Enzyme Recycling Research Articles

Protein digestion and amino acid absorption mechanisms along the midgut of Musca domestica larvae

A model of protein digestion and peptide and amino acid absorption along the midgut of Musca domestica larvae was proposed and supported by RNA-Seq analyses, protein bioinformatics, microvillar-membrane-enriched midgut proteomics, and enzymatic activities. Peptidase genes are highly expressed in the posterior midgut (PM), whereas those for cathepsins have expression limited to the middle midgut (MM). MM has the lowest levels of gene expression of almost all peptidases but has high expression of genes for membrane-bound serine endopeptidases. The anterior midgut (AM) has intermediate expression values of serine endopeptidase and aminopeptidase (AP) genes and low expression of carboxypeptidases (CPs). Gene expression and peptidase activities were usually consistent for putative intracellular and membrane-bound enzymes. However, secreted peptidase gene expression and activities have divergent values, especially in the PM, which may be due to the countercurrent water flux causing enzyme recycling, thus decreasing their excretion. Data suggest that Trys and APs act in the AM. In the acidic MM, lysozymes kill microorganisms found in the diet, releasing proteins digested by cathepsins D, which may also digest Trys coming from the AM. Finally, highly active serine endopeptidases, CPs, dipeptidases, and APs complete protein digestion in PM. Absorption of peptides and amino acids coupled to protons may occur along the midgut, especially in PM, as occurs for facilitated amino acid transport. Absorption with sodium ions is probably restricted to AM and PM. Our findings provide valuable insights into the protein digestion and amino acid absorption mechanism in M. domestica larvae.

The FAM114A proteins are adaptors for the recycling of Golgi enzymes.

Golgi-resident enzymes remain in place while their substrates flow through from the endoplasmic reticulum to elsewhere in the cell. COPI-coated vesicles bud from the Golgi to recycle Golgi residents to earlier cisternae. Different enzymes are present in different parts of the stack, and one COPI adaptor protein, GOLPH3, acts to recruit enzymes into vesicles in part of the stack. Here, we used proximity biotinylation to identify further components of intra-Golgi vesicles and found FAM114A2, a cytosolic protein. Affinity chromatography with FAM114A2, and its paralogue FAM114A1, showed that they bind to Golgi-resident membrane proteins, with membrane-proximal basic residues in the cytoplasmic tail being sufficient for the interaction. Deletion of both proteins from U2OS cells did not cause substantial defects in Golgi function. However, a Drosophila orthologue of these proteins (CG9590/FAM114A) is also localised to the Golgi and binds directly to COPI. Drosophila mutants lacking FAM114A have defects in glycosylation of glue proteins in the salivary gland. Thus, the FAM114A proteins bind Golgi enzymes and are candidate adaptors to contribute specificity to COPI vesicle recycling in the Golgi stack.

Two‐Step Biocatalytic Synthesis of (1S)‐Nor(pseudo)ephedrine Catalyzed by Immobilized Enzymes

AbstractNor(pseudo)ephedrines [N(P)Es] are naturally occurring compounds showing sympathomimetic activity that have many applications especially in the chemical industry and in the pharmaceutical field. Recently a two‐step biocatalytic cascade for the preparation of (1S)‐N(P)E was designed, consisting of a benzoin‐type condensation catalyzed by the (S)‐selective acetoin:dichlorophenolindophenol oxidoreductase (Ao : DCPIP OR) followed by a transamination mediated by either a (S)‐ or (R)‐selective amine transaminase (ATA). In this study, we successfully immobilized both Ao : DCPIP OR and two ATAs of opposite enantioselectivity and used them in the biosynthetic cascade to N(P)Es. Immobilization yield, activity recovery, and stability of the immobilized enzymes, both after use (enzyme recycling) and storage (shelf‐life), were assessed. Ao : DCPIP OR immobilized on glyoxyl‐agarose exhibited remarkable stability under storage conditions throughout the 30‐days monitoring period. Moreover, it was successfully reused in the synthesis of (S)‐phenylacetylcarbinol [(S)‐PAC] yielding high conversion (>94 %) and enantiomeric excess (>99 %). The (R)‐selective At‐ATA from Aspergillus terreus, immobilized on Eupergit® C, demonstrated a slightly higher storage stability when compared to the (S)‐selective Sbv333‐ATA from Streptomyces sp. Bv333 immobilized on the same carrier. Remarkably, both enzymes were effectively reused for ten reaction cycles in the synthesis of N(P)Es, starting from the enzymatically synthesized (S)‐PAC, achieving complete conversions and excellent diastereoselectivity (>99 %).

Key Takeaways on the Cost-Effective Production of Cellulosic Sugars at Large Scale

The production of cellulosic sugars in lignocellulose biorefinery presents significant economic and environmental challenges due to the recalcitrant nature of biomass. The economic and facile production of renewable sugars with high yield and productivity is pivotal for the success of biorefinery. The cellulosic sugars are valorized either by biochemical routes or chemical routes or by hybrid (biological and chemical) routes into renewable chemicals, fuels, and materials. This manuscript focuses on the critical parameters affecting the economic viability of cellulosic sugar production at large scale, including biomass-specific pretreatment strategies and enzyme cost efficiency. High pretreatment costs, carbohydrate loss, and inhibitors production during pretreatment are identified as major contributors to overall production costs. To address these issues, we highlight the importance of developing cost-effective and efficient pretreatment methods tailored to specific biomass types and strategies for enzyme reuse and recycling. Future research should focus on innovations in pretreatment technologies, improved logistics for high-density feedstocks, biomass feeding systems, and advancements in enzyme technology to enhance the economic and environmental sustainability of lignocellulosic biorefineries. The findings highlight the need for continued innovation and optimization to make the commercial-scale production of cellulosic sugars more viable and sustainable.

Transport of enzymes by lymph flow digestive glands

The exosecretes of the digestive glands contain two pools of hydrolytic enzymes - newly formed in the secretory cycle of glandulocytes and recreted from blood flowing to the glands. The second pool of enzymes is repeated many times due to the incretor transport and resorption of hydrolases and zymogens from the secretions of the ductal system of the glands and the chyme of the gastrointestinal tract into the composition of the interstitial fluid and the lymph formed by it. These processes ensure the recycling of digestive gland enzymes, reusable delivery of hydrolases and their inclusion in the composition of exosecretes. The key role in providing enzyme digestion is played by the transport of resorbed and increted enzymes by the lymph flow. This is the digestive role of lymph and lymph flow.

Immobilized enzyme cascade for targeted glycosylation

Glycosylation is a critical post-translational protein modification that affects folding, half-life and functionality. Glycosylation is a non-templated and heterogeneous process because of the promiscuity of the enzymes involved. We describe a platform for sequential glycosylation reactions for tailored sugar structures (SUGAR-TARGET) that allows bespoke, controlled N-linked glycosylation in vitro enabled by immobilized enzymes produced with a one-step immobilization/purification method. We reconstruct a reaction cascade mimicking a glycosylation pathway where promiscuity naturally exists to humanize a range of proteins derived from different cellular systems, yielding near-homogeneous glycoforms. Immobilized β-1,4-galactosyltransferase is used to enhance the galactosylation profile of three IgGs, yielding 80.2–96.3% terminal galactosylation. Enzyme recycling is demonstrated for a reaction time greater than 80 h. The platform is easy to implement, modular and reusable and can therefore produce homogeneous glycan structures derived from various hosts for functional and clinical evaluation.

Intelligent chemiluminescent system for wash-free and ultrasensitive assay of aflatoxin B1 in coix seed and decoction pieces

Aflatoxin B1 (AFB1) is one of the most harmful mycotoxins. Convenient and reliable determination of trace AFB1 is highly desired. Herein, dual hemin labeled DNA hairpin was designed as a chemiluminescence (CL) switch and combined with aptamer of AFB1 and its partial complementary strand for homogenous assay of AFB1. Due to the judicious design of three DNA sequences and the help of nicking enzyme, the AFB1 recognition, signal transduction and recycling amplification can be implemented during one-step reaction. The whole protocol only involved the simple mixing of samples with homogeneous solution, avoiding the preparation of sophisticated solid-phase sensor and eliminating the multi-step washing and resulting errors, which provided a unique tool for on-site rapid testing. Importantly, when the operation process was simplified, the detection sensitivity can be enhanced facilely due to the enzyme recycling. Under optimal conditions, the intelligent CL system for sensing AFB1 exhibited a wide dynamic range (1.0 × 10-2 ∼ 100 ng mL−1) with a low detection limit (down to 1.5 pg mL−1), suitable for detecting trace AFB1 in complex matrix. The methodology was validated in coix seed and decoction pieces. The superiority including free of washing and separation, high sensitivity, acceptable accuracy, and greenness, showed good applicability of the proposed CL system in safety evaluation of food and medicine.

Direct purification and immobilization of his-tagged enzymes using unmodified nickel ferrite NiFe2O4 magnetic nanoparticles

Purification of valuable engineered proteins and enzymes can be laborious, costly, and generating large amount of chemical waste. Whilst enzyme immobilization can enhance recycling and reuse of enzymes, conventional methods for immobilizing engineered enzymes from purified samples are also inefficient with multiple-step protocols, regarding both the carrier preparation and enzyme binding. Nickel ferrite magnetic nanoparticles (NiFe2O4 MNPs) offer distinct advantages in both purification and immobilization of enzymes. In this work, we demonstrate the preparation of NiFe2O4 MNPs via a one-step solvothermal synthesis and their use in direct enzyme binding from cell lysates. These NiFe2O4 MNPs have showed an average diameter of 8.9 ± 1.7 nm from TEM analysis and a magnetization at saturation (Ms) value of 53.0 emu g–1 from SQUID measurement. The nickel binding sites of the MNP surface allow direct binding of three his-tagged enzymes, d-phenylglycine aminotransferase (d-PhgAT), Halomonas elongata ω-transaminase (HeωT), and glucose dehydrogenase from Bacillus subtilis (BsGDH). It was found that the enzymatic activities of all immobilized samples directly prepared from cell lysates were comparable to those prepared from the conventional immobilization method using purified enzymes. Remarkably, d-PhgAT supported on NiFe2O4 MNPs also showed similar activity to the purified free enzyme. By comparing on both carrier preparation and enzyme immobilization protocols, use of NiFe2O4 MNPs for direct enzyme immobilization from cell lysate can significantly reduce the number of steps, time, and use of chemicals. Therefore, NiFe2O4 MNPs can offer considerable advantages for use in both enzyme immobilization and protein purification in pharmaceutical and other chemical industries.

Label-free and Colorimetric Sensitive Detection of SNPs Based on Catalytic Beacon and RNase Cleavage Reaction

Background: Single nucleotide polymorphisms (SNPs) are important hallmarks in various pathological activities, especially genetic and inherited diseases, and detecting them with accuracy, high throughput and low cost becomes increasingly necessary. Methods: Herein, we have developed a new label-free and sensitive detection method for SNPs assay. Due to its favorable traits, the method presents an excellent performance. Briefly, the peroxidase- mimicking catalytic activity of G-quadruplex-hemin DNAzymes ensures label-free and colorimetric SNPs detection. At the same time, the RNA enzyme of the specific cleavage action can easily achieve the recycling of RNA enzyme and signal amplification. Results: In this study, the P-hemin DNAzyme with target DNA could catalyze the H2O2-mediated oxidation of ABTS to cause an observed color change compared to mutant DNA. The sensitivity and detection range of the DNA biosensor was achieved through the signal amplification program of special binding and cleavage of RNase H. A linear dependence of the absorbance at 420 nm on the concentrations between 0.5 and 50 nM was obtained (R2=0.965), and the detection limit was 8.76 nM. Conclusion: A new strategy for signal amplification process based on RNase cleavage reaction and Catalytic Beacon was constructed. Collectively, the developed SNPs assay might be extended to a broad range of clinical early diagnosis and treatment of genetic diseases.

Histochemistry and transcriptomics of mucins and peritrophic membrane (PM) proteins along the midgut of a beetle with incomplete PM and their complementary function

The midgut of Zabrotes subfasciatus (Coleoptera) and other insects may have regions lacking a peritrophic membrane (matrix, PM) and covered with a jelly-like material known as peritrophic gel. This work was undertaken to test the hypothesis that the peritrophic gel is a vertebrate-like mucus. By histochemistry we identified mucins along the whole midgut, which contrasts with the known occurrence of PM only at the posterior midgut. We also analyzed the expression of the genes coding for mucus-forming mucins (Mf-mucins), peritrophins, chitin synthases and chitin deacetylases along the midgut and carcass (insect without midgut) by RNA-seq. Mf-mucins were identified as proteins with high O-glycosylation and multiple tandem repeats of Pro/Thr/Ser residues. Peritrophins were separated into PM proteins, cuticular proteins analogous to peritrophins (CPAPs) and ubiquitous-chitin-binding domain-(CBD)-containing proteins (UCBPs). PM proteins have at least 3, CPAP one or 3, and UCBPs have a varied number of CBDs. PM proteins are more expressed at midgut, CPAP at the carcass, and UCBP at both. The results showed that most PM proteins are mainly expressed at the posterior midgut, together with midgut chitin synthase and chitin deacetylase, and in agreement with the presence of PM only at the posterior midgut by visual inspection. The excretion of most midgut chitinase is avoided, suggesting that the shortened PM is functional. Mf-mucins are expressed along the whole midgut, probably forming the extracellular mucus layer observed by histochemistry. Thus, the lack of PM at anterior and middle midgut causes the exposure of a mucus, which may correspond to the previously described peritrophic gel. The putative functional interplay of mucus and PM is discussed. The major role of mucus is proposed to be tissue protection and of PM to enhancing digestive efficiency by allowing enzyme recycling.

Immobilization of Cellulase Enzymes on Single-Walled Carbon Nanotubes for Recycling of Enzymes and Better Yield of Bioethanol Using Computer Simulations.

The utilization of microbial cellulase enzymes for transforming plant biomass into biofuel or bioethanol, which can serve as a substitute for fossil fuel, is a subject of growing interest. Nonetheless, large-scale production of biofuel using cellulases is not economically feasible as the extraction of these enzymes from diverse microorganisms is an expensive process. To address this issue, immobilizing the enzyme to a substrate material, e.g., carbon nanotubes (CNTs), to recycle without a significant decline in its catalytic activity is a promising solution. Due to the hydrophobic nature of CNTs, we employed molecular docking and network analysis methodologies to identify potential CNT-binding sites on the outer surface of a wild-type cellulase enzyme, CelS. Classical molecular dynamics simulations of CNT-bound CelS through one of the selected binding sites resulted in negligible changes in the secondary structure of the enzyme and its catalytic domain, implying the least possible effect on the catalytic activity post-immobilization. Furthermore, our study reveals that while the unfolding near the CNT-binding region in CelS is more pronounced when the enzyme is interacting with a wider CNT, resulting in enhanced contact area and improved binding affinity, its impact on the overall CelS structure is relatively less significant when compared to thinner CNTs. Particularly, CNTs of diameter ∼12 Å can serve as a favorable option for substrate materials in cellulase immobilization. Our study also provides critical insights into the binding mechanisms between cellulase and CNTs, which could lead to the development of more efficient biocatalysts for biofuel production.

Economic analysis of enzyme recycling during enzymatic hydrolysis of sugar beets for soluble sugars production

AbstractEnzymatic hydrolysis of sugar beets is an efficient alternative to conventional hot water extraction where overall soluble extraction is valued. The economic analysis of a sugar beet soluble sugar production plant was designed and developed using SuperPro Designer software. The commercial scale operation was sized to process 100 million metric ton (MT)/day of sugar beets. The base case process includes transportation, grinding, thermal pretreatment, enzymatic hydrolysis, solid–liquid separation, and evaporation. The final product from the model was assumed to be a sugar syrup stream at 65% total solids content. Since the breakeven selling price was sensitive to the cost of enzymes, alternative scenario A added an ultrafiltration unit operation to separate the enzymes and reuse them in subsequent batches of hydrolysis. Steam was another major contributor to the operating cost. Alternative scenario B included a natural gas boiler to generate steam from biogas produced from a co‐located dairy manure digester. The breakeven selling price of the sugar syrup from the base case, alternative scenario A, and alternative scenario B were $965, $911, and $955 per metric ton, respectively. Sensitivity analysis showed that the breakeven selling price is sensitive to the raw material cost, especially for the pectic enzymes.

IR780-doped cobalt ferrite nanoparticles@poly(ethylene glycol) microgels as dual-enzyme immobilized micro-systems: Preparations, photothermal-responsive dual-enzyme release, and highly efficient recycling

To solve the problems of separating dual enzymes from the carriers of dual-enzyme immobilized micro-systems and greatly increase the carriers’ recycling times, photothermal-responsive micro-systems of IR780-doped cobalt ferrite nanoparticles@poly(ethylene glycol) microgels (CFNPs-IR780@MGs) are prepared. A novel two-step recycling strategy is proposed based on the CFNPs-IR780@MGs. First, the dual enzymes and the carriers are separated from the reaction system as a whole via magnetic separation. Second, the dual enzymes and the carriers are separated through photothermal-responsive dual-enzyme release so that the carriers can be reused. Results show that CFNPs-IR780@MGs is 281.4 ± 9.6 nm with a shell of 58.2 nm, and the low critical solution temperature is 42 °C, and the photothermal conversion efficiency increases from 14.04% to 58.41% by doping 1.6% of IR780 into the CFNPs-IR780 clusters. The dual-enzyme immobilized micro-systems and the carriers are recycled 12 and 72 times, respectively, and the enzyme activity remains above 70%. The micro-systems can realize whole recycling of the dual enzymes and carriers and further recycling of the carriers, thus providing a simple and convenient recycling method for dual-enzyme immobilized micro-systems. The findings reveal the micro-systems’ important application potential in biological detection and industrial production.

Application of Plant Proteases in Meat Tenderization: Recent Trends and Future Prospects.

Papain, bromelain, and ficin are commonly used plant proteases used for meat tenderization. Other plant proteases explored for meat tenderization are actinidin, zingibain, and cucumin. The application of plant crude extracts or powders containing higher levels of compounds exerting tenderizing effects is also gaining popularity due to lower cost, improved sensory attributes of meat, and the presence of bioactive compounds exerting additional benefits in addition to tenderization, such as antioxidants and antimicrobial effects. The uncontrolled plant protease action could cause excessive tenderization (mushy texture) and poor quality due to an indiscriminate breakdown of proteins. The higher cost of separation and the purification of enzymes, unstable structure, and poor stability of these enzymes due to autolysis are some major challenges faced by the food industry. The meat industry is targeting the recycling of enzymes and improving their stability and shelf-life by immobilization, encapsulation, protein engineering, medium engineering, and stabilization during tenderization. The present review critically analyzed recent trends and the prospects of the application of plant proteases in meat tenderization.

Lactic Acid: A Comprehensive Review of Production to Purification

Lactic acid (LA) has broad applications in the food, chemical, pharmaceutical, and cosmetics industries. LA production demand rises due to the increasing demand for polylactic acid since LA is a precursor for polylactic acid production. Fermentative LA production using renewable resources, such as lignocellulosic materials, reduces greenhouse gas emissions and offers a cheaper alternative feedstock than refined sugars. Suitable pretreatment methods must be selected to minimize LA cost production, as the successful hydrolysis of lignocellulose results in sugar-rich feedstocks for fermentation. This review broadly focused on fermentative LA production from lignocellulose. Aspects discussed include (i). low-cost materials for fermentative LA production, (ii). pretreatment methods, (iii). enzymatic hydrolysis of cellulose and hemicellulose, (iv). lactic acid-producing microorganisms, including fungi, bacteria, genetically modified microorganisms, and their fermentative pathways, and (v). fermentation modes and methods. Industrial fermentative lactic acid production and purification, difficulties in using lignocellulose in fermentative LA production, and possible strategies to circumvent the challenges were discussed. A promising option for the industrial production and purification of LA that contains enzyme and cell recycling continuous simultaneous saccharification and fermentation coupled with membrane-based separation was proposed. This proposed system can eliminate substrate-, feedback-, and end-product inhibition, thereby increasing LA concentration, productivity, and yield.

Self-sufficient biocatalysts constructed using chitin-based microspheres

The concomitant recycling of enzymes and co-factors is a big challenge in biocatalysis. In this study, co-immobilization of the enzyme and the co-factor pyridoxal 5′-phosphate was achieved using chitin-based microspheres as the support. Chitin/chitosan composite microspheres were prepared and then modified with polyethyleneimine. Three pyridoxal 5′-phosphate-independent enzymes were used to demonstrate that the self-sufficient biocatalysts based on chitin/chitosan composite microspheres and polyethyleneimine modified microspheres displayed good generalizability for high recycling efficiency of both enzyme and pyridoxal 5′-phosphate. Furthermore, the self-sufficient biocatalysts were applied in the continuous flow production of cadaverine and maintained good continuous catalysis without the exogenous addition of pyridoxal 5′-phosphate, suggesting that this co-immobilization platform shows promise for application in industrial biocatalysis.

Development of an Integrated System for Highly Selective Photoenzymatic Synthesis of Formic Acid from CO2.

Herein, a Zr-based dual-ligand MOFs with pre-installed Rh complex was employed for NADH regeneration in situ and also used for immobilization of formic acid dehydrogenase (FDH) in order to realize a highly efficient CO2 fixation system. Then, based on the detailed investigations into the photochemical and electrochemical properties, it is demonstrated that the introduction of the photosensitive meso-tetra(4-carboxyphenyl) porphin (TCPP) ligands increased the catalytic active sites and improved photoelectric properties. Furthermore, the electron mediator Rh complex, anchored on the zirconium-based dual-ligand MOFs, enhanced the efficiency of electron transfer efficiency and facilitated the separation of photogenerated electrons and holes. Compared with UiO-66-NH2 , Rh-H2 TCPP-UiO-66-NH2 exhibits an optimized valence band structure and significantly improved photocatalytic activity for NAD+ reduction, resulting the synthesis of formic acid from CO2 increased from 150 μg mL-1 (UiO-66-NH2 ) to 254 μg mL-1 (Rh-H2 TCPP-UiO-66-NH2 ). Moreover, the assembled photocatalyst-enzyme coupled system also allows facile recycling of expensive electron mediator, enzyme, and photocatalyst.

Laccase–copper phosphate hybrid nanoflower as potent thiazole remediation agent

The increase in the global population in recent years has caused a fast increase in pollution problems worldwide. One of these problems is emerging pollutants, which are a class of human-made organic compounds that have been detected in various water bodies. To develop a recyclable, reusable, and efficient material for the degradation of these pollutants, we report the synthesis, characterization and application of hybrid nanoflowers imbedded with laccase enzymes. These copper phosphate-laccase nanomaterials had a spherical flower-like shape, a surface area of 53.46 m2/g, and a mean pore size of 10.58 nm. When tested against a panel of diverse compounds, the hybrid nanoflowers were able to degrade a number of emerging pollutants, including 2-mercaptobenzothiazole (MBT), caffeic acid, thiabendazole, cimetidine, and sulfamethoxazole, with efficiencies of 97 %, 97.0 %, 78.1 %, 72.3 %, and 51.3 %, respectively. For 2-mercaptobenzothiazole (MBT), we identified the intermediate produced during the degradation process. In addition, the nanoflowers were able to degrade >90 % MBT in a spiked ground water sample. Furthermore, we tested the recyclability and storage stability of the hNFs for the degradation of MBT and found that they could be reused for five cycles and stored for 21 days at 4 °C. Our results suggest that laccase-embedded hybrid nanoflowers to be potent remediation agents for the degradation of thiazole as well as other emerging pollutants. Also, the successful immobilization of laccase on hybrid nanoflowers shown in the present study can allow for the efficient recycling of laccase enzymes for multiple degradation cycles leading to potential scaling up and building of a bioreactor.

Surface display of (R)-carbonyl reductase on Escherichia coli as biocatalyst for recycling biotransformation of 2-hydroxyacetophenone

The purified (R)-Carbonyl reductase (RCR) is widely applied in the synthesis of chiral compounds, but often involves tedious procedures and high production costs of protein separation and purification. A surface immobilization of biocatalyst on cell surface for biotransformation is an option for ease enzyme separation and recycling. In this study, the RCR from Candida parapsilosis was immobilized on the surface of E. coli using the Lpp–OmpA and EhaA anchors, showing that the Lpp–OmpA is better for displaying the target molecules in terms of enzyme activities, product formation, and display efficiency. By deleting the NAD+ degrading enzyme UshA, the whole-cell biocatalytic activity was two-fold higher than that of the original strain. The created biocatalyst can retain about 80% of its catalytic activity after four rounds of recycling, reached the conversion rate of 83.5% within 30 mins with the substrate (R)-PED concentration of 1 mM. This study provides a novel strategy for 2-hydroxy acetophenone synthesis using the surface display of the enzyme on the cell while avoiding enzyme purification and side reactions from the host.

Process strategies to reduce cellulase enzyme loading for renewable sugar production in biorefineries

Second-generation (2G) sugar is the principal building block in biorefineries producing sustainable fuels, green chemicals, and materials. Its production involves a pre-treatment strategy, which can remove either hemicellulose or lignin, followed by a cellulase cocktail-mediated hydrolysis of pre-treated biomass applying numerous modifications. The high production cost of sugar from biomass using high-solid loadings with the desired purity is the important limiting factor for their economic conversion into bio-renewables. Pre-treatment and cellulase-mediated enzymatic hydrolysis of recalcitrant biomass are among the major technical and economic impediments to the overall success of biorefineries. Critical parameters such as the high amount of cellulase required for biomass hydrolysis, high cellulase cost, unproductive binding of cellulase to lignin, inhibitive reactions, and lower substrate applicability directly influence the recovery of sugar at a competitive price. Understanding the interaction of the relationship of the enzymes and substrates with the interactions of the reducing cellulase amount and effective hydrolysis rate should be positioned for the recovery of 2G sugar at a competitive price. Thus, effective strategies are needed to minimize cellulase dosage to overcome the cost of the enzyme. Further, on-site enzyme production, the addition of pre-treated biomass in fed-batch mode, and the washing of pre-treated biomass can play a pivotal role in the economic production of 2G sugar. This review provides a comprehensive analysis of the specific strategies such as applying extraneous proteins and additives, enzyme recycling, and effect of washing pre-treated biomass on 2G sugar production at a large scales in lignocellulose biorefineries.