Enzymatic Hydrolysis Efficiency Research Articles

Influence of biphasic phenoxyethanol-alkaline pretreatment on the correlation between inter-structure and enzymatic hydrolysis in bamboo residues

The effective promotion of delignification (67.6 %) and xylan removal (44.8 %) from bamboo residues using a 2-phenoxyethanol/sodium hydroxide solution (P/A) system is demonstrated, while simultaneously enriching oligosaccharides contents of the pre-hydrolysate to 10.2 g/L. Increasing the P/A ratio from 0:1 to 4:1 improves the enzymatic digestibility of the substrates from 55.7 % to 70.1 % at 100 °C and from 73.8 % to 83.7 % at 120 °C. Furthermore, partial correlation analysis demonstrates that the physiochemical properties, including delignification, xylan removal, and crystallinity, show a significant positive correlation with enzymatic hydrolysis efficiency. Higher temperatures and P/A ratios during alkaline biphasic pretreatment promote the shrinkage of plant cells and delignification, with temperature being a particularly significant driver. These findings provide valuable insights into the alkaline and biphasic pretreatment of biomass and facilitate the optimization of the bio-refining system.

Efficient Enzymatic Hydrolysis of the Whey Protein Fraction from Bovine Milk

Studies were conducted to optimize the hydrolysis of the major whey proteins with the serine proteases Alcalase and Protozyme, and of the immunoglobulin fraction of colostrum with Alcalase. The protein-peptide composition of the enzymatic hydrolysates of whey and colostrum was determined using high-performance liquid chromatography and electrophoresis data under denaturing conditions. The hydrolysis conditions, in conjunction with the application of a heat treatment of the substrate, were designed to enhance the efficiency of whey proteolysis using Protozyme. An increase in the proteolysis degree of the native immunoglobulin fraction of colostrum with Alcalase was also achieved. The results of the experimental work are intended for the production of a hypoallergenic protein component derived from bovine milk

Corn-derived Expansin synergistically promotes enzymatic hydrolysis of corn cob

The enzymatic hydrolysis of lignocellulose is hindered by challenges such as high enzyme usage and associated costs. It is essential to explore effective approaches to improve the efficiency of enzymatic hydrolysis while reducing costs. Expansins are non-enzymatic proteins that can interact with lignocellulose and facilitate the loosening of plant cell walls. Given their natural affinity to plant cell walls, we hypothesized that a corn Expansin could enhance the enzymatic hydrolysis of corn lignocellulose. In this study, we expressed a corn (Zea mays) Expansin, EXPA17, in yeast cells and explored the synergistic effect between EXPA17 and commercial cellulase, and found that EXPA17 exhibited a pronounced synergistic effect on the enzymatic hydrolysis of corn cobs. The addition of 0.015 mg/mL EXPA17 resulted in a 14.00 % increase in glucose yield. Fourier-transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM) analysis revealed that EXPA17 proteins disrupted hydrogen bonds in the amorphous regions of corn cobs, leading to a more porous and looser structure, thereby enhancing cellulose accessibility. Our work leveraged the synergistic effect between Expansin and lignocellulose from the same source of corn, providing a novel strategy to improve the efficiency of enzymatic hydrolysis of corn lignocellulose while potentially reducing the associated costs.

Enhancing reed cellulose conversion to glucose with octyl glucoside and tea saponin pretreatment

Low pretreatment temperatures are beneficial for enhancing the activity of both soluble lignin and lignin in pretreated solids. To achieve high-activity lignin and a higher glucose yield at a lower lignin removal rate and reduced enzymatic loading, the synergistic effects of octyl glucoside and tea saponin during low-temperature ammonia‑oxygen pretreatment and enzymatic hydrolysis were explored. Utilizing a moderated lignin extraction at 38 %, with pretreatment at 110 °C, and enriched with 1 % octyl glucoside and 0.06 g/g tea saponin, the biochemical conversion was galvanized. This manifested in an enzymatic hydrolysis efficiency of 96 % and a glucose yield in excess of 85 % at an enzyme loading of 5 FPU/g of pretreated-solids. Conversely, the lignin removal rate without surfactants was 26 %, resulting in 70 % hydrolysis efficiency even at a higher enzyme loading of 15 FPU/g of pretreated-solids. In order to minimize the pretreatment temperature and enzyme load as much as possible without affecting the efficiency of enzymatic hydrolysis and glucose yield, the goal of this research is to investigate the roles of octyl glucoside in the pretreatment process and tea saponin in the enzymatic hydrolysis process. This study provides a new approach for refining lignocellulosic biomass.

Aminated lignin improved enzymatic hydrolysis of cellulosic substrate treated by p-toluenesulfonic acid

Lignin can affect the enzymatic hydrolysis efficiency of lignocellulose. In this study, the lignin isolated from sugarcane bagasse (SCB) pretreated with p-toluenesulfonic acid (PL) was firstly aminated, and then the effects of PL and aminated PL (APL) on the bagasse enzymatic hydrolysis efficiency (EHE) were investigated. The results showed that the addition of PL and APL promoted the EHE, and EHE with APL (73.82 %) was higher than PL (51.39 %). To explore the reason, the data were further analyzed including cellulase adsorption capacity, enzyme activity, cellulase-lignin interaction, and molecular docking. It was found that APL adsorbed more cellulase (27.83 mg protein/g lignin) than PL (4.96 mg protein/g lignin), resulting from the greater interaction force and lower binding free energy between APL and cellulase. The addition of APL more remarkably enhanced the cellobiohydrolase and endoglucanase activities than PL due to more effectively inducing cellulase conformation optimization.

Enhancing enzymatic conversion of castor stalk through dual-functional ethanolamine pretreatment

Castor stalk from hemp plants is an attractive lignocellulosic feedstock for biomass refining valorization due to its similar chemical composition to hardwoods. In this study, the castor stalk fibers were pretreated with efficient dual-functional ethanolamine to achieve delignification and swelling of the cellulosic fibers, followed by cellulase enzymatic digestion for biomass conversion. Experimental results showed that ethanolamine pretreatment at 160 °C for 1 h effectively removed 69.20 % of lignin and 43.18 % of hemicellulose. In addition to efficient delignification and removal of hemicellulose, the study also revealed that supramolecular structure of cellulose was another major factor affecting enzymatic hydrolysis performance. The lowered crystallinity (60–70 %) and swelled crystal sizes (2.95–3.04 nm) promoted enzymatic hydrolysis efficiency during the heterogeneous reaction process. Under optimal conditions (160 °C, 1 h; enzyme loading: 15 FPU/g substrate), promoted yields of 100 % glucose and over 90 % xylose were achieved, which were significantly higher than those obtained from untreated castor stalk. These findings highlighted the effectiveness of the dual-functional ethanolamine pretreatment strategy for efficient bioconversion of lignocellulosic feedstocks. Overall, this study provides valuable insights into the development of new strategies for the efficient utilization of biomass resources, which is essential for the sustainable development of our society.

Extraction and characterization of valuable compounds from chicken sternal cartilage: Type II collagen and chondroitin sulfate

Type II collagen (Col II) and chondroitin sulfate (CS) are the main macromolecules in the extracellular matrix. This study investigated the characteristics of Col II and CS obtained from chicken sternal cartilage (CSC) via enzymatic hydrolysis for various treatment times. For Col II and CS, the highest efficiency of enzymatic hydrolysis was achieved after 24 and 6 h of treatment, respectively. The average molecular weights were α1 chain-130 kDa, β chain-270 kDa for Col II, and 80.27 kDa for CS. Fourier transform infrared spectroscopy revealed that the Col II samples maintained their triple-helical structure and that the predominant type of CS was chondroitin-4-sulfate. Scanning electron microscopy revealed that the Col II and CS samples possessed fibrillar and clustered structures, respectively. This study suggests that collagen and CS obtained from CSC can be used as promising molecules for application in food and pharmaceutical industries.

Green Extraction of Reed Lignin: The Effect of the Deep Eutectic Solvent Composition on the UV-Shielding and Antioxidant Properties of Lignin.

Lignin, the second most abundant natural polymer, is a by-product of the biorefinery and pulp and paper industries. This study was undertaken to evaluate the properties and estimate the prospects of using lignin as a by-product of the pretreatment of common reed straw (Phragmites australis) with deep eutectic solvents (DESs) of various compositions: choline chloride/oxalic acid (ChCl/OA), choline chloride/lactic acid (ChCl/LA), and choline chloride/monoethanol amine (ChCl/EA). The lignin samples, hereinafter referred to as Lig-OA, Lig-LA, and Lig-EA, were obtained as by-products after optimizing the conditions of reed straw pretreatment with DESs in order to improve the efficiency of subsequent enzymatic hydrolysis. The lignin was studied using gel penetration chromatography, UV-vis, ATR-FTIR, and 1H and 13C NMR spectroscopy; its antioxidant activity was assessed, and the UV-shielding properties of lignin/polyvinyl alcohol composite films were estimated. The DES composition had a significant impact on the structure and properties of the extracted lignin. The lignin's ability to scavenge ABTS+• and DPPH• radicals, as well as the efficiency of UV radiation shielding, decreased as follows: Lig-OA > Lig-LA > Lig-EA. The PVA/Lig-OA and PVA/Lig-LA films with a lignin content of 4% of the weight of PVA block UV radiation in the UVA range by 96% and 87%, respectively, and completely block UV radiation in the UVB range.

Efficient Enzymatic Hydrolysis and Polyhydroxybutyrate Production from Non-Recyclable Fiber Rejects from Paper Mills by Recombinant Escherichia coli

Non-recyclable fiber rejects from paper mills, particularly those from recycled linerboard mills, contain high levels of structural carbohydrates but are currently landfilled, causing financial and environmental burdens. The aim of this study was to develop efficient and sustainable bioprocess to upcycle these rejects into polyhydroxybutyrate (PHB), a biodegradable alternative to degradation-resistant petroleum-based plastics. To achieve high yields of PHB per unit biomass, the specific objective of the study was to investigate various approaches to enhance the hydrolysis yields of fiber rejects to maximize sugar recovery and evaluate the fermentation performance of these sugars using Escherichia coli LSBJ. The investigated approaches included size reduction, surfactant addition, and a chemical-free hydrothermal pretreatment process. A two-step hydrothermal pretreatment, involving a hot water pretreatment (150 °C and 15% solid loading for 10 min) followed by three cycles of disk refining, was found to be highly effective and resulted in an 83% cellulose conversion during hydrolysis. The hydrolysate obtained from pretreated biomass normally requires a detoxification step to enhance fermentation efficiency. However, the hydrolysate obtained from the pretreated biomass contained minimal to no inhibitory compounds, as indicated by the efficient sugar fermentation and high PHB yields, which were comparable to those from fermenting raw biomass hydrolysate. The structural and thermal properties of the extracted PHB were analyzed using various techniques and consistent with standard PHB.

Efficient enzymatic hydrolysis of active oxygen and solid alkali/dilute sulfuric acid-pretreated corn cob

This work proposed a two-step pretreatment method of cooking with active oxygen and solid alkali (CAOSA) followed by dilute sulfuric acid treatment (DA), to contribute enzymatic hydrolysis of corn cob. The first step, CAOSA pretreatment, removed 83.16 % of the lignin, while the second step, a milder pretreatment with DA further eliminated hemicellulose. After the two-step pretreatment, the cellulose content of the residue reached 66.74 %, and with a solid loading of 5 %, the glucose yield was remarkably high at 98.35 % with a concentration of 36.47 g/L. The optimal conditions of the process were determined as follows: DA with 1 % H2SO4 at a solid-to-liquid ratio of 1:10, a temperature of 110 ℃, and a pretreatment time of 60 min. To confirm the efficacy of the two-step pretreatment, we characterized the samples treated with different methods using X-ray diffraction (XRD), Fourier-transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). The developed method proposes an effective pretreatment and results in the complete enzymatic hydrolysis of corn cob.

Enhancing efficiency in ethanol production from high solid corn stover: Insights into enzymatic hydrolysis parameters and cellulase recovery

BackgroundThe simultaneous saccharification and co-fermentation (SSCF) at high solid loading is an effective approach to improve the economic feasibility of bioethanol. MethodsIn this study, the high-solid SSCF of superfine grinding corn stover was treated with cheap and readily available deionized water (DW) instead of sodium citrate buffer solution (SCBS). Additionally, the evaluation of temperature, enzyme loading, solid loading, and cellulose recovery were carried out to determine their impact on ethanol production. Significant FindingsThe results of the analysis indicated that replacing SCBS with DW had negligible effects on the SSCF of ethanol and byproducts (acetic acid and glycerol). The ethanol yield increased by 18 % at 35 °C compared to the yield at 30 °C. As enzyme loading increased, more cellulose active sites in corn stover were bound by cellulase, which increased the ethanol yield by improving the efficiency of enzymatic hydrolysis. The SSCF process didn't incorporate any buffer, resulting in an even higher ethanol titer (55.42 g/L). Furthermore, the cellulase productivity increased by 71 % after recovery, compared to the non-recovery. The results indicated that the optimized SSCF process is a potential technique for producing bioethanol on a commercial level because it improves the production efficiency while keeping costs low.

Enhancing cellulose hydrolysis via cellulase immobilization on zeolitic imidazolate frameworks using physical adsorption.

This study investigates the immobilization of cellulase on zeolitic imidazolate frameworks (ZIFs) by physical adsorption, specifically the ZIF-8-NH2 and Fe3O4@ZIF-8-NH2, to enhance enzymatic hydrolysis efficiency. The immobilization process was thoroughly analyzed, including optimization of conditions and characterization of ZIF carriers and immobilized enzymes. The impacts on the catalytic activity of cellulase under various temperatures, pH levels, and storage conditions were examined. Additionally, the reusability of the immobilized enzyme was assessed. Results showed the cellulase immobilized on Fe3O4@ZIF-8-NH2 exhibited a high loading capacity of 339.64 mg/g, surpassing previous studies. Its relative enzymatic activity was found to be 71.39%. Additionally, this immobilized enzyme system demonstrates robust reusability, retaining 68.42% of its initial activity even after 10 cycles. These findings underscore the potential of Fe3O4@ZIF-8-NH2 as a highly efficient platform for cellulase immobilization, with promising implications for lignocellulosic biorefinery.

A designed ZrOCl2/ethylene glycol deep eutectic solvent for efficient lignocellulose valorization

Deep eutectic solvents (DESs) hold great potential in biorefining because they can efficiently deconstruct the recalcitrant structure of lignocellulose. In particular, inorganic salts with Lewis acids have been proven to be effective at cleaving lignin–carbohydrate complexes. Herein, a Zr-based DES system composed of metal chloride hydrate (ZrOCl2·8H2O) and ethylene glycol (EG) was designed and used for poplar powder pretreatment. Zr4+-based salts provide sufficient acidity for lignocellulose depolymerization. The acidity of the DES was analysed by the Kamlet-Taft solvatochromic parameter, and the results demonstrated that the acidity can be regulated by the DES composition. Under the optimum conditions (ZrOCl2·8H2O:EG molar ratio of 1:2), the DES pretreatment removes nearly 100 % hemicellulose and 94.7 % lignin. The recovered lignin exhibited a low polydispersity of 1.7. The cellulose residues deliver an efficiency of 94.4 % upon enzymatic digestion. Moreover, the DES can be easily recovered with high yield and purity, and the recycled DES still maintains high delignification and enzymatic hydrolysis efficiencies. The proposed DES pretreatment technology is promising for biomass valorization.

Dissolving Chitin by Novel Deep Eutectic Solvents for Effectively Enzymatic Hydrolysis.

Chitin is the most productive nitrogen-containing polysaccharide in nature with immense potential for transforming into a range of chemicals. However, its dense crystal structure poses a challenge for depolymerization, limiting its applications. To overcome these challenges, a novel series of deep eutectic solvents (DESs) based on benzyltrimethylammonium chloride (TMBAC) as the hydrogen bond acceptor was developed. These TMBAC-based DESs, in combination with lactic acid, oxalic acid, and malic acid as the hydrogen bond donor demonstrated efficient chitin dissolution, achieving a solubility of up to 12% and an 88% recovery rate of regenerated chitin. The regenerated chitin was characterized using XRD, FT-IR, SEM, and 13C CP-MAS NMR, which indicated the preservation of chitin's chemical structure, a significant decrease in crystallinity, and a reduction in the molecular weight. Furthermore, the enzymatic hydrolysis efficiency of chitin was nearly doubled after treatment with TMBAC-based DESs, surpassing the effectiveness of untreated chitin. This approach holds promise for facilitating subsequent transformation and utilization of chitin.

Waste to wealth: A novel low temperature eco-friendly lignocellulose pretreatment strategy for glucose production

Acid pretreatment is considered as one of the most promising pretreatment strategies. However, the operation temperature of acid pretreatment is commonly higher, and acid pretreatment is difficult to remove lignin. Herein, a novel pretreatment strategy (dilute sulfuric acid coupling K2S2O8 (DA-PDS) pretreatment) was proposed to remove lignin at lower temperature. The feasibility of DA-PDS pretreatment was predicted by means of density functional theory (DFT). Then, the optimal operation conditions of DA-PDS pretreatment were studied. Temperature was proved to be the most critical factor affecting the DA-PDS pretreatment. The optimal reaction conditions of DA-PDS pretreatment were 5 % DA and 6 % PDS at 80 °C for 2 h. The operation temperature was 40 °C lower than common acid pretreatment. On this condition, the cellulose content and enzymatic hydrolysis efficiency by DA-PDS pretreatment were about 59 % and 73 %, which were 11 % and 13 % higher than that of DA pretreatment, respectively. In addition, this study revealed the mechanism of DA-PDS pretreatment. DA not only accelerated radical generation rate, but also improved the contact probability between radical and lignin. Radicals generated by PDS destroyed the structure of lignin. This made DA more easily to remove hemicellulose. This work presented a novel and energy-saving strategy for lignocellulose pretreatment.

INCREASING THE EFFICIENCY OF ENZYMATIC HYDROLYSIS OF LIGNOCELLULOSIC MATERIALS BY FREEZING PRETREATMENT

This paper describes the differences between the efficiency of enzymatic hydrolysis of selected lignocellulosic materials after pretreatment by cyclic freezing and thawing. It also discusses the analysis of the positive effect of alkaline conditions on pretreatment. The selected materials were Populus alba L., wheat straw from Triticum aestivum L. and Cannabis sativa. Three pretreatment methods were used to compare the efficiency of enzymatic decomposition of cellulose and cellulose accessibility. The best results show the wheat straw pre-treated at -20°C in the freezer with NaOH addition with a concentration of monosaccharides of 56.6 g.l-1 compared to initial hydrolysed material with a concentration of monosaccharides of 24.4 g.l-1. The results show better digestibility of grass compared to wood dendromass

Study on the mechanism of lignin non-productive adsorption on cellobiohydrolase

Enzymatic hydrolysis is important for lignocellulosic biomass conversion into fermentable sugars. However, the nonproductive adsorption of enzyme on lignin was major hinderance for the enzymatic hydrolysis efficiency. In this study, non-productive adsorption mechanism of cellulase component cellobiohydrolase (CBH) onto lignin was specific investigated. Research revealed that the adsorption behavior of CBH on eucalyptus alkali lignin (EuA) was affected by reaction conditions. As study on the adsorption kinetic, it was indicated that the adsorption cellulose binding domain (CBD) of CBH onto EuA well fitted with Langmuir adsorption model and pseudo second-order adsorption kinetics model. And the tyrosine site related to the adsorption of CBD onto lignin was proved by the fluorescence and UV spectra analysis. The results of this work provide a theoretical guidance to understanding the nonproductive adsorption mechanism and building method to reduce the adsorption of cellulase on the lignin.

Developing a new ethylene glycol/H2O pretreatment system to achieve efficient enzymatic hydrolysis of sugarcane bagasse cellulose and recover highly active lignin: Countercurrent extraction

Improving pretreatment efficiency is a critical premise in achieving efficient biomass conversion, and obtaining high-performance natural polymers is the guarantee of high-value conversion of biomass. In this study, a new pilot-scale continuous countercurrent pretreatment reaction unit about ethylene glycol-alkali solution was designed for pretreating sugarcane bagasse in order to achieve efficient separation of the three major components of lignocellulose when expanding the scale of pretreatment, reduce lignin deposition on the fiber surface, and obtain highly active lignin and excellent enzymatic hydrolysis efficiency of cellulose. X-ray diffractometer (XRD), X-ray photoelectron spectrometer (XPS), brunauer-emmett-teller (BET) and scanning electron microscope (SEM) methods are used to analyze the structural properties of sugarcane bagasse before and after pretreatment, and high-performance liquid chromatography (HPLC) is used to analyze the monosaccharide components in the enzymatic solution. In addition, the structural properties of the recovered lignin are analyzed by gel permeation chromatography (GPC), 31P NMR and 2D-HSQC-NMR methods. The results indicate that the system can gain a high cellulose recovery of 92.99% along with a lignin removal of 95.33%, and recovered lignin has low lignin carbohydrate complexes, low condensation, and rich in phenolic hydroxyl groups for 1.95 mmol/g. Meanwhile, the countercurrent pretreatment system can effectively reduce the deposition of lignin on the cellulose surface, which is evidently superior to the non-countercurrent pretreatment and facilitates the efficiency of enzymatic saccharification of substrate, achieving a high glucose yield of 99% as well as a total sugar yield of 91.11%. The method efficiently separates biomass in a green manner, and solid residues are easily hydrolyzed, showing potential for industrial-scale production.

Blocking miR396 activity by overexpression MIM396 improved switchgrass tiller number and biomass yield.

MicroRNA396 (miR396) plays an important role in the regulation of plant growth and development by repressing the expression level of its target growth-regulating factor (GRF) family genes. In our previous study, we found that overexpression of miR396 negatively regulated both tillering and biomass yield in switchgrass (Panicum virgatum L.). We, therefore, speculated that blocking the expression of miR396 could enhance switchgrass tillering and biomass yield. Here, we produced transgenic switchgrass plants overexpressing a target mimicry form of miR396 (MIM396) in wild type (WT) and Os-MIR319b overexpressing switchgrass plant (with higher enzymatic hydrolysis efficiency, but reduced tillering), in which the expression of miR396 was blocked. The phenotype and biological yields of these plants were analyzed. Blocking miR396 to improve its target PvGRFs expression in switchgrass improved the tiller number and dry weight of transgenic plants. Further morphological analysis revealed that MIM396 plants increased the number of aerial branches and basal tillers compared to those of wild-type plants. The enzymatic efficiency of MIM396 plants was reduced; however, the total sugar production per plant was still significantly higher than that of wild-type plants due to the increase in biomass. In addition, blocking miR396 in a transgenic switchgrass plant overexpressing Os-MIR319b (TG21-Ms) significantly increased the PvGRF1/3/5 expression level and tiller number and biomass yield. The miR156-target gene PvSPL4, playing a negative role in aerial and basal buds outgrowth, showed significant downregulated in MIM396 and TG21-Ms. Those results indicate that miR396-PvGRFs, through disrupting the PvSPL4 expression, are involved in miR319-PvPCFs in regulating tiller number, at least partly. MIM396 could be used as a molecular tool to improving tiller number and biomass yield in switchgrass wild type and miR319b transgenic plants. This finding may be applied to other graminaceous plants to regulate plant biological yield.

Analysis of the relationship between starch molecular conformation and enzymatic hydrolysis efficiency

Resistant starch (RS) is important in controlling diabetes. The primary objective of this study is to examine the impact of molecular conformation on the enzymatic hydrolysis efficiency of starch by α-amylase. And the interactions between starch molecules with different conformations and α-amylase were analysed by using molecule dynamics simulation and molecular docking. It was found, the natural conformational starch molecule was hydrolysed from the middle of the starch chain by α-amylase, producing polysaccharides. The bent PS-conformational starch molecules with multiple O2-O3 intramolecular hydrogen bonds produced by high-pressure was hydrolysed from the head of the starch chain to produce glucose, which is not conducive to RS formation. The stretched H-conformation without intramolecular hydrogen bonds produced by heat treatment was not hydrolysed by α-amylase. However, it occupied the active groove and formed strong interactions with α-amylase, which prevented other starch molecules from binding to α-amylase, thus reducing hydrolysis efficiency. Moreover, the total interaction energies between the three starch molecules and α-amylase were approximately 78 kJ/mol. And several hydrogen bonds were formed between the starch molecules and α-amylase, which provides evidence for the continuous sliding hydrolysis hypothesis of α-amylase. Moreover, these results provide an important reference for elucidating the mechanism of RS formation.