Cellobiose fermentation by Saccharomyces cerevisiae: Comparative analysis of intra versus extracellular sugar hydrolysis
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
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…
Revalorization of cellulosic wastes from Posidonia oceanica and Arundo donax as catalytic materials based on affinity immobilization of an engineered β-galactosidase
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.
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…
Structural and functional consequences of the replacement of proximal residues Cys172 and Cys192 in the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii
Proximal Cys(172) and Cys(192) in the large subunit of the photosynthetic enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39) are evolutionarily conserved among cyanobacteria, algae and higher plants. Mutation of Cys(172) has been shown to affect the redox properties of Rubisco in vitro and to delay the degradation of the enzyme in vivo under stress conditions. Here, we report the effect of the replacement of Cys(172) and Cys(192) by serine on the catalytic properties, thermostability and three-dimensional structure of Chlamydomonas reinhardtii Rubisco. The most striking effect of the C172S substitution was an 11% increase in the specificity factor when compared wi…
Cysteines 449 and 459 modulate the reduction-oxidation conformational changes of ribulose 1.5-bisphosphate carboxylase/oxygenase and the translocation of the enzyme to membranes during stress
The role of cysteines 449 (Cys449) and 459 (Cys459) from the large subunit (LS) of ribulose 1-5-bisphosphate carboxylase/oxygenase (Rubisco) in the reduction-oxidation (redox) regulation of the enzyme was assessed by site-directed mutagenesis of these residues and chloroplast transformation of Chlamydomonas reinhardtii. In vitro studies indicated that mutations C449S, C459S or C449S/ C459S do not affect the activity and proteolytic susceptibility of the enzyme in the reduced state. However, when oxidized, the mutant enzymes differed from the wild type (WT), showing an increased resistance to inactivation and, in the case of the double mutant (DM), an altered structural conformation as refle…
Redox modulation of Rubisco conformation and activity through its cysteine residues
Treatment of purified Rubisco with agents that specifically oxidize cysteine-thiol groups causes catalytic inactivation and increased proteolytic sensitivity of the enzyme. It has been suggested that these redox properties may sustain a mechanism of regulating Rubisco activity and turnover during senescence or stress. Current research efforts are addressing the structural basis of the redox modulation of Rubisco and the identification of critical cysteines. Redox shifts result in Rubisco conformational changes as revealed by the alteration of its proteolytic fragmentation pattern upon oxidation. In particular, the augmented susceptibility of Rubisco to proteases is due to increased exposure…
Comprehensive transcriptional analysis of the oxidative response in yeast
The oxidative stress response in Saccharomyces cerevisiae has been analyzed by parallel determination of mRNA levels and transcription rates for the entire genome. A mathematical algorithm has been adapted for a dynamic situation such as the response to stress, to calculate theoretical mRNA decay rates from the experimental data. Yeast genes have been grouped into 25 clusters according to mRNA level and transcription rate kinetics, and average mRNA decay rates have been calculated for each cluster. In most of the genes, changes in one or both experimentally determined parameters occur during the stress response. 24% of the genes are transcriptionally induced without an increase inmRNAlevels…
Modification of the proteolytic fragmentation pattern upon oxidation of cysteines from ribulose 1,5-bisphosphate carboxylase/oxygenase.
The proteolytic susceptibility of the native CO 2 -fixing photosynthetic enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) has been shown to increase in vitro after oxidative treatments that affect cysteine thiols. A limited incubation of oxidized (pretreated with the disulfide cystamine) Rubisco from Chlamydomonas reinhardtii with subtilisin or proteinase K generated fragments of molecular mass about 53 kDa (band I in SDS-PAGE) and 47 kDa (band II) derived from the large subunit (55 kDa) of the enzyme. In contrast, proteolysis of the reduced Rubisco (pretreated with the free thiol cysteamine) produced only the 53 kDa band. The same fragmentation pattern was repr…
REDOX PROPERTIES ARE CONSERVED IN RUBISCOS FROM DIATOMS AND GREEN ALGAE THROUGH A DIFFERENT PATTERN OF CYSTEINES1
Eukaryotic RUBISCO appears in two sequence-diverging forms, known as red-like (present in nongreen algae) and green-like (of green algae and higher plants) types. Oxidation of cysteines from green-like RUBISCOs is known to result in conformational changes that inactivate the enzyme and render a relaxed structure more prone to proteolytic attack. These changes may have regulatory value for green algae and higher plants, promoting RUBISCO catabolism under stress conditions. We compare here red-like RUBISCOs from several diatoms with a representative green-like RUBISCO from Chlamydomonas reinhardtii, paying special attention to the cysteine-dependent redox properties. Purified diatom RUBISCO p…
Development of enzymatically-active bacterial cellulose membranes through stable immobilization of an engineered beta-galactosidase
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…