Analysis of the rate limiting steps in enzymatic hydrolysis of lignocellulose : the effect of lignin and enzyme characteristics

Physicochemical lignocellulose pretreatment and subsequent enzymatic conversion of polysaccharides to platform sugars is an important technology in the valorisation of various lignocellulosic biomass streams. This technology is needed in the bio- and circular economy. Lignin, one of the main components in lignocellulose, is known to inhibit enzymatic hydrolysis by non-productively binding enzymes and sterically preventing enzymes to access cellulose. In this work, the aim was to elucidate how lignin in herbaceous plants and softwood is modified during pretreatment and what is the effect of pretreatment severity on lignin-derived inhibition in the enzymatic hydrolysis. Characteristics of cellulases and hemicellulases contributing to binding and inactivation on lignin were investigated. Spruce and wheat straw were hydrothermally pretreated with or without an acid catalyst at increasing severities, followed by isolation of the lignin to explore the inhibitory effects. Lignin inhibition in the enzymatic hydrolysis of microcrystalline cellulose Avicel increased with increasing pretreatment severity. When spruce and wheat straw were pretreated at the same severity, as assessed by the combined severity factor, lignins from both biomasses were equally inhibitory. Furthermore, lignin from mild pretreatment severities did not have a significant effect on the hydrolysis of Avicel. This indicate that the changes in lignin structure during pretreatment are the main reasons for the inhibitory effect of lignin. A decrease in β-O-4 aryl ether bond as well as a change in molecular weight of lignin was observed after pretreatment. The molecular weight of spruce lignin decreased, whereas the molecular weight of wheat straw increased after pretreatment. Degradation and polymerisation reactions competed during pretreatment and the net effect depended on biomass type and pretreatment severity. Lignin-derived inhibition in Avicel hydrolysis strongly correlated with the binding and inactivation of the cellobiohydrolase TrCel7A to lignin. TrCel7A is the main component in the Trichoderma reesei cellulase cocktails. The correlation between enzyme binding to lignin and inhibition in hydrolysis was studied using six purified enzymes common in cellulase cocktails, cellobiohydrolases TrCel7A, TrCel6A, endoglucanases TrCel7B and TrCel5A, a xylanase TrXyn2 from T. reesei and a β-glucosidase AnCel3A from Aspergillus niger. The cellobiohydrolases, an endoglucanase and a xylanase were all inhibited by isolated lignin. Interestingly, the most thermostable enzyme AnCel3A exhibited minor binding to lignin and was, in fact, activated by lignin. The activation of AnCel3A was the result of soluble lignin-derived compounds. The enzymes TrCel6A and TrCel7B, which exhibited strong binding to thin lignin films as analysed by quartz crystal microbalance (QCM), were also the enzymes that were most inhibited by lignin in hydrolysis assays. The interactions contributing to enzyme binding to lignin were enzyme-specific, however, some common interactions were identified. Enzymes containing a carbohydrate binding module from family 1 (CBM1), TrCel7A, TrCel6A, TrCel7B and TrCel5A, exhibited greater adsorption to lignin than the enzymes without a CBM. Furthermore, enzymes having a net positive surface charge bound to lignin more than enzymes with a net negative surface charge in the experimental pH. Enzyme surface hydrophobicity was computationally determined. Enzymes containing large uniform hydrophobic patches on the enzyme surface had stronger binding to lignin as only a low amount of enzyme was released from the lignin surface during rinsing with buffer. Thermal stability had a profound effect on lignin tolerance for family GH11 xylanases. Two T. reesei xylanases TrXyn1 and TrXyn2 as well as two forms of a metagenomic xylanase Xyl40 were all inhibited by lignin in hydrolysis assays and bound to lignin after incubation with enzymatically isolated lignin from steam pretreated spruce. Interestingly, the thermostable xylanases Xyl40 produced in Escherichia coli and the catalytic domain of Xyl40 produced in T. reesei remained partially active on the lignin surface, whereas the thermolabile TrXyn1 and TrXyn2 became inactive. N-glycosylation of the catalytic domain of Xyl40 did not affect the hydrolysis yield but had a significant effect on lignin tolerance. The glycosylated xylanase achieved higher hydrolysis yields in the presence of lignin than the deglycosylated xylanase. High thermal stability and structural glycans improved the lignin tolerance of the xylanases studied.Kasvibiomassan biotekninen konversio liikenteen polttoaineeksi tai kemikaaleiksi on tärkeä teknologia bio- ja kiertotaloudessa. Lignoselluloosapitoisen kasvibiomassan muuntaminen hyödynnettäviksi sokereiksi vaatii esikäsittelyn avaamaan kasvisolun rakennetta. Sen jälkeen rakennepolysakkaridit, selluloosa sekä hemiselluloosa, pilkotaan entsyymiproteiinien avulla sokerimonomeereiksi. Syntyvästä sokeriliuoksesta voidaan mikrobien avulla tuottaa joko biopolttoainetta tai kemikaaleja. Ligniini on aromaattinen polymeeri ja se on selluloosan ja hemiselluloosan lisäksi yksi pääkomponentti lignoselluloosapitoisessa materiaaleissa. Ligniini on yksi entsymaattista hydrolyysiä rajoittava tekijä, sillä selluloosan pinnalla oleva ligniini voi steerisesti estää entsyymien pääsyn selluloosalle tai ligniini voi epäspesifisesti sitoa entsyymejä pinnalleen rajoittaen niiden toimintaa. Työn tavoitteena oli selvittää miten termokemiallisen esikäsittelyn vahvuus vaikuttaa entsyymi-ligniini-vuorovaikutuksiin ja mitkä entsyymiominaisuudet vaikuttavat entsyymien epäspesifiseen sitoutumiseen ligniinin pinnalle. Työssä käytettiin eristettyjä ligniinejä sekä puhdistettuja entsyymejä. Kuusipurulle ja vehnän oljelle tehtiin sarja termokemiallisia esikäsittelyjä eri vahvuuksissa ja ligniini eristettiin käytettäväksi entsyymihydrolyysikokeissa mallisubstraatin kanssa. Esikäsittelyn vahvuudella oli selkeä vaikutus ligniiniperäiseen inhibitioon. Ligniiniperäinen inhibitio kasvoi esikäsittelyn vahvuuden kasvaessa ja esikäsittelemättömästä tai miedosti esikäsitellystä biomassasta eristetyillä ligniineillä ei ollut huomattavaa vaikutusta entsyymien hydrolyysisaantoon. Ligniinin aiheuttama inhibitio entsymaattiseen hydrolyysiin korreloi Trichoderma reesei -homeen entsyymiseoksen pääkomponentin, sellobiohydrolaasin TrCel7A, sitoutumiseen ligniinin pinnalle. Näiden huomioiden perusteella voidaan päätellä, että ligniiniperäinen inhibitio oli seuraus esikäsittelystä ja että esikäsittely aiheutti entsyymin epäspesifistä sitoutumista ligniiniin haitaten entsyymin toimintaa hydrolyysissä. Eri entsyymiominaisuuksien vaikutusta ligniiniin sitoutumiseen tutkittiin mittaamalla T. reesei sekä Aspergillus niger -homeiden entsyymiseoksien kuuden yleisen entsyymikomponentin sitoutumista ohuille ligniinikalvoille QCM-tekniikkaa käyttäen. T. reesei -homeen sellulaasit TrCel6A ja TrCel7B, jotka sitoutuivat ligniiniin eniten, myös inhiboituivat ligniinistä eniten hydrolyysikokeissa. Molekyylitason interaktiot, jotka vaikuttivat entsyymien ligniiniin sitoutumiseen, olivat entsyymispesifejä, mutta myös yhteisiä tekijöitä tunnistettiin. Entsyymit, joilla oli hiilihydraatteihin sitoutuva moduuli (CBM) sitoutuivat ligniiniin enemmän kuin entsyymit, joilla ei ollut CBM-moduulia. Lisäksi entsyymin positiivinen pintavaraus lisäsi entsyymin sitoutumista negatiivisesti varautuneeseen ligniiniin ja hydrofobiset alueet entsyymin pinnalla tekivät sitoutumisesta voimakkaampaa. Entsyymien lämpöstabiiliuden vaikutusta ligniininperäiseen inhibitioon tutkittiin kolmella perheen GH11 ksylanaasilla. Kaikki kylanaasit sitoutuivat ligniiniin, mutta lämpöstabiili Xyl40 pysyi osittain aktiivisena ligniinin pinnalla, kun taas lämpökestävyydeltään epästabiilimmat ksylanaasit inaktivoituivat. Lisäksi Xyl40 ksylanaasin rakenteelliset sokerit vähensivät ligniiniperäistä inhibitiota hydrolyysikokeissa. Näin ollen voidaan olettaa, että ksylanaaseilla korkea lämpöstabiilius ja rakenteelliset sokerit estävät entsyymin inaktivoitumisen vähentämällä proteiinin rakenteen purkautumista ligniinin pinnalle

Tillage System and Crop Sequence Affect Soil Disease Suppressiveness and Carbon Status in Boreal Climate

Correction FRONTIERS IN MICROBIOLOGY Volume: 12 Article Number: 693341 DOI: 10.3389/fmicb.2021.693341 Published: MAY 20 2021The soil-borne plant pathogens cause serious yield losses and are difficult to control. In suppressive soils, disease incidence remains low regardless of the presence of the pathogen, the host plant, and favorable environmental conditions. The potential to improve natural soil disease suppressiveness through agricultural management practices would enable sustainable and resilient crop production systems. Our aim was to study the impact of autumn tillage methods and crop sequence on the soil carbon status, fungistasis and yield in boreal climate. The disease suppression was improved by the long-term reduced and no tillage management practices with and without crop rotation. Compared to the conventional plowing, the non-inversion tillage systems were shown to change the vertical distribution of soil carbon fractions and the amount of microbial biomass by concentrating them on the soil surface. Crop sequence and the choice of tillage method had a combined effect on soil organic carbon (SOC) sequestration. The improved general disease suppression had a positive correlation with the labile carbon status and microbial biomass. From the most common Fusarium species, the predominantly saprophytic F. avenaceum was more abundant under non-inversion practice, whereas the opposite was true for the pathogenic ones. Our findings furthermore demonstrated the correlation of the soil fungistasis laboratory assay results and the prevalence of the pathogenic test fungus Fusarium culmorum on the crop cereals in the field. Our results indicate that optimized management strategies have potential to improve microbial related soil fungistasis in boreal climate.Peer reviewe

Inhibitory effect of lignin on the hydrolysis of xylan by thermophilic and thermolabile GH11 xylanases

BACKGROUND: Enzymatic hydrolysis of lignocellulosic biomass into platform sugars can be enhanced by the addition of accessory enzymes, such as xylanases. Lignin from steam pretreated biomasses is known to inhibit enzymes by non-productively binding enzymes and limiting access to cellulose. The effect of enzymatically isolated lignin on the hydrolysis of xylan by four glycoside hydrolase (GH) family 11 xylanases was studied. Two xylanases from the mesophilic Trichoderma reesei, TrXyn1, TrXyn2, and two forms of a thermostable metagenomic xylanase Xyl40 were compared. RESULTS: Lignin isolated from steam pretreated spruce decreased the hydrolysis yields of xylan for all the xylanases at 40 and 50 °C. At elevated hydrolysis temperature of 50 °C, the least thermostable xylanase TrXyn1 was most inhibited by lignin and the most thermostable xylanase, the catalytic domain (CD) of Xyl40, was least inhibited by lignin. Enzyme activity and binding to lignin were studied after incubation of the xylanases with lignin for up to 24 h at 40 °C. All the studied xylanases bound to lignin, but the thermostable xylanases retained 22–39% of activity on the lignin surface for 24 h, whereas the mesophilic T. reesei xylanases become inactive. Removing of N-glycans from the catalytic domain of Xyl40 increased lignin inhibition in hydrolysis of xylan when compared to the glycosylated form. By comparing the 3D structures of these xylanases, features contributing to the increased thermal stability of Xyl40 were identified. CONCLUSIONS: High thermal stability of xylanases Xyl40 and Xyl40-CD enabled the enzymes to remain partially active on the lignin surface. N-glycosylation of the catalytic domain of Xyl40 increased the lignin tolerance of the enzyme. Thermostability of Xyl40 was most likely contributed by a disulphide bond and salt bridge in the N-terminal and α-helix regions. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13068-022-02148-4

Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce

Abstract Background The recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs). Results LMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis. Conclusions LMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes

Peltomaan tautisuppressiivisuuden tehostaminen muokkausmenetelmän ja viljelykierron avulla

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Improved general plant pathogen suppression by agricultural management practices

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Peltomaan tautisuppressiivisuuden tehostaminen viljelytoimenpiteiden avulla

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