Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase

Abstract

BackgroundThe recycling of agro-industrial wastes is at present limited by the availability of efficient and low-cost enzyme cocktails. The use of these materials as culture media to produce the enzymes can contribute to the profitability of the recycling process and to the circular economy. The aim of this work is the construction of a recombinant yeast strain efficient to grow in mixed whey (residue of cheese making) and beet molasses (residue of sugar manufacture) as culture medium, and to produce heterologous α-galactosidase, an enzyme with varied industrial applications and wide market.ResultsThe gene MEL1, encoding the α-galactosidase of Saccharomyces cerevisiae, was integrated (four copies) in the LAC4 locus of the Kluyveromyces lactis industrial strain GG799. The constructed recombinant strain produces high levels of extracellular α-galactosidase under the control of the LAC4 promoter, inducible by lactose and galactose, and the native MEL1 secretion signal peptide. K. lactis produces natively beta-galactosidase and invertase thus metabolizing the sugars of whey and molasses. A culture medium based on whey and molasses was statistically optimized, and then the cultures scaled-up at laboratory level, thus obtaining 19 U/mL of heterologous α-galactosidase with a productivity of 0.158 U/L h, which is the highest value reported hitherto from a cheap waste-based medium.ConclusionsA K. lactis recombinant strain was constructed and a sustainable culture medium, based on a mixture of cheese whey and beet molasses, was optimized for high productivity of S. cerevisiae α-galactosidase, thus contributing to the circular economy by producing a heterologous enzyme from two agro-industrial wastes.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0988-6) contains supplementary material, which is available to authorized users.

Bioconversion of Beet Molasses to Alpha-Galactosidase and Ethanol

“…In addition, the stability of the plasmid expressing ScAGal is not a problem since 98% of the cells growing in molasses retained the plasmid at the end of the cultures. In fact, we obtained a production of 30 U/mL of ScAGal which is 1.6-fold higher than the recently reported with a Kluyveromyces lactis engineered strain growing in a mixture of cheese whey and molasses (Álvarez-Cao et al, 2018) and higher than the 10.4 U/mL obtained for Aspergillus fumigatus alpha-galactosidase expressed in A. sojae in an optimized synthetic medium in the presence of 10.5% (w/v) molasses (Gurkok et al, 2011). The alpha-galactosidase yield reached is among the highest of those obtained in submerged fermentation for the alpha-galactosidase enzyme of different origins on various carbon sources (Anisha, 2017).…”

“…The cost of the enzyme actually limits the profitability of most of the above cited applications. The use of cheap sub-products, such as molasses and whey, as substrates for production of ScAGal, was previously reported and might favor the economy of the processes (Álvarez-Cao et al, 2018). The aim of this work is to develop a system for the valuation of beet molasses, based on their use as substrate for ScAGal and bioethanol production by S. cerevisiae in two-steps.…”

High-yield production and biochemical characterization of α-galactosidase produced from locally isolated Penicillium sp.

“…Generally, metal ions have been shown to be relevant in enzyme activity. It has been reported that some metal ions such as Ag + , Cr +3 , Cu +2 , Fe +3 , Hg +2 , Mn +2 , Pb +2 , and Zn +2 inhibited α-galactosidase activity (Gote et al 2006;Álvarez-Cao et al 2018).…”

“…The huge amounts of low-cost byproducts are generated by agro-industrial activities and these are susceptible to be transformed in culture media for the production of highvalue-added bio-products such as ethanol and other biofuels, enzymes, and heterologous proteins (Álvarez-Cao et al 2018;Álvarez-Cao et al 2019).…”

“…The cost of the enzyme actually limits the profitability of most of the above cited applications. The use of cheap sub-products, such as molasses and whey, as substrates for production of Saccharomyces cerevisiae αgalactosidase (ScAGal) was previously reported and might favor the economy of the processes (Álvarez-Cao et al 2018). The use of residues as culture media to produce the required enzymes can help with the economy of the process.…”