糖的效果研究  

Heterologous Expression of α-Galactosidase Gene from Aspergillus sulphureus and Its Hydrolytic Effect on Soybean Oligosaccharides

作　　者：曹恒 杨美玉 刘安国 张梦杰 曹云鹤[1] 陆文清[1] CAO Heng;YANG Meiyu;LIU Anguo;ZHANG Mengjie;CAO Yunhe;LU Wenqing(State Key Laboratory of Animal Nutrition,College of Animal Science and Technology,China Agricultural University,Beijing 100193,China)

机构地区：[1]中国农业大学动物科学技术学院动物营养学国家重点实验室,北京100193

出　　处：《动物营养学报》2021年第12期7092-7104,共13页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：国家科技支撑计划(2013BAD10B01)。

摘　　要：本试验旨在研究硫色曲霉α-半乳糖苷酶基因(As-gal1)在毕赤酵母中的表达及其对豆浆中大豆寡糖的酶解效果。以本实验室前期对硫色曲霉进行全基因组测序为依据,选取1条功能鉴定为α-半乳糖苷酶的cDNA序列As-gal1进行基因分析,并根据毕赤酵母密码子偏好性进行序列优化,构建优化型菌株进行72 h摇瓶发酵,测定酶学性质。对该工程菌株进行30 L发酵罐高密度发酵,用发酵罐所得粗酶酶解豆浆中的大豆寡糖。结果表明:1)该α-半乳糖苷酶基因As-gal1全长1 626 bp,编码541个氨基酸,诱导72 h后优化型工程菌株的α-半乳糖苷酶活性为3.20 U/mL。2)发酵得到的α-半乳糖苷酶的最适pH为5.0,最适温度为55℃;除pH 6.0外,在pH 4.0~8.0稳定性良好;耐热性较差,60℃10 min和70℃5 min即可近乎全部失活;该酶对大部分金属离子具有抗性,但其被MnSO_(4)抑制,被FeCl_(3)促进;30 L发酵罐高密度发酵结果显示,甲醇诱导177 h酶活性可达最高,为190 U/mL,比摇瓶提高了约58倍。3)大豆寡糖酶解试验结果显示,55℃下加酶量为2 U/mL反应3 h时,豆浆中总寡糖的降解率为49.07%。综上所述,本试验选取的As-gal1异源表达后获得的α-半乳糖苷酶对大豆中寡糖具有较好的降解潜力。This experiment was conducted to study the expression ofα-galactosidase gene from Aspergillus sulphureus(As-gal1)in Pichia pastoris and its enzymatic hydrolysis effect on soybean oligosaccharides in soybean milk.Based on the whole genome sequencing of Aspergillus sulphureus in our laboratory,a cDNA sequence as As-gal1,whose function was identified asα-galactosidase,was selected for gene analysis,and the sequence was optimized according to the codon preference of Pichia pastoris.The optimized strain was constructed for 72 h shaking-flask fermentation,and the enzymatic properties were determined.The engineered strain was fermented in a 30 L ferment tank with high density,and the soybean oligosaccharides in soybean milk were enzymolyzed with the crude enzyme obtained from the fermenter.The results showed as follows:1)the total length of thisα-galactosidase gene As-gal1 was 1 626 bp and encoded 541 amino acids.The activity ofα-galactosidase in the optimized engineered strain was 3.20 U/mL after 72 h of induction.2)The optimum pH ofα-galactosidase obtained by fermentation was 5.0 and the optimum temperature was 55 ℃ . Except for pH6.0,it had good stability in pH from 4.0 to 8.0.The heat resistance was poor,and almost all of them were deactivated at 60 ℃ for 10 min and 70 ℃ for 5 min.The enzyme was resistant to most metal ions,but it was inhibited by MnSO_(4) and promoted by FeCl_(3).The results of high-density fermentation in 30 L fermenter showed that the maximum enzyme activity was 190 U/mL induced by methanol for 177 h,which was about 58 times higher than that of shaking flask.3)The results of enzymatic hydrolysis of soybean oligosaccharides showed that the degradation rate of total oligosaccharides in soybean milk was 49.07% after 3 h of 2 U/mL of enzyme at 55 ℃ . In conclusion,theα-galactosidase obtained from the heterologous expression of As-gal1 in this experiment has a good degradation potential for oligosaccharides in soybean.

关 键 词：硫色曲霉 Α-半乳糖苷酶 毕赤酵母 高密度发酵 寡糖酶解 

分 类 号：S816[农业科学—饲料科学]