Construction of a Tandem Repeat Peptide Sequence with Pepsin Cutting Sites to Produce Recombinant α-Melanocyte-Stimulating Hormone.

The production of α-melanocyte-stimulating hormone (α-MSH), a peptide hormone composed of 13 amino acids, is attempted by recombinant expression using E. coli as the host. To achieve this aim, a synthetic gene containing eight tandem repeats of msh gene (8msh) was designed for ribosomal synthesis of 8 α-MSH. The merit of the strategy is to diminish the peptide toxicity against the host cell and to achieve a higher production yield. Pepsin cleavage sites are introduced between the peptides for enzymatic proteolysis to obtain the monomeric peptide of α-MSH. The constructed plasmid was transformed into different strains of E. coli hosts, and E. coli XL1-Blue with gene 8msh revealed the highest yield of 8 α-MSH. Although 8 α-MSH was fractionalized in the insoluble pellets after cell lysis, pepsin cleavage was able to produce soluble α-MSH peptide, as analyzed and confirmed by mass spectrometry and peptide activity assays. The production of α-MSH was quantified using HPLC with a yield of 42.9 mg/L of LB culture. This study demonstrates the feasibility of producing α-MSH using recombinant expression of tandem repeat gene. The production procedure involves minimal post-treatment and processing and can be scaled up for industrial application.

Engineering a Trypsin-Resistant Thermophilic α-Galactosidase to Enhance Pepsin Resistance, Acidic Tolerance, Catalytic Performance, and Potential in the Food and Feed Industry.

α-Galactosidase has potential applications, and attempts to improve proteolytic resistance of enzymes have important values. We use a novel strategy for genetic manipulation of a pepsin-sensitive region specific for a pepsin-sensitive but trypsin-resistant high-temperature-active Gal27B from Neosartorya fischeri to screen mutants with enhanced pepsin resistance. All enzymes were produced in Pichia pastoris to identify the roles of loop 4 (Gal27B-A23) and its key residue at position 156 (Gly156Arg/Pro/His) in pepsin resistance. Gal27B-A23 and Gly156Arg/Pro/His elevated pepsin resistance, thermostability, stability at low pH, activity toward raffinose (5.3-6.9-fold) and stachyose (about 1.3-fold), and catalytic efficiencies (up to 4.9-fold). Replacing the pepsin cleavage site Glu155 with Gly improved pepsin resistance but had no effect on pepsin resistance when Arg/Pro/His was at position 156. Thus, pepsin resistance could appear to occur through steric hindrance between the residue at the altered site and neighboring pepsin active site. In the presence of pepsin or trypsin, all mutations increased the ability of Gal27B to hydrolyze galactosaccharides in soybean flour (up to 9.6- and 4.3-fold, respectively) and promoted apparent metabolizable energy and nutrient digestibility in soybean meal for broilers (1.3-1.8-fold). The high activity and tolerance to heat, low pH, and protease benefit food and feed industry in a cost-effective way.