Search

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 thermal stability: they could be reused at 75 °C for complete hydrolysis of 5% (w/v) lactose solution at least six consecutive times without significant loss of activity., This work was funded by grant AGL2016-75245-R from Spain's 'Secretaría de Estado de Investigación, Desarrollo e Innovación'.MJF was supported by a Ramon y Cajal contract (RYC2014-158) from the Spanish Ministerio de Economia; Industria y Competitividad.

Xylanases can boost pulp bleachability in Elemental Chlorine Free (ECF) processes, but their industrial implementation for producing bleached kraft pulps is not straightforward. It requires enzymes to be active and stable at the extreme conditions of alkalinity and high temperature typical of this industrial process; most commercial enzymes are unable to withstand these conditions. In this work, a novel highly thermo and alkaline-tolerant xylanase from Pseudothermotoga thermarum was overproduced in E. coli and tested as a bleaching booster of hardwood kraft pulps to save chlorine dioxide (ClO2) during ECF bleaching. The extremozyme-stage (EXZ) was carried out at 90 °C and pH 10.5 and optimised at lab scale on an industrial oxygen-delignified eucalyptus pulp, enabling us to save 15% ClO2 to reach the mill brightness, and with no detrimental effect on paper properties. Then, the EXZ-assisted bleaching sequence was validated at pilot scale under industrial conditions, achieving 25% ClO2 savings and reducing the generation of organochlorinated compounds (AOX) by 18%, while maintaining pulp quality and papermaking properties. Technology reproducibility was confirmed with another industrial kraft pulp from a mix of hardwoods. The new enzymatic technology constitutes a realistic step towards environmentally friendly production of kraft pulps through industrial integration of biotechnology., This work was supported by WoodZymes project funded by the Bio-based Industries Joint Undertaking (BBI JU) under GA 792070. The BBI JU received support from the EU’s H2020 research and innovation programme and the Bio Based Industries Consortium.

Endoxylanases active under extreme conditions of temperature and alkalinity can replace the use of highly pollutant chemicals in the pulp and paper industry. Searching for enzymes with these properties, we carried out a comprehensive bioinformatics study of the GH10 family. The phylogenetic analysis allowed the construction of a radial cladogram in which protein sequences putatively ascribed as thermophilic and alkaliphilic appeared grouped in a well-defined region of the cladogram, designated TAK Cluster. One among five TAK sequences selected for experimental analysis (Xyn11) showed extraordinary xylanolytic activity under simultaneous conditions of high temperature (90 °C) and alkalinity (pH 10.5). Addition of a carbohydrate binding domain (CBM2) at the C-terminus of the protein sequence further improved the activity of the enzyme at high pH. Xyn11 structure, which has been solved at 1.8 Å resolution by X-ray crystallography, reveals an unusually high number of hydrophobic, ionic and hydrogen bond atomic interactions that could account for the enzyme’s extremophilic nature., This work was funded as part of the European Project WOODZYMES, by the Bio Based Industries Joint Undertaking, under the European Union’s Horizon 2020 research and innovation program (Grant Agreement H2020-BBI-JU-792070) and by grants BIO2016-76601-C3-3-R and PID2019-105838RB-C33 from the Spanish Government.

Thermostable β-galactosidase (TmLac) has been immobilized as hybrid inorganic-protein nanoflowers using salts of Cu2+, Mn2+, Zn2+, Co2+ and Ca2+ as the inorganic component. The incorporation efficiency of enzyme into the nanoflowers was higher than 95% for a protein concentration of 0.05 mg/mL. The structure, activity and recyclability of the nanoflowers with different chemical composition were analyzed. Ca2+, Mn2+ and Co2+ nanoflowers showed a level of lactase activity equivalent to their same content of free enzyme. Cu2+nanoflowers showed only marginal enzyme activity in agreement with the inhibitory effect of this cation on the enzyme. TmLac nanoflowers provide an efficient methodology for enzyme immobilization and recyclability. TmLac-Ca2+ nanoflowers presented the best properties for lactose hydrolysis both in buffered and in milk, and could be reused in five consecutive cycles., This work was funded by grant AGL2016-75245-R from Spain's ‘Secretaría de Estado de Investigación, Desarrollo e Innovación’. MJF was supported by a Ramon y Cajal contract (RYC2014-158) from the Spanish Ministerio de Economía, Industria y Competitividad.

Lactose intolerance is a common digestive disorder that affects a large proportion of the adult human population. The severity of the symptoms is highly variable, depending on the susceptibility to the sugar and the amount digested. For that reason, enzymes that can be used for the production of lactose-free milk and milk derivatives have acquired singular biotechnological importance. One such case is

10 pags., 6 figs., 1 tab., The synthesis of multivalent pyrrolidine iminosugars via CuAAC click reaction between different pyrrolidine-azide derivatives and tri- or hexavalent alkynyl scaffolds is reported. The new multimeric compounds, together with the monomeric reference, were evaluated as inhibitors against two homologous GH1 β-glucosidases (BglA and BglB from Paenibacillus polymyxa). The multivalent inhibitors containing an aromatic moiety in the linker between the pyrrolidine and the scaffold inhibited the octameric BglA (µM range) but did not show affinity against the monomeric BglB, despite the similarity between the active site of both enzymes. A modest multivalent effect (rp/n = 12) was detected for the hexavalent inhibitor 12. Structural analysis of the complexes between the monomeric and the trimeric iminosugar inhibitors (4 and 10) and BglA showed the insertion of the inhibitors at the active site of BglA, confirming a competitive mode of inhibition as indicated by enzyme kinetics. Additionally, structural comparison of the BglA/4 complex with the reported BglB/2F-glucose complex illustrates the key determinants responsible for the inhibitory effect and explains the reasons of the inhibition of BglA and the no inhibition of BglB. Potential inhibition of other β-glucosidases with therapeutic relevance is discussed under the light of these observations., This work was supported by the Spanish Ministry of Economyand Competitiveness (CTQ2016-77270-R, BIO2016-76601-C3-3-R,AGL2016-75245-R), the Junta de Andalucía (FQM-345), MIUR-Italy(“Progetto Dipartimenti di Eccellenza 2018-2022” allocated toDepartment of Chemistry “Ugo Schiff”) and Ente Cassa di Risparmio di Firenze (grant n°. 2016/0845). M. Martínez-Bailén acknowledges theSpanish government for a FPU fellowship. We thank CITIUS-Universityof Seville (MS and NMR facilities) and the Synchrotron RadiationSource at Alba (Barcelona, Spain) for assistance at BL13-XALOCbeamline

In this work, enzymatically active polysaccharide-based hydrogels and aerogels have been developed. To this end, a thermostable β-galactosidase (TmLac) enzyme from Thermotoga maritima embedded in nanoflowers’ format was used to evaluate the capacity of the hydrogel matrices to preserve the hydrolytic activity of the enzyme and the reusability of the hydrogels formed. Commercial agar, unpurified agar and agarose were compared as supporting materials. Although the developed hydrogel capsules can be used at high temperature (75 °C) and reused for the digestion of lactose to a greater extent than the free nanoflowers, loaded hydrogel capsules behaved differently depending on the type of polysaccharide used. Commercial agar was the most promising one since these hydrogel capsules could be reused, maintaining the structural integrity and reaching higher enzymatic activity (after seven cycles at 75 °C) than the free TmLac-Ca2+ nanoflowers. To facilitate handling and storage, aerogels were developed by freeze-drying the hydrogel capsules. Aerogels of agarose and unpurified agar underwent structural changes during freeze-drying that adversely affected their subsequent use, losing their integrity after being rehydrated. However, commercial agar aerogels were successfully developed and reused thanks to the existing interactions with TmLac-Ca2+ nanoflowers (confirmed by FTIR), which resulted in better capsule integrity and enzyme protection. The hydrolytic activity of enzymatically active aerogels based on commercial agar was in the same range of the free TmLac and TmLac-Ca2+ nanoflowers, being significantly higher to their counterparts in the hydrated form (hydrogels based on commercial agar)., This research was supported by grants from Spain's ‘Secretaría de Estado de Investigación, Desarrollo e Innovación' (AGL2016-75245-R), Agencia Estatal de Investigación (AEI, Grant PCI2018-092886) and cofunded by the European Union's Horizon 2020 research and innovation programme (ERA-Net SUSFOOD2). MJF was supported by a Ramon y Cajal contract (RYC2014-15842) from the Spanish Ministerio de Economia; Industria y Competitividad.

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 Pyrococcus furiosus, to cellulosic material from aquatic biomass wastes (such as Posidonia oceanica and Arundo donax) has been assayed. Both species generate environmental wastes that could be revalorized if converted into bioactive materials. Cellulose cryogels, but not films, from P. oceanica were able to bind the TmLac-CBM2 hybrid, with a higher immobilization yield (90%) than that from A. donax cellulose cryogels (60%). However, fractions containing also hemicellulose were less effective as immobilization supports in both cases, with yields of 47% and 30%, respectively. Cellulose cryogels loaded with β-galactosidase were able to hydrolyse lactose with the same efficiency as the free form of the enzyme. In contrast, enzyme-loaded cellulose films were inactive. This study represents a proof of concept for the valorisation of cellulosic wastes as bioactive materials. Furthermore, it provides information about the interaction specificity between the binding module and the cellulosic support, useful for other enzymes., This research was supported by grants from Spain's ‘Secretaría de Estado de Investigación, Desarrollo e Innovación’ (AGL2016-75245-R), Agencia Estatal de Investigación (AEI, Grant PCI2018-092886) and cofunded by the European Union's Horizon 2020 research and innovation programme (ERA-Net SUSFOOD2). MJF was supported by a Ramon y Cajal contract (RYC2014-158) from the Spanish Ministerio de Economia; Industria y Competitividad. The authors thank the Central Support Service for Experimental Research (SCSIE) of the University of Valencia for the electronic microscopy service.

10 pags., 5 figs., 1 tab., Lactose intolerance is a common digestive disorder that affects a large proportion of the adult human population. The severity of the symptoms is highly variable, depending on the susceptibility to the sugar and the amount digested. For that reason, enzymes that can be used for the production of lactose-free milk and milk derivatives have acquired singular biotechnological importance. One such case is Thermotoga maritima β-galactosidase (TmLac). Here, we report the cryo-EM structure of TmLac at 2.0 Å resolution. The protein features a newly solved domain at its C-Terminus, characteristic of the genus Thermotoga, which promotes a peculiar octameric arrangement. We have assessed the constraints imposed by the quaternary protein structure on the construction of hybrid versions of this GH2 enzyme. Carbohydrate binding modules (CBM) from the CBM2 and CBM9 families have been added at either the amino or carboxy terminus, and the structural and functional effects of such modifications have been analyzed. The results provide a basis for the rational design of hybrid enzymes that can be efficiently attached to different solid supports., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness BIO2016-76601-C3-3-R (to J.S.A.), BFU2017-87316 (to R.F.L.), and AGL2016-75245-R (to J.P.). We thank the staff of the Synchrotron Radiation Source at Barcelona (Alba, Spain) for providing access and for technical assistance at the BL13-XALOC beamline. We thank the Midlands Regional Cryo-EM Facility at LIBSCB, major funder MRC, and C. Savva for assistance during data acquisition. We thank the EM unit from CNIO and the cryo-EM unit from the CNB−CIB (CSIC) for support using the EM facilities.

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% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness (grant number AGL2016-75245-R). Berta N. Estevinho thanks Fundação para a Ciência e a Tecnologia (FCT), Portugal, for postdoctoral grant SFRH/BPD/73865/2010. M.J. Fabra is recipient of a Ramon y Cajal (RYC-2014-158) contract from the Spanish Ministry of Economy, Industry and Competitiveness.

Enzymes are proteins that act as facilitators or catalysts of biochemical reactions associated with the function of living organisms. Enzymes work as highly specialized molecular machines able to break, ensemble, or transform molecules. Although enzymes have been used by humans since prehistoric times, it was late in nineteenth century and in the first half of the twentieth that the chemical nature of enzymes and their mechanism of function were elucidated. In addition to their basic function to sustain life, enzymes have become irreplaceable tools in many industries and activities of daily living.

Oligosaccharides are compounds of great interest in the food and pharmaceutical industry, mainly due to their prebiotic action and other health-promoting effects, in addition to other properties, acting as low-caloric sweeteners, antioxidants, or antimicrobial agents. Oligosaccharide functionality depends on their chemical structure, which relies on the nature of the building sugar residues, the linkage type, and the degree of polymerization. Their production is based either in the partial hydrolysis of polysaccharides or in the synthesis from other sugars with lower degree of polymerization. This methodology is described along the first part of this chapter, making special emphasis on the enzymatic-based technology, which shows clear advantages over chemical methods. Glycoside hydrolases and glycosyltransferases are the two enzyme classes involved in oligosaccharide production. They operate as highly specialized molecular tools for the generation of the different types of oligosaccharides. In the second part of the chapter, the strategies employed for improving the properties of these enzymes are explained. Enzyme engineering, through site-directed mutagenesis or directed evolution, has succeeded in delivering enzymatic variants with enhanced oligosaccharide yields, a different product profile or a wider substrate specificity. Moreover, immobilization of enzymes on solid supports through different methods has allowed their reutilization during repeated batches or their implementation in continuous systems, and, in some instances, it was also accompanied by an increased activity or stability. Overall, this chapter provides an overview about the facts and potentials of oligosaccharide production methodologies.

Manuscript of the article published in print 13 April 2016. The Supporting Information is available free of charge on the ACS Publications website at DOI:10.1021/acs.jafc.6b00222 ., Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3′-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3′-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold., This work was funded by grant BIO2013-48779-C4-3-R, from Spain's 'Secretaría de Estado de Investigación, Desarrollo e Innovación'. D T-P was supported by a FPU fellowship from 'Ministerio de Economía y Competitividad'.

We describe a simple, efficient process for the production of 6-kestose, a trisaccharide with well-documented prebiotic properties. A key factor is the use of a yeast transformant expressing an engineered version of Saccharomyces invertase with enhanced transfructosylating activity. When the yeast transformant was grown with 30 % sucrose as the carbon source, 6-kestose accumulated up to ca. 100 g/L in the culture medium. The 6-kestose yield was significantly enhanced (up to 200 g/L) using a two-stage process carried out in the same flask. In the first stage, the culture was grown in 30 % sucrose at physiological temperature (30 °C) to allow overexpression of the invertase. In the second stage, sucrose was added to the culture at high concentration (60 %) and the temperature shifted to 50 °C. In both cases, 6-kestose was synthesized with high specificity, representing more than 95 % of total FOS.

Thermoresistant, recombinant β-galactosidase from Thermotoga maritima was purified and immobilized on the surface of epoxy-coated magnetic beads. The enzyme, which has hexameric quaternary structure as shown by gel filtration chromatography, attaches to the resin through multiple covalent linkages that involve different subunits. The bound enzyme shows higher stability than the free form. The immobilized enzyme showed to be efficient for the hydrolysis of lactose and the biosynthesis of galactooligosaccharides (GOS). The chemical structure of synthesized GOS has been determined by NMR revealing that the main product was β-3′-galactosyl lactose. Although β-galactosidases from different sources have been used for the same purposes, the distinct advantage of the methodology described in this communication is that the enzyme can be easily produced, purified and immobilized in large quantities.