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  • In this study an galactosidase gene GalA from a Aspergillus


    In this study, an α-galactosidase gene, GalA, from a Aspergillus oryzae strain RIB40 [14] was artificially synthesized. To produce high levels of secretory expression in Pichia and facilitate its industrial application, we systematically investigated the gene dosages and the role of the ERSAs, namely, HAC1, PDI, Hsp40, and Ero1, in the secretory expression of GalA in Pichia cells. We also conducted bench-top bioreactor cultivation and expression of GalA and optimized the parameters for α-galactosidase hydrolysis of two types of galactooligosaccharide antinutritional factors, raffinose and stachyose.
    Materials and methods
    Discussion Galactooligosaccharides are the main antinutritional polysaccharides in soybean meal feed. Among these antinutritional galactooligosaccharides, raffinose and stachyose are the two main components, constituting 7% of the soybean meal [19]. As the digestive tracts of animals lack the endogenous enzymes, these galactooligosaccharides generally cannot be digested and absorbed by animals, although they can be degraded and utilized by intestinal microbes. However, this dexamethasone acetate synthesis fermentation process made it easy to produce CO2, CH4 and other gases. This process produces flatulence in the intestinal tissues, thereby affecting the health of the animals [20]. α-Galactosidase is capable of catalyzing the hydrolysis of α-1,6-linked terminal galactose residues from galactooligosaccharide substrates such as raffinose and stachyose [1]. In the feed industry, the addition of α-galactosidase to feed could improve soybean nutrient utilization and avoid the generation of flatulence for animals. In our study, under the optimal conditions, 5% raffinose could be efficiently degraded by our galactosidase. For stachyose, although the hydrolysis ratio is lower than that for raffinose, approximately 80% of the stachyose could be hydrolyzed after 4 h of digestion (Fig. 5). Obtaining the recombinant strains that could high-level express the thermally stable α-galactosidase was the prerequisite for achieving larger scale applications of α-galactosidase in the feed industry. Increasing the gene dosage in the host genome is generally used to improve the secretory expression level of enzymes. By constructing a multicopy expression vector of human brain-derived neurotrophic factor, Liang et al. [7] found that the expression level of 6-copy transformants was 83% higher than that of a single-copy transformant. By constructing the K6W-ubiquitin recombinant expression vector, Nordén [8] found that the expression levels of the proteins were positively correlated with the copy number of the corresponding expression cassette. In our study, a series of plasmids carrying single-copy, two-copy and three-copy GalA gene expression cassettes were constructed. Due to the reasons that the expression cassettes could inserted into the Pichia genome by homologous recombination with AOX1 gene for several times [6], after being transformed into the Pichia cells, recombinants carrying two, four and six copies of the GalA gene in their genome were obtained. After methanol-induced expression of these recombinants, we found that the gene copy number increased, the expression of GalA was improved. When the copy number in the Pichia cell genome was four, the activity reached the maximal level of 3520 U/mL. However, the secretory expression level was not linearly related to the copy number. When the copy number was six, the expression level dramatically declined. One possible reason was that the overexpressed exogenous gene allowed posttranslational processes of the cell to fold and process in time, causing accumulation of unfolded or misfolded proteins. Thus, the exogenous protein could not be secreted into the extracellular space and reduced the secretory expression level of the six copies of GalA carrying Pichia recombinants [9,21]. The endoplasmic reticulum is critical for successful extracellular secretion through complex posttranslational processes (Fig. 6). Generally, the newly synthesized secretory proteins will first enter the endoplasmic reticulum, in which they are folded and modified under the action of folding enzymes and molecular chaperones. Initially, the Ire1 (Inositol-requiring enzyme 1) protein is kept inactive by binding with the Kar2 protein [10,22,23]. When the unfolded and misfolded proteins accumulate excessively in the endoplasmic reticulum, the endoplasmic reticulum senses this stress and triggers the cells to initiate the unfolded protein response (UPR) to make Kar2 and Ire1 separate. Kar2 then binds with the unfolded proteins and the misfolding proteins to initiates Ire1 forms a polymer to produce self-phosphorylation. The phosphorylated Ire1 initiates the expression of the HAC1 protein, which can bind UPREs and promote the expression of UPR genes such as Bip, PDI, Ero1, Hsp40, and Hsp70. PDI can promote disulfide bond formation and isomerization, causing the protein to fold correctly. Ero1 in the endoplasmic reticulum can cause PDI to be converted into an oxidation state. The oxidation state of PDI promotes the formation of disulfide bond in proteins [11]. The molecular chaperones of the Hsp40 family mainly bind misfolded proteins and transfer these proteins to Hsp70 for refolding or degradation (Fig. 6).