showing 8 related works from this author
Cellobiose fermentation by Saccharomyces cerevisiae: Comparative analysis of intra versus extracellular sugar hydrolysis
2018
Abstract A prevalent procedure for the production of second generation bioethanol makes use of engineered yeast strains capable to hydrolyze cellobiose either in the cytosol or extracellularly. These two approaches have been compared in this study. For intracellular cellobiose hydrolysis, we initially tested three recombinant Saccharomyces cerevisiae strains that produced different cytosolic β-glucosidases and the cellodextrin transporter Po_CdtC from Penicillium oxalicum . The strain coexpressing Po_CdtC and the β-glucosidase from Neurospora crassa (NcBgl) showed the highest cellobiase activity but its growth in cellobiose was limited by sugar intake. A search of alternative cellobiose per…
Synthesis of Isomaltooligosaccharides by Saccharomyces cerevisiae Cells Expressing Aspergillus niger α‑Glucosidase
2017
The α-glucosidase encoded by the aglA gene of Aspergillus niger is a secreted enzyme belonging to family 31 of glycoside hydrolases. This enzyme has a retaining mechanism of action and displays transglycosylating activity that makes it amenable to be used for the synthesis of isomaltooligosaccharides (IMOs). We have expressed the aglA gene in Saccharomyces cerevisiae under control of a galactose-inducible promoter. Recombinant yeast cells expressing the aglA gene produced extracellular α-glucosidase activity about half of which appeared cell bound whereas the other half was released into the culture medium. With maltose as the substrate, panose is the main transglycosylation product after 8…
Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A
1996
Mutations enhancing the thermostability of β-glucosidase A of Bacillus polymyxa, a family 1 glycosyl hydrolase, have been obtained after hydroxylamine mutagenesis of a plasmid containing the bglA gene, transformation of Escherichia coli with the mutagenized plasmid, and identification of transformant colonies that showed β-glucosidase activity after a thermal treatment that inactivated the wild-type enzyme. Two additive mutations have been characterized that cause replacement of glutamate at position 96 by lysine and of methionine at position 416 by isoleucine respectively. The thermoresistant mutant enzymes showed increased resistance to other denaturing agents, such as pH and urea, while …
Revalorization of cellulosic wastes from Posidonia oceanica and Arundo donax as catalytic materials based on affinity immobilization of an engineered…
2020
Catalytic materials obtained by enzyme immobilization have multiple potential applications in the food industry. The choice of the immobilization method and support may be critical to define the properties of the immobilized enzyme compared to the soluble form. Although the use of immobilized enzymes shows multiple advantages, their catalytic efficiency is compromised in many instances. Molecular engineering techniques have been used to generate hybrid proteins where the enzyme of interest is fused to a module with affinity to a specific biopolymer. Binding of the hybrid TmLac-CBM2 protein, in which the β-galactosidase from Thermotoga maritima is fused to a carbohydrate-binding module from …
Matryoshka enzyme encapsulation: Development of zymoactive hydrogel particles with efficient lactose hydrolysis capability.
2019
This report describes an efficient procedure for enzyme encapsulation and its application for the hydrolysis of lactose. The enzymatic material that has been developed consists of hydrogel particles (ca. 3–4 mm of diameter) composed of either alginate or an alginate-agarose combination, in which bacterial cells loaded with a thermostable β-galactosidase are embedded. The cells were rendered fully permeable to the substrate, either chromogenic p-nitrophenyl galactose or lactose, by thermal treatment at 75 °C. Hydrogel particles made of a mixture of alginate and agarose displayed high catalytic activity (i.e. 1 g of beads hydrolyze the lactose equivalent of 100 mL of milk in 15 min) and therm…
Self-diploidization in Saccharomyces cerevisiae kar2 heterokaryons
1993
Zygotes isolated by micromanipulation from crosses of Saccharomyces cerevisiae strains, one of which carries a kar mutation, give rise most frequently to cytoductant colonies showing the nuclear constitution of either one of the two haploid parental strains. In crosses of kar2-1 strains to wild-type, about 10% of the cytoductants of both mating types are homozygous autodiploids. There is evidence indicating that self-diploidization occurs by fusion between sibling nuclei in the heterokaryotic zygote. Here we describe this phenomenon and propose to take advantage of it for the construction of genotypically-defined diploids able to mate, and of polyploid strains, which are useful tools in gen…
Rtp1p Is a Karyopherin-Like Protein Required for RNA Polymerase II Biogenesis
2013
The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its bi…
Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered beta-galactosidase
2018
Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% l…