米曲霉来源α-半乳糖苷酶基因gal A在毕赤酵母中的表达及其对豆浆中大豆寡糖的酶解效果  

Expression of α-Galactosidase Gene gal A from Aspergillus oryzae in Pichia pastoris and Its Enzymolysis Effect on Soybean Oligosaccharides in Soymilk

作　　者：郭昱含 杨勇智[1] 郭贺楠 王剑 曹云鹤[1] GUO Yuhan;YANG Yongzhi;GUO Henan;WANG Jian;CAO Yunhe(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》2018年第11期4569-4579,共11页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金："十二五"国家科技支撑计划项目(2013BAD10B01)

摘　　要：本试验旨在研究米曲霉来源α-半乳糖苷酶基因gal A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NCBI数据库中米曲霉来源α-半乳糖苷酶基因gal A的mRNA序列(GenBank登录号:XP_001817311.1)为依据,利用PCR扩增得到gal A基因并根据毕赤酵母使用密码子的偏好性优化基因序列,构建野生型和优化型毕赤酵母工程菌株,摇瓶发酵120 h,测定酶学性质。设置25和45℃2个温度,每个温度下各设置0.6、1.2和2.4 U 3个加酶量,用发酵得到的α-半乳糖苷酶酶解10 mL豆浆中的大豆寡糖。结果显示:该α-半乳糖苷酶基因gal A全长1 605 bp,不含内含子,编码534个氨基酸,诱导120 h后优化型工程菌株的α-半乳糖苷酶活性为1.952 U/mL,比野生型工程菌株提高了285%。发酵得到的α-半乳糖苷酶的最适pH为4.33,最适温度为55℃;在pH 3.00～8.00间稳定性良好,在55℃条件下保持40 min后残余α-半乳糖苷酶相对活性为60%;该酶对大部分金属离子具有抗性,但其被MnSO4抑制;以对硝基苯基-β-D-吡喃半乳糖苷(p NPG)为底物时的酶动力学参数米氏常数(Km)为0.024 3 mol/L,最大反应速度(Vmax)为1.0×10-7mol/(L·s)。酶解试验结果显示,45℃下加酶量为2.4 U反应12 h后,大豆寡糖中棉籽糖的降解率为50.0%,水苏糖的降解率为31.9%。由此可见,本试验中生产的α-半乳糖苷酶对豆浆中的大豆寡糖有一定的降解作用。The aim of this experiment was to express the α-galactosidase gene gal-A from Aspergillus oryzae in Pichia pastoris and study its enzymolysis effect on soybean oligosaccharides in soymilk.The specific primers were designed on the basis of mRNA sequence published in NCBI database（GenBank accession number：XP_001817311.1）,and were used in polymerase chain reaction（PCR） to obtain the sequence of gal-A gene.Codon sequence was optimized according to the codon bias of Pichia pastoris.Wild type and optimized type Pichia pastoris engineered strains were constructed,fermented in shaking flask for 120 h and the enzyme properties were determined.Ten mL soymilk were treated with different concentrations（0.6,1.2 and 2.4 U） of α-galactosidase under different temperatures（25 and 45 ℃）.The results showed that the full length of α-galactosidase gene gal-A was 1 605 bp without intron,coding 534 amino acids.After inducing 120 h,the α-galactosidase activity of optimized type engineered strain was 1.952 U/mL,which improved 285% compared with the wild type engineered strain.Optimal pH and temperature of this α-galactosidase was 4.33 and 55 ℃,respectively.The enzyme showed good pH stability at the range of pH 3.00 to 8.00.For temperature stability,after incubating 40 min in 55 ℃,the relative activity of residue α-galactosidase was 60%.The α-galactosidase showed resistance to most of metal ions detected,while was inhibited by M nSO4.For enzyme kinetic characteristics,the M ichaelis constant（Km） and the maximum reaction velocity（Vmax） of α-galactosidase using 4-nitrophenyl-β-D-galactoside（p NPG） as substrate were 0.024 3 mol/L and 1.0×10^-7 mol/（L ·s）,respectively.Enzymatic hydrolysis results showed that soybean oligosaccharides were degradated under 45 ℃ with 2.4 U α-galactosidase,after 12 h,the degradation rate of raffinose and stachyose were 50.0% and 31.9%,respectively.These results indicate that α-galactosidase obtained in this experiment can degrade soybean oligosaccharides in s

关 键 词：米曲霉 Α-半乳糖苷酶 密码子优化 毕赤酵母 大豆寡糖 

分 类 号：Q786[生物学—分子生物学]