A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

Abstract

Abstract:The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and expressed under the control of the GAL1 promoter. α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

Efficient expression of a Paenibacillus barcinonensis endoglucanase in Saccharomyces cerevisiae

“…Cellulase-expressing recombinant Saccharomyces cerevisiae strains have potential applications in bioethanol production from cellulosic substrates, as part of a consolidated bio-processing strategy [16, 33-35, 62, 64], in wine production, as a way to improve the efficiency of the maceration process [31,61], and in beer brewing, by reducing the content of barley b-glucans in fermenting wort and enhancing filterability as a result [4,21,23,32]. The aim of the work we present was to evaluate the possibility of expressing the celA (GenBank Access No.…”

“…Correct targeting of the C1-coded fusion proteins to the cell wall was confirmed by cell wall purification and extraction of the fusion protein by reducing agents. Immobilization to the cell wall of recombinant enzymes by fusing them to the cell wall-retaining domains of S. cerevisiae cell wall proteins, apart from facilitating the recovery of the enzyme and its reusability, offers enzymes a physical support that improves their stability [19,21,39,42,50,54]. However, in our case, Western-blot experiments also showed the presence of fusion proteins or their degradation product in the growth medium of the C1-transformed strains, a result that was confirmed by zymograms and cellulase activity determinations, suggesting the existence of at least partial leakage to the growth medium of the C1-derived fusion proteins targeted to the cell wall.…”

“…Other potential, and more traditional, fields of application of S. cerevisiae strains expressing recombinant cellulases are those of wine-making [12,31,49,61] and beer brewing [4,21,23,32]. The expression of an endo-b-1,4-glucanase, among other enzymes, in a recombinant wine yeast strain, decreased turbidity after maceration and increased free-flow wine, showing the potential of recombinant cellulase-secreting wine yeast strains in the commercial-scale processing and clarification, color extraction and stabilization and aroma enhancement of wine [31,61].…”

Production of conjugated linoleic acids by Lactobacillus plantarum strains isolated from naturally fermented Chinese pickles

“…The isolation and characterization of L. plantarum strain lp15 with a relatively high LA conversion rate and strong LA tolerance from naturally fermented pickles, not only added new bacterial strain source of producing beneficial compound CLAs, but also provided some premises for the future in production of fermented products enriched in CLAs in human diets. Next we will focus on enhancing the CLA-producing ability of this strain by optimizing the culture conditions, as well as mutagenesis or genetic engineering methods, such as whole-cell biocatalysis (Chen et al, 2011) or cell-surface displaying (Guo et al, 2010), and will try to expand its application in milk and vegetable fermentation.…”

Construction of the yeast whole-cell Rhizopus oryzae lipase biocatalyst with high activity

“…To display ROL on the yeast cell surface, the wild-type and optimized pro-ROL-linker genes were inserted into the NheI-SacII section of the expression cassette pGMAK (Guo et al, 2010), respectively, which was constructed in our previous study. The resulting plasmids were named pGMRAK (wild pro-ROL) and pGMRAK (optimized pro-ROL) ( Fig.…”