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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2019, Vol. 39 Issue (10): 75-81    DOI: 10.13523/j.cb.20191009
技术与方法     
共表达N-乙酰转移酶提高Aspergillus nidulans α-葡糖苷酶在毕氏酵母中的表达研究 *
任莉琼,吴敬,陈晟()
1 江南大学食品科学与技术国家重点实验室 生物工程学院工业生物技术教育部重点实验室 江南大学教育部食品安全国际合作联合实验室 无锡 214122
Co-Expression of N-Acetyltransferase Enhances the Expression of Aspergillus nidulans α-Glucosidase in Pichia pastoris
REN Li-qiong,WU Jing,CHEN Sheng()
1 State Key Laboratory of Food Science and Technology, School of Biotechnology and Key Laboratory of Industrial Biotechnology Ministry of Education, Joint Laboratory of Food Safety International Cooperation of the Ministry of Education, Jiangnan University,Wuxi 214122,China
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摘要：

α-葡糖苷酶以低聚寡糖为底物,通过切割非还原末端α-1, 4糖苷键以获得葡萄糖基,同时转苷生成α-1, 6糖苷键,广泛应用于低聚异麦芽糖生产、代谢生理研究、疾病预防治疗等各个领域。Aspergillus nidulans来源的α-葡糖苷酶在毕氏酵母中外源表达时存在酶活较低、蛋白质降解等问题,为进一步提高α-葡糖苷酶表达量,共表达N-乙酰转移酶(Mpr1)以降低发酵过程细胞受到的氧化胁迫,提高酶活。以实验室保藏的P.pastoris KM71/pPIC9K-AgbB为出发菌株,构建共表达菌株Pichia pastoris KM71/pPIC9K-AgbB/pPICZA-Mpr1,经过摇瓶发酵120h,α-葡糖苷酶转苷酶酶活和蛋白质含量可达22.56U/ml和0.52mg/ml,分别是出发菌株摇瓶产酶的1.92倍和1.27倍。在此基础上进行3.6L罐发酵温度和甲醇诱导浓度优化,在25℃,以1%的甲醇浓度诱导发酵最高酶活和蛋白质含量可达128.12U/ml和1.81mg/ml,分别是起始菌株上罐产酶的1.96倍和1.50倍。
关键词： N-乙酰转移酶(Mpr1);  α-葡糖苷酶;;  毕氏酵母;  共表达;  发酵优化    
Abstract:

α-Glucosidase can cleave α-1,4 glucosidic linkages from the non-reducing end of oligosaccharide substrates to release glucoses, the enzyme also catalyzes transglucosylation, synthesizing α-1,6 glucosidic linkages. α-Glucosidase can be used in various fields such as isomaltooligosaccharide production, metabolic physiology research, disease prevention and treatment. Aspergillus nidulans-derived α-glucosidase has low enzymatic activity and degradation of protein in exogenous expression in Pichia pastoris. In order to improve the exogenous expression of Aspergillus nidulans-derived α-glucosidase in Pichia pastoris. A recombinant strain P.pastoris KM71/pPIC9K-AgbB/pPICZA-Mpr1 was constructed based on P.pastoris KM71/pPIC9K-AgbB which has been recombinantly expressed, finally optimized for 3.6L tank fermentation. The high-copy recombinant co-expressing strain obtained by screening showed that α-glucosidase transglucoside activity and protein content can reach 22.56U/ml and 0.52mg/ml in shake flask fermentation, respectively, which was 1.92 times and 1.27 times of the original strain in shake flask fermentation. After optimizing the temperature and methanol induction concentration of the recombinant co-expressing strain in 3.6L tank fermentation, the optimum fermentation conditions was 1% methanol concentration at 25℃.The enzyme activity and protein content of the co-expressed strains can reach 128.12U/ml and 1.81mg/ml, respectively, which was 1.96 times and 1.50 times of the original strain in 3.6L tank fermentation.
Key words: N-acetyltransferase (Mpr1)    α-Glucosidase    P. pastoris KM71    Co-expression    Fermentation optimization
收稿日期: 2019-03-05 出版日期: 2019-11-12
ZTFLH:  Q591.4  
基金资助: * 国家杰出青年基金(31425020);国家自然科学基金(31571776);中央高校基本科研业务费专项资金(JUSRP51706A);江苏省重点研发计划(社会发展)(BE2015751)资助项目
通讯作者: 陈晟     E-mail: chensheng@jiangnan.edu.cn
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引用本文:

任莉琼,吴敬,陈晟. 共表达N-乙酰转移酶提高Aspergillus nidulans α-葡糖苷酶在毕氏酵母中的表达研究 *[J]. 中国生物工程杂志, 2019, 39(10): 75-81.

REN Li-qiong,WU Jing,CHEN Sheng. Co-Expression of N-Acetyltransferase Enhances the Expression of Aspergillus nidulans α-Glucosidase in Pichia pastoris. China Biotechnology, 2019, 39(10): 75-81.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20191009    或    https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I10/75

图1  P. pastoris KM71/ pPIC9K-AgdB在3.6L罐中的发酵情况
图2  PCR核酸电泳验证
图3  重组质粒酶切验证图
图4  P. pastoris KM71/ pPIC9K-AgdB/pPICZA-Mpr1在不同温度下的产酶情况
发酵过程参数 诱导阶段温度(℃)
25 28 30
诱导干重(g/L) 37.2±0.5 37.2±0.5 37.2±0.5
最终干重(g/L) 148.7±3.3 118.9±1.2 92.9±1.1
最终酶活(U/ml) 107.9±4.8 96.7±4.8 87.7±1.3
甲醇消耗总量(g/L) 890±11.4 765±11.3 615±10.3
菌株对甲醇得率(g/g) 0.17 0.16 0.15
α-葡糖苷酶生产强度[U/(ml·h)] 0.82±0.2 0.73±0.2 0.66±0.1
表1  P.pastoris KM71/ pPIC9K-AgdB/pPICZ-Mpr1 在不同诱导温度下的发酵情况
图5  P. pastoris KM71/ pPIC9K-AgdB/pPICZA-Mpr1在不同甲醇浓度下的产酶情况
参数 诱导阶段浓度(%)
0.5 1.0 1.5
诱导干重(g/L) 37.2 ± 0.5 37.2 ± 0.5 37.2 ± 0.5
最终干重(g/L) 96.3 ± 1.3 118.9 ± 1.2 76.6 ± 1.1
最终酶活(U/ml) 91.2 ± 3.6 96.7 ± 4.8 82.3 ± 3.3
甲醇消耗总量(g/L) 645 ± 11.4 765 ± 11.3 710 ± 10.3
菌株对甲醇得率(g/g) 0.15 0.16 0.11
α-葡糖苷酶生产强度[U/(ml·h)] 0.70 ± 0.2 0.73 ± 0.2 0.62 ±0.1
表2  P.pastoris KM71/ pPIC9K-AgdB/pPICZ-Mpr1 在不同甲醇诱导浓度下的发酵情况
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