Novel Approach to Prepare Ultrathin Lignocellulosic Film for Monitoring Enzymatic Hydrolysis Process by Quartz Crystal Microbalance

BackgroundThe presence of lignin normally affects enzymatic saccharification of lignocellulose detrimentally. However, positive effects of lignin on enzymatic hydrolysis have been recently reported. Enzyme–lignin interactions could be the key to reveal the underlying mechanism of their discrepant behaviors. In this study, to elucidate the positive effects of extractable lignin (EL) on enzymatic hydrolysis of ethanol organosolv-pretreated wood sawdust, two lignin fractions, EL and milled wood lignin (MWL), were isolated sequentially from pretreated substrates. Quartz crystal microbalance with dissipation (QCM-D) was then used to investigate the lignin aggregation effects on enzyme adsorption.ResultsWe found that both EL and MWL had a narrow molecular weight distribution. However, MWL had an obviously higher molecular weight than EL. This indicated that EL and MWL likely represent two distinct lignin fractions from ethanol organosolv-pretreated substrates. HSQC NMR analysis revealed that less β-O-4, β-β, and β-5 linkages and a higher S/G ratio was present in EL, as compared to MWL. QCM-D analysis showed that the enzyme adsorption on lignin was highly relevant to these lignin structural characteristics. An obviously lower maximum enzyme adsorption capacity was observed on EL films (152.63–168.09 ng/cm2) compared to MWL films (196.71–224.73 ng/cm2). Furthermore, enzyme desorption on lignin films was determined. A significantly lower irreversible enzyme adsorption was observed on EL (75.40 ng/cm2) compared to MWL (137.35 ng/cm2). More importantly, two reconstructed lignin films were designed to investigate lignin assembly on enzyme adsorption. The results indicated that the presence of EL reduced irreversible enzyme adsorption on the reconstructed lignin films by 39.2–45.0%.ConclusionsLignin structure determined the interaction between enzyme and lignins. A positive correlation was observed between molecular weight, the content of β-5 linkages, and enzyme adsorption on lignin. EL, which was more depolymerized and less condensed, had the lower enzyme adsorption of the two preparations tested. Additionally, the presence of EL reduced enzyme adsorption on reconstructed lignin films, perhaps through a mechanism involving the blocking of non-productive enzyme binding sites on the MWL. This could be the mechanism for the positive effects of EL on enzymatic hydrolysis.Electronic supplementary materialThe online version of this article (10.1186/s13068-019-1402-2) contains supplementary material, which is available to authorized users.

Section: Methodsmentioning

confidence: 99%

“…The morphology and roughness of the lignin films were characterized by atomic force microscopy (AFM) (Dimesion Edge, Brucker, Saarbrücken, Germany). The images were scanned in tapping mode, and analyzed using NanoScope analysis software according to a previous report [ 35 ].…”

Section: Methodsmentioning

confidence: 99%

New strategy to elucidate the positive effects of extractable lignin on enzymatic hydrolysis by quartz crystal microbalance with dissipation

Lai

Yang

Lin

et al. 2019

“…The dynamics of enzymatic hydrolysis showed that when pseudo-lignin and residual lignin were mixed with cellulose, the maximum glucose release was reduced and the time to reach the maximum enzymatic hydrolysis efficiency was longer than that of pure cellulose. Jiang et al [ 165 ] prepared a full-component lignocellulose ultrathin film using the LiCl/DMSO solvent system. The in situ enzymatic hydrolysis of the film in QCM-D verified the non-productive adsorption of cellulase onto lignin.…”

Section: Interaction Between Lignin and Cellulasementioning

confidence: 99%

Recent advances in understanding the effects of lignin structural characteristics on enzymatic hydrolysis

Yuan

Jiang

Chen

et al. 2021

Self Cite

143

Enzymatic hydrolysis of lignocellulose for bioethanol production shows a great potential to remit the rapid consumption of fossil fuels, given the fact that lignocellulose feedstocks are abundant, cost-efficient, and renewable. Lignin results in low enzymatic saccharification by forming the steric hindrance, non-productive adsorption of cellulase onto lignin, and deactivating the cellulase. In general, the non-productive binding of cellulase on lignin is widely known as the major cause for inhibiting the enzymatic hydrolysis. Pretreatment is an effective way to remove lignin and improve the enzymatic digestibility of lignocellulose. Along with removing lignin, the pretreatment can modify the lignin structure, which significantly affects the non-productive adsorption of cellulase onto lignin. To relieve the inhibitory effect of lignin on enzymatic hydrolysis, enormous efforts have been made to elucidate the correlation of lignin structure with lignin–enzyme interactions but with different views. In addition, contrary to the traditional belief that lignin inhibits enzymatic hydrolysis, in recent years, the addition of water-soluble lignin such as lignosulfonate or low molecular-weight lignin exerts a positive effect on enzymatic hydrolysis, which gives a new insight into the lignin–enzyme interactions. For throwing light on their structure–interaction relationship during enzymatic hydrolysis, the effect of residual lignin in substrate and introduced lignin in hydrolysate on enzymatic hydrolysis are critically reviewed, aiming at realizing the targeted regulation of lignin structure for improving the saccharification of lignocellulose. The review is also focused on exploring the lignin–enzyme interactions to mitigate the negative impact of lignin and reducing the cost of enzymatic hydrolysis of lignocellulose.

“…Dry Masson pine was milled in a planetary ball mill (Fritsch Pulverisette 7, Idar-Oberstein, Germany) at 40 °C as described previously [34]. The milled Masson pine was dispersed in a dioxane/water (96/4, v/v) mixture in a 1:25 ratio of wood to solvent and mechanically stirred.…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme adsorption/desorption behaviors on lignin films were monitored in situ using quartz crystal microgravimetry with dissipation monitoring (QCM-D) (QCM-D E4 model, Biolin Corp., Gothenburg, Sweden) at 4 °C as previously described [34]. The purified MMPL was dissolved in dioxane at a concentration of 1% (w/v).…”

Section: Methodsmentioning

confidence: 99%

Altering the linker in processive GH5 endoglucanase 1 modulates lignin binding and catalytic properties

Wang

Zhang

Long

et al. 2018

BackgroundThe non-productive adsorption of cellulases onto lignin in biomass is a key issue for the biofuel process economy. It would be helpful to reduce the inhibitory effect of lignin on enzymatic hydrolysis by engineering weak lignin-binding cellulases. Cellulase linkers are highly divergent in their lengths, compositions, and glycosylations. Numerous studies have revealed that linkers can facilitate optimal interactions between structured domains. Recently, efforts have focused on the contributions and mechanisms of carbohydrate-binding modules and catalytic domains that affect lignin affinity and processivity of cellulases, but our understanding of the effects of the linker regions on lignin adsorption and processivity of GH5 processive endoglucanases is still limited.ResultsEight GH5 endoglucanase 1 variants of varying length, flexibility, and sequence in the linker region were constructed. Their characteristics were then compared to the wild-type enzyme (EG1). Remarkably, significant differences in the lignin adsorption profiles and processivities were observed for EG1 and other variants. Our studies suggest that either the length or the specific amino acid composition of the linker has a prominent influence on the lignin-binding affinity of the enzymes. Comparatively, the processivity may depend primarily on the length of the linker and less so on the specific amino acid composition. EG1-ApCel5A, a variant with better performance in enzymatic hydrolysis in the presence of lignin, was obtained by replacing a longer, flexible linker. In total, up to between 28.2 and 30.1% more reducing sugars were generated from filter paper by EG1-ApCel5A in the presence of lignin compared to EG1.ConclusionsOur results highlight the relevance of the linker region in the lignin adsorption and processivity of a processive endoglucanase. Our findings suggest that the linker region may be used as a target for the design of more active and weaker lignin-binding cellulases.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1333-3) contains supplementary material, which is available to authorized users.