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Cellulases adsorb reversibly on biomass lignin.

ABSTRACT: Adsorption of cellulases onto lignin is considered a major factor in retarding enzymatic cellulose degradation of lignocellulosic biomass. However, the adsorption mechanisms and kinetics are not well understood for individual types of cellulases. This study examines the binding affinity, kinetics of adsorption, and competition of four monocomponent cellulases of Trichoderma reesei during adsorption onto lignin. TrCel7A, TrCel6A, TrCel7B, and TrCel5A were radiolabeled for adsorption experiments on lignin-rich residues (LRRs) isolated from hydrothermally pretreated spruce (L-HPS) and wheat straw (L-HPWS), respectively. On the basis of adsorption isotherms fitted to the Langmuir model, the ranking of binding affinities was TrCel5A?>? TrCel6A?>? TrCel7B?>? TrCel7A on both types of LRRs. The enzymes had a higher affinity to the L-HPS than to the L-HPWS. Adsorption experiments with dilution after 1 and 24?hr and kinetic modeling were performed to quantify any irreversible binding over time. Models with reversible binding parameters fitted well and can explain the results obtained. The adsorption constants obtained from the reversible models agreed with the fitted Langmuir isotherms and suggested that reversible adsorption-desorption existed at equilibrium. Competitive binding experiments showed that individual types of cellulases competed for binding sites on the lignin and the adsorption data fitted the Langmuir adsorption model. Overall, the data strongly indicate that the adsorption of cellulases onto lignin is reversible and the findings have implications for the development of more efficient cellulose degrading enzymes.

SUBMITTER: Djajadi DT

PROVIDER: S-EPMC6282830 | biostudies-other | 2018 Dec

REPOSITORIES: biostudies-other

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Cellulases adsorb reversibly on biomass lignin.

Djajadi Demi T DT Pihlajaniemi Ville V Rahikainen Jenni J Kruus Kristiina K Meyer Anne S AS

Biotechnology and bioengineering 20181016 12

Adsorption of cellulases onto lignin is considered a major factor in retarding enzymatic cellulose degradation of lignocellulosic biomass. However, the adsorption mechanisms and kinetics are not well understood for individual types of cellulases. This study examines the binding affinity, kinetics of adsorption, and competition of four monocomponent cellulases of Trichoderma reesei during adsorption onto lignin. TrCel7A, TrCel6A, TrCel7B, and TrCel5A were radiolabeled for adsorption experiments o ...[more]

PMID: 30132790

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Project description:BackgroundNon-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.ResultsIn this study, we have utilized an FPLC (fast protein liquid chromatography)-based methodology to quantify free Trichoderma reesei cellulases (namely CBH I, CBH II, and EG I) concentration within a complex hydrolyzate mixture during the varying time course of biomass saccharification. Three pretreated corn stover (CS) samples were included in this study: Ammonia Fiber Expansion(a) (AFEX™-CS), dilute acid (DA-CS), and ionic liquid (IL-CS) pretreatments. The relative fraction of bound individual cellulases varied depending not only on the pretreated biomass type (and lignin abundance) but also on the type of cellulase. Acid pretreated biomass had the highest levels of non-recoverable cellulases, while ionic liquid pretreated biomass had the highest overall cellulase recovery. CBH II has the lowest thermal stability among the three T. reesei cellulases tested. By preparing recombinant family 1 carbohydrate binding module (CBM) fusion proteins, we have shown that family 1 CBMs are highly implicated in the non-productive binding of full-length T. reesei cellulases to lignin.ConclusionsOur findings aid in further understanding the complex mechanisms of non-productive binding of cellulases to pretreated lignocellulosic biomass. Developing optimized pretreatment processes with reduced or modified lignin content to minimize non-productive enzyme binding or engineering pretreatment-specific, low-lignin binding cellulases will improve enzyme specific activity, facilitate enzyme recycling, and thereby permit production of cheaper biofuels.

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Cellulases adsorb reversibly on biomass lignin.

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Cellulases adsorb reversibly on biomass lignin.

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