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

CAO Heng; YANG Meiyu; LIU Anguo; ZHANG Mengjie; CAO Yunhe; LU Wenqing

doi:10.3969/j.issn.1006-267x.2021.12.049

2021 , Vol. 33 >Issue 12: 7092 - 7104

DOI: https://doi.org/10.3969/j.issn.1006-267x.2021.12.049

MOLECULAR AND CELLULAR NUTRITION

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

CAO Heng ,
YANG Meiyu ,
LIU Anguo ,
ZHANG Mengjie ,
CAO Yunhe ,
LU Wenqing

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State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China

Received date: 2021-04-27

Online published: 2021-12-16

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Abstract

This experiment was conducted to study the expression of α-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 as α-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 this α-galactosidase gene As-gal1 was 1 626 bp and encoded 541 amino acids. The activity of α-galactosidase in the optimized engineered strain was 3.20 U/mL after 72 h of induction. 2) The optimum pH of α-galactosidase obtained by fermentation was 5.0 and the optimum temperature was 55℃. Except for pH 6.0, 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 MnSO₄ and promoted by FeCl₃. 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, the α-galactosidase obtained from the heterologous expression of As-gal1 in this experiment has a good degradation potential for oligosaccharides in soybean.

Key words： Aspergillus sulphureus; α-galactosidase; Pichia pastoris; high density fermentation; oligosaccharide enzymolysis

Cite this article

CAO Heng , YANG Meiyu , LIU Anguo , ZHANG Mengjie , CAO Yunhe , LU Wenqing . Heterologous Expression of α-Galactosidase Gene from Aspergillus sulphureus and Its Hydrolytic Effect on Soybean Oligosaccharides[J]. Chinese Journal of Animal Nutrition, 2021 , 33(12) : 7092 -7104 . DOI: 10.3969/j.issn.1006-267x.2021.12.049

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