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  • Studies have shown that coexpression of the HAC gene in


    Studies have shown that coexpression of the HAC1 gene in P. pastoris improved the 5416 level of foreign proteins [24,25]. By coexpression with PDI and Ero1, the expression level of the coexpressed strains was increased by 60% [26]. In our study, the α-galactosidase gene was coexpressed with four endoplasmic reticulum proteins: HAC1, PDI, Hsp40, and Ero1. The results showed that the activity of recombinant strains coexpressing HAC1, PDI and Ero1 increased by 14.06%, 25.89% and 29.58%, respectively. These results coincide with the function and roles of HAC1, PDI, and Ero1 in protein expression and secretion. In contrast, the activity of recombinant strains coexpressing Hsp40 with GalA had their activity decreased by 43.80%. We speculated that the Hsp40 protein itself might be misfolded. This unfolded or misfolded Hsp40 family protein may not correctly combine with misfolded proteins to transfer them to Hsp70 family proteins. By systematically comparing Pichia recombinants carrying multiple copies of the GalA gene and co-expressing GalA with ERSAs, we finally screened out the Pichia recombinants with the highest secretory expression potential. After methanol-induced expression in a 14-L bench top bioreactor, the enzyme activity reached 3520 U/mL. This level was significantly higher than that recorded in a mutated Aspergillus strain after solid-state fermentation [27] and was also higher than the heterologous expression of Talaromyces emersonii and Rhizomucor miehei α-galactosidases in P. pastoris [28,29].
    Conclusions In this study, the effects of gene dosage and endoplasmic reticulum secretion factors on the secretory expression of a galactosidase gene derived from a A. niger strain RIB40 were studied by constructing multicopy expression cassette recombinants and coexpressing the galactosidase gene with endoplasmic reticulum secretion protein-related genes. Four copies of the GalA gene in the Pichia genome were optimal for its secretory expression. The proteins HAC1, PDI and Ero1 improved the secretory expression levels of the galactosidase gene, but Hsp40 decreased its expression. We further optimized the parameters for the galactosidase hydrolysis of two antinutritional factors: raffinose and stachyose. This study has fulfilled the scale-up production of galactosidase and thus will facilitate its industrial applications.
    Acknowledgments We thank G J Chen, X B Peng, X You, and Q C Chen for their help with performing the experiments. This work was financially supported by the Science and Technology Supporting Program of Wuhan Science and Technology Bureau (2016020101010084). Dr. J K Yang is an incumbent member of the Chutian Scholar Program.
    Introduction In certain populations, there are many individuals with lactose intolerance, occurring due to the lack of the enzyme β Galactosidase which is required to break down lactose. Consequently, the lactose is fermented in the intestine, where it can create digestive disorders (Adhikari, Dooley, Chambers, & Bhumiratana, 2010). The application of β-Galactosidase has received much attention in the hydrolysis of lactose in dairy products such as milk and whey (Husain, Ansari, Alam, & Azam, 2011). According to the present scientific knowledge, soluble enzymes cannot be used in industrial and environmental applications due to the inhibition, instability, and difficult recovery of the product (Homaei, Sariri, Vianello, & Stevanato, 2013). In order to overcome such limitations, enzyme immobilization has been considered as one of the most successful methods (Dwevedi & Kayastha, 2009). Incorporation of enzymes into/onto nano-scale materials has gained considerable attention due to high ratio of surface area-to-volume of nanomaterials, increasing accessible surface area for enzyme incorporation and catalytic efficiency (Wong, Dai, Talbert, Nugen, & Goddard, 2014). Generally, nanofibers are defined as fibers of less than 100 nm in diameter, representing a commercially-scalable technology in nanomaterial fabrication (Z. M. Huang, Zhang, Kotaki, & Ramakrishna, 2003). Recently, electrospinning has been emerged as one of the most generally-used techniques to fabricate nanofibers from variety of materials such as polymers, inorganic materials and composite structures (Aytac, Dogan, Tekinay, & Uyar, 2014). Electrospun fibers have several remarkable properties such as very large surface-to-volume ratios, and high porosity with small pore sizes, making them the best candidate for many important applications including tissue engineering, wound dressing, drug delivery systems, and many others (Canbolat, Celebioglu, & Uyar, 2014; Hu et al., 2017; Huang et al., 2003; Li et al., 2017). A considerable number of synthetic and natural biopolymers are electrospun. However, food biopolymers such as proteins and polysaccharides are generally recognized as safe and widely used in food products (Chen, Remondetto, & Subirade, 2006).