Co-Expression of N-Acetyltransferase Enhances the Expression of Aspergillus nidulans α-Glucosidase in Pichia pastoris

doi:10.13523/j.cb.20191009

China Biotechnology

2019, Vol. 39

Issue (10): 75-81 DOI: 10.13523/j.cb.20191009

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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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

Received: 05 March 2019 Published: 12 November 2019

ZTFLH:

Q591.4

Corresponding Authors: Sheng CHEN E-mail: chensheng@jiangnan.edu.cn

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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.

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

Fig.1 P. pastoris KM71/ pPIC9K-AgdB fermentation in 3.6L tank

Fig.2 Nucleotides from PCR electrophoresis verification M: DNA2000 maker; 1, 2: Mpr1 PCR band

Fig.3 Assay of recombinant plasmid digestion M:DNA5000 maker;1,2:Double enzyme digestion verification

Fig.4 Production of enzymes by P. pastoris KM71/ pPIC9K-AgdB/pPICZ-Mpr1 at induced different temperatures

Table 1 P.pastoris KM71/ pPIC9K-AgdB/pPICZ-Mpr1 fermentation at different induced temperatures

Fig.5 P. Pastoris KM71/ pPIC9K-AgdB/pPICZA-Mpr1 production at different methanol concentrations

Table 2 P.pastoris KM71/ pPIC9K-AgdB/pPICZ-Mpr1 fermentation at different methanol concentrations


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