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Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications.

ABSTRACT: Kluyveromyces lactis ?-galactosidase (Kl-?-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half-lives at 45?°C compared to Kl-?-Gal (2.2 and 6.8 fold increases, respectively). Likewise, Tm values of R116C/T270C and R116C/T270C/G818C were 2.4 and 8.5?°C, respectively, higher than Kl-?-Gal Tm. Enrichment in enzymatically active oligomeric forms in these mutant variants also increased their catalytic efficiency, due to the reinforcement of the interface contacts. In this way, using an artificial substrate (p-nitrophenyl-?-D-galactopyranoside), the Vmax values of the mutants were ~1.4 (R116C/T270C) and 2 (R116C/T270C/G818C) fold higher than that of native Kl-?-Gal. Using the natural substrate (lactose) the Vmax for R116C/T270C/G818C almost doubled the Vmax for Kl-?-Gal. Validation of these mutant variants of the enzyme for their use in applications that depend on prolonged incubations at high temperatures was achieved at the laboratory scale by monitoring their catalytic activity in GOS synthesis.

SUBMITTER: Rico-Diaz A

PROVIDER: S-EPMC5374532 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications.

Rico-Díaz Agustín A Álvarez-Cao María-Efigenia ME Escuder-Rodríguez Juan-José JJ González-Siso María-Isabel MI Cerdán M Esperanza ME Becerra Manuel M

Scientific reports 20170331

Kluyveromyces lactis β-galactosidase (Kl-β-Gal) is one of the most important enzymes in the dairy industry. The poor stability of this enzyme limits its use in the synthesis of galactooligosaccharides (GOS) and other applications requiring high operational temperature. To obtain thermoresistant variants, a rational mutagenesis strategy by introducing disulphide bonds in the interface between the enzyme subunits was used. Two improved mutants, R116C/T270C and R116C/T270C/G818C, had increased half ...[more]

PMID: 28361909

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Project description:Nutraceutical formulations based on probiotic microorganisms have gained significant attention over the past decade due to their beneficial properties on human health. Yeasts offer some advantages over other probiotic organisms, such as immunomodulatory properties, anticancer effects and effective suppression of pathogens. However, one of the main challenges for their oral administration is ensuring that cell viability remains high enough for a sustained therapeutic effect while avoiding possible substrate inhibition issues as they transit through the gastrointestinal (GI) tract. Here, we propose addressing these issues using a probiotic yeast encapsulation strategy, Kluyveromyces lactis, based on gelatin hydrogels doubly cross-linked with graphene oxide (GO) and glutaraldehyde to form highly resistant nanocomposite encapsulates. GO was selected here as a reinforcement agent due to its unique properties, including superior solubility and dispersibility in water and other solvents, high biocompatibility, antimicrobial activity, and response to electrical fields in its reduced form. Finally, GO has been reported to enhance the mechanical properties of several materials, including natural and synthetic polymers and ceramics. The synthesized GO-gelatin nanocomposite hydrogels were characterized in morphological, swelling, mechanical, thermal, and rheological properties and their ability to maintain probiotic cell viability. The obtained nanocomposites exhibited larger pore sizes for successful cell entrapment and proliferation, tunable degradation rates, pH-dependent swelling ratio, and higher mechanical stability and integrity in simulated GI media and during bioreactor operation. These results encourage us to consider the application of the obtained nanocomposites to not only formulate high-performance nutraceuticals but to extend it to tissue engineering, bioadhesives, smart coatings, controlled release systems, and bioproduction of highly added value metabolites.

Dataset Information

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications.

Publications

Rational mutagenesis by engineering disulphide bonds improves Kluyveromyces lactis beta-galactosidase for high-temperature industrial applications.

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