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

GUO Yuhan, YANG Yongzhi, GUO Henan, WANG Jian, CAO Yunhe

doi:10.3969/j.issn.1006-267x.2018.11.033

2018 , Vol. 30 >Issue 11: 4569 - 4579

DOI: https://doi.org/10.3969/j.issn.1006-267x.2018.11.033

Molecular Nutrition

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

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

Received date: 2018-04-13

Online published: 2018-11-20

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Abstract

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 MnSO₄. For enzyme kinetic characteristics, the Michaelis constant (K_m) and the maximum reaction velocity (V_max) of α-galactosidase using 4-nitrophenyl-β-D-galactoside (pNPG) 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 soymilk to some extent.

Key words： Aspergillus oryzae; α-galactosidase; codon optimization; Pichia pastoris; soybean oligosaccharides

Cite this article

GUO Yuhan, YANG Yongzhi, GUO Henan, WANG Jian, CAO Yunhe . Expression of α-Galactosidase Gene gal A from Aspergillus oryzae in Pichia pastoris and Its Enzymolysis Effect on Soybean Oligosaccharides in Soymilk[J]. Chinese Journal of Animal Nutrition, 2018 , 30(11) : 4569 -4579 . DOI: 10.3969/j.issn.1006-267x.2018.11.033

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