Enzymatic Hydrolysis Production Research Articles

Effect of ultrasonic pretreatment on antifreeze activity of gluten antifreeze polypeptide

In this study, the effects of thermal hysteresis activity, ice recrystallization inhibition, amino acid composition and surface hydrophobicity of different ultrasonic pretreatments on gluten antifreeze polypeptide was studied.The results of the thermal hysteresis activity showed that the collaborative (flat-panel combined probe) ultrasonic working mode was superior to the single-frequence ultrasound mode. In the optimal working mode (15 min, 100 W/L), the results of polarized light microscope and gel chromatography column showed that ultrasonic pretreatment could effectively inhibit the growth of ice crystals, reduced the size of ice crystals, and to a certain extent, transfer the molecular weight distribution of peptides to small molecular peptides. The molecular weight was mainly concentrated at about 200–1000 u. The contents of Valine, Methionine, Leucine, Proline and Glutamic acid residues in the gluten antifreeze polypeptide pretreated by ultrasonic wave were relatively high and more hydrophobic groups were exposed. The secondary structure results showed that the molecular conformation of the polypeptide was mainly α-helix and β-sheet conformations.This paper systematically revealed the intrinsic correlation mechanism between the changes of key domains of substrate proteins and the antifreeze activity of enzymatic hydrolysis products assisted by ultrasoud, providing a theoretical basis for the preparation of antifreeze polypeptide.

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

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

New Derivatives of Modified Starch for Food Technology.

The food industry extensively uses chemically modified starches and their hydrolysates, which is mainly due to their emulsification ability. Therefore, it becomes inevitable to develop new starch derivatives, including modified starch hydrolysates, and effective preparation methods to meet the increasing demands of producers, consumers, and technology. This study comprehensively researches the physical, chemical, and functional properties (such as the water-binding capacity, swelling power, solubility, and fat absorption capacity) of chemically modified biopolymers and their enzymatic hydrolysis products. We utilized oxidized and acetylated potato and waxy-corn starches with varying degrees of substitution by carboxyl and acetyl groups in our research. The process of enzymatic hydrolysis was performed in a recirculated membrane reactor (CRMR). Our findings indicated that the physicochemical properties of starch derivatives and their hydrolysates depended on the biological origin of the biopolymer and the type and degree of modification. However, the presence of carboxyl groups in the modified starch molecules is critical and affects the rheological properties and water-binding capacity of the starch preparations. For example, in the case of waxy-corn starch preparations with a lower content of carboxyl groups (i.e., derivatives with a low degree of oxidation), the water-binding capacity (WBC) increases when compared to native starch. The highest WBC value of 206.3% was noted for the doubly modified waxy-corn starch with an oxidation degree of 0.2% and an acetylation degree of 2.5%, while native waxy-corn starch shows a WBC of 161.4%. In contrast, it was observed that preparations with a higher content of carboxyl groups, i.e., derivatives with an oxidation degree of 2.5%, show a lower swelling power compared to native waxy starch.

Microbial Protein Production Using Lignocellulosic Biomass (Switchgrass) and Klebsiella oxytoca M5A1—A Nitrogen Fixer

The expanding global population has increased the demand for sustainable protein sources, and microbial protein (MP) has emerged as a promising alternative. However, conventional carbon (glucose) and nitrogen (ammonia, urea) sources needed for MP production pose environmental and economic issues. This study aims to produce protein using lignocellulosic biomass (LCB) as a carbon source and the nitrogen fixation ability of Klebsiella oxytoca M5A1 as a nitrogen source. The study investigates the pretreatment of LCB (switchgrass), enzymatic hydrolysis, protein quantification, nitrogen fixation, glucose utilization and organic acids production. K. oxytoca M5A1 harnessed free nitrogen from the atmosphere and used abundant, cheap glucose from LCB to produce MP and organic acids as by-products. Protein production occurred in two phases: first within the initial 8 h and secondly, within the last 16 h. The highest protein concentration was at 40 h, with approximately 683.46 µg/mL protein content. High-performance liquid chromatography system (HPLC) analysis revealed a dynamic profile of glucose utilization and organic acids (Lactic acid, Propionic acid, Acetic acid, and Succinic acid) production. K. oxytoca M5A1 exhibited an early high rate of glucose consumption, and conversion to organic acids, that were later used for second-phase protein production. The acids profile revealed intra-conversion from one acid to another via metabolic pathways (glycolysis and tricarboxylic acid cycle). Overall, leveraging LCB and the nitrogen-fixing ability of K. oxytoca M5A1 for MP production offers an eco-friendly and cost-effective alternative to traditional protein sources, contributing to a sustainable circular economy.

A dual-signal triple-readout optical sensing platform for α-glucosidase and β-glucosidase activity monitoring and inhibitor screening based on luminescent covalent organic framework

BackgroundAs promising biomarkers of diabetes, α-glucosidase (α-Glu) and β-glucosidase (β-Glu) play a crucial role in the diagnosis and management of diseases. However, there is a scarcity of techniques available for simultaneously and sensitively detecting both enzymes. What's more, most of the approaches for detecting α-Glu and β-Glu rely on a single-mode readout, which can be affected by multiple factors leading to inaccurate results. Hence, the simultaneous detection of the activity levels of both enzymes in a single sample utilizing multiple-readout sensing approaches is highly attractive. ResultsIn this work, we constructed a facile sensing platform for the simultaneous determination of α-Glu and β-Glu by utilizing a luminescent covalent organic framework (COF) as a fluorescent indicator. The enzymatic hydrolysis product common to both enzymes, p-nitrophenol (PNP), was found to affect the fluorometric signal through an inner filter effect on COF, enhance the colorimetric response by intensifying the absorption peak at 400 nm, and induce changes in RGB values when analyzed using a smartphone-based color recognition application. By combining fluorometric/colorimetric measurements with smartphone-assisted RGB mode, we achieved sensitive and accurate quantification of α-Glu and β-Glu. The limits of detection for α-Glu were determined to be 0.8, 1.22, and 1.85 U/L, respectively. Similarly, the limits of detection for β-Glu were 0.16, 0.42, and 0.53 U/L, respectively. SignificanceApplication of the proposed sensing platform to clinical serum samples revealed significant differences in the two enzymes between healthy people and diabetic patients. Additionally, the proposed sensing method was successfully applied for the screening of α-Glu inhibitors and β-Glu inhibitors, demonstrating its viability and prospective applications in the clinical management of diabetes as well as the discovery of antidiabetic medications.

A novel tyrosinase inhibitory peptide obtained from Sipunculus nudus gelatin hydrolysate: Preparation, identification, and action mechanism

Tyrosinase inhibitory peptides (TIPs) derived from food resources have attracted immense attention in the food, cosmetic, and pharmaceutical industries owing to their excellent biological safety and ease of absorption. In this study, single-factor experiment and response surface optimization were used to prepare the Sipunculus nudus gelatin enzymatic hydrolysis (SNGH) product. Subsequently, a novel tyrosinase inhibitory peptide was screened out through ultrafiltration classification, biological activity prediction, molecular docking, and molecular dynamics simulation, and its inhibitory mechanism was analyzed. The results revealed that the optimal enzymatic hydrolysis process was: the enzyme addition was 4768.87 U/g, the enzymatic hydrolysis time was 4.72 h, and the enzymatic hydrolysis temperature was 42.76 °C. A total of 287 peptides were detected in the ultrafiltration component Ⅰ (SNGH-Ⅰ) with a high tyrosinase inhibitory activity, and 161 peptides with binding energies < −7.0 kcal/mol were screened out through molecular docking. Subsequently, 91 peptides with a biological activity score >0.3 were obtained after biological activity screening. Finally, 5 possible TIPs were screened out according to their hydrophobicity, peptide score, and molecular weight. Among them, IIAPPERKY, VWDESFKVF, and FAGDDAPRAVFPS exhibited a superior tyrosinase inhibitory activity. Molecular dynamics simulations revealed that IIAPPERKY had better conformational stability and belonged to the group of reversible competitive inhibitors.

Triple Sensing Modes for Triggered β-Galactosidase Activity Assays Based on Kaempferol-Deduced Silicon Nanoparticles and Biological Imaging of MCF-7 Breast Cancer Cells.

β-Galactosidase (β-Gala) is an essential biomarker enzyme for early detection of breast tumors and cellular senescence. Creating an accurate way to monitor β-Gala activity is critical for biological research and early cancer detection. This work used fluorometric, colorimetric, and paper-based color sensing approaches to determine β-Gala activity effectively. Via the sensing performance, the catalytic activity of β-Gala resulted in silicon nanoparticles (SiNPs), fluorescent indicators obtained via a one-pot hydrothermal process. As a standard enzymatic hydrolysis product of the substrate, kaempferol 3-O-β-d-galactopyranoside (KOβDG) caused the fluorometric signal to be attenuated on kaempferol-silicon nanoparticles (K-SiNPs). The sensing methods demonstrated a satisfactory linear response in sensing β-Gala and a low detection limit. The findings showed the low limit of detection (LOD) as 0.00057 and 0.098 U/mL for fluorometric and colorimetric, respectively. The designed probe was then used to evaluate the catalytic activity of β-Gala in yogurt and human serum, with recoveries ranging from 98.33 to 107.9%. The designed sensing approach was also applied to biological sample analysis. In contrast, breast cancer cells (MCF-7) were used as a model to test the in vitro toxicity and molecular fluorescence imaging potential of K-SiNPs. Hence, our fluorescent K-SiNPs can be used in the clinic to diagnose breast cellular carcinoma, since they can accurately measure the presence of invasive ductal carcinoma in serologic tests.

Analysis of Aroma Components of Enzymatic Hydrolysis Products of Oak Lignin from Different Producing Areas

The morphology and structure of lignin in French oak, American oak and Chinese Mongolian oak were observed and compared by scanning electron microscopy (SEM). The results showed that the surface of lignin after enzymatic hydrolysis was more irregular and the contact area was obviously increased. GC-MS detected 3 kinds of lignin, 4 kinds of components and 30 kinds of volatile substances. The lignin content of French oak was the highest (8911.5 ± 512.2 μg/L, 29 kinds), followed by that of American oak (8071.7 ±541.9 μg/L, 26 kinds), and that of Mongolian oak was the least (7903.3 ± 492.5 μg/L, 26 kinds). The results of aroma characteristics analysis, principal component analysis and heat map analysis showed that the aroma components of the three kinds of oak lignin were significantly different. French oak lignin could provide more volatile phenols, with obvious smoky, woody and sweet flavor. Mongolian oak lignin carries more phenolic and ester compounds, providing a relatively soft floral, fruity flavor. American oak lignin has a relatively average aroma substance content, which can bring strong and complex complex aroma characteristics.

Online prediction of the enzymatic hydrolysis efficiency of crop straw

The extent of removal of lignin and hemicellulose are crucial indicators for evaluating the efficiency of enzymatic hydrolysis of crop straw. Numerous factors influence these two indices. Establishing a quantitative model that correlates these factors with hydrolysis efficiency is essential, as it can guide efficient hydrolysis. In this study, a predictive method for enzymatic hydrolysis efficiency in crop straw was proposed using Grey relational analysis (GRA), Kernel principal component analysis (KPCA), and a least squares support vector machine (LSSVM). The authors collected a dataset from actual production data and developed an efficiency predictive model using GRA for variable selection, KPCA for dimensionality reduction, and LSSVM for model training. This model allows for the direct estimation of the final enzymatic hydrolysis efficiency based on production condition variables, which can include enzyme amount, temperatures, pH, time, agitation, and straw dimensions. Extensive experimental testing validated the effectiveness of the proposed method, resulting in minimal errors, a high degree of fit, and exceptional performance. The methodology described in this study can serve as a foundation for optimising the design of efficient enzymatic hydrolysis production processes for crop straw. Additionally, it offers valuable soft measurements to support efficient control of the enzymatic hydrolysis process.

A novel NiO/CeO2-mSiO2 nanocages with peroxidase-mimetic activity for ultra-sensitive biosensing

A novel NiO/CeO2−mSiO2 nanocages with superior peroxidase−like activities due to the synergetic effect between NiO and CeO2 were fabricated by a simple etching−precipitation method with core−shell structured Cu2O@mSiO2 as a sacrificial template. Based on the peroxidase−like activity of the NiO/CeO2−mSiO2 nanocages, an enzyme cascade−triggered colorimetric sensing platform was exploited for alkaline phosphatase (ALP) detection, owing to the fact that ascorbic acid (AA) that is the ALP enzymatic hydrolysis product of ascorbic acid 2−phosphate (AAP) can efficiently reduce the oxidation product of TMB (oxTMB) generated under the catalysis of the NiO/CeO2−mSiO2. For the sensing of ALP, a good linear response was observed in the range of 0.0005–1.25 mU mL−1 with the limit of detection (LOD) as low as 0.00048 mU mL−1. Furthermore, the proposed enzyme cascade−triggered colorimetric sensing platform could be further explored for sensing of tyrosinase, according to the fact that dopamine (DA) the catalytic product of tyramine (Tyr) by tyrosinase could initiate the redox reaction with the oxTMB acquired under the catalysis of the NiO/CeO2−mSiO2. A wide linear response was found for tyrosinase detection from 0.02 mU mL−1 to 3.4 U mL−1 with a LOD as low as 0.002 U mL−1. This work not only offers a facile and effective approach to fabricate nanozymes with excellent catalytic activity, but also provides a promising enzyme cascade−triggered colorimetric method for the ultra−sensitive detection of biological enzymes.

A reaction–diffusion coupled kinetic model for hydrothermal acid pretreatment of wheat straw

Hydrothermal acid pretreatment is a promising method to overcome the recalcitrance of lignocellulosic biomass for subsequent efficient enzymatic hydrolysis and biofuel production. However, little is known about heterogeneous catalysis reactions involving the proton diffusion in the pore of lignocellulosic biomass coupled with the hydrolysis of hemicellulose and cellulose catalyzed by proton. In this study, a reaction–diffusion coupled kinetic model for hydrothermal acid pretreatment of wheat straw was developed based on the visualization of particle geometry and pore features. The increase rate of average proton concentration in the pores is higher at a higher temperature or a smaller particle size. A mathematical model was successfully developed to describe the time courses of average proton concentration dictated by initial acid concentration and an unsteady diffusion fitting parameter. The kinetic parameters of each reaction step for hydrothermal acid pretreatment of wheat straw were obtained by fitting experimental data. The activation energies for the hydrolysis of slow-hydrolyzing cellulose, fast-hydrolyzing cellulose, and hemicellulose were determined of 937.8 kJ·mol−1, 90.7 kJ·mol−1, and 39.8 kJ·mol−1, respectively, indicating the temperature dependence of slow-hydrolyzing cellulose with a high crystalline index is strongest. The prediction accuracy of this model can be further improved at higher stirring speeds and initial acid concentrations. Additionally, the model demonstrates high prediction accuracy across three different particle sizes. Generally, this model can gratifyingly correlate data of formation and degradation of monosaccharides and furfural in the hydrothermal acid pretreatment of wheat straw with fair accuracy.

Catalytic action of hydronium-ion in rice straw pretreatment and enhancement of enzymatic hydrolysis and ethanol production

The present investigation will focus on the catalytic effect of hydronium ion during liquid-water/ dilute sulfuric acid/ferric chloride pretreatment (LWP/ SAP/ FCP) on the hydrolysate and pretreated rice straw. The overall pretreatment process has been represented as a sequential catalytic reactions. The temperature range of pretreatment is varied in the range of 140–180 °C and the concentration of dilute sulfuric acid and ferric chloride is 0.1 M. The first order rate constants of xylan and glucan degradation have been determined for LWP/ SAP/ FCP. The maximum xylan and glucan conversion of 96.8% w/wand 31.4%w/w respectively are achieved for FCP at 180 °C. Xylose, glucose, furfural, 5-hydroxymethyl furfural, acetic acid and formic acid appear as the main products for all pretreatments. Only in case of FCP levulinic acid also appears as a hydrolysis product. The thermal characteristics, functional groups, crystallinity, surface morphology of untreated and pretreated rice straw have been evaluated. Among all pretreatment processes under study, the highest values of glucose yield of 78.96% (w/w) using enzymatic hydrolysis and 83.79% of theoretical yield of ethanol using Saccharomyces cerevisiae have been achieved for ferric chloride pretreatment.

Application of Aromatic Ring Quaternary Ammonium and Phosphonium Salts–Carboxylic Acids-Based Deep Eutectic Solvent for Enhanced Sugarcane Bagasse Pretreatment, Enzymatic Hydrolysis, and Cellulosic Ethanol Production

Deep eutectic solvents (DESs) with a hydrophobic aromatic ring structure offer a promising pretreatment method for the selective delignification of lignocellulosic biomass, thereby enhancing enzymatic hydrolysis. Further investigation is needed to determine whether the increased presence of aromatic rings in hydrogen bond receptors leads to a more pronounced enhancement of lignin removal. In this study, six DES systems were prepared using lactic acid (LA)/acetic acid (AA)/levulinic acid (LEA) as hydrogen bond donors (HBD), along with two independent hydrogen bond acceptors (HBA) (benzyl triethyl ammonium chloride (TEBAC)/benzyl triphenyl phosphonium chloride (BPP)) to evaluate their ability to break down sugarcane bagasse (SCB). The pretreatment of the SCB (raw material) was carried out with the above DESs at 120 °C for 90 min with a solid–liquid ratio of 1:15. The results indicated that an increase in the number of aromatic rings may result in steric hindrance during DES pretreatment, potentially diminishing the efficacy of delignification. Notably, the use of the TEBAC:LA-based DES under mild operating conditions proved highly efficient in lignin removal, achieving 85.33 ± 0.52% for lignin removal and 98.67 ± 2.84% for cellulose recovery, respectively. The maximum digestibilities of glucan (56.85 ± 0.73%) and xylan (66.41 ± 3.06%) were attained after TEBAC:LA pretreatment. Furthermore, the maximum ethanol concentration and productivity attained from TEBAC:LA-based DES-pretreated SCB were 24.50 g/L and 0.68 g/(L·h), respectively. Finally, the comprehensive structural analyses of SCB, employing X-rays, FT-IR, and SEM techniques, provided valuable insights into the deconstruction process facilitated by different combinations of HBDs and HBAs within the DES pretreatment.

Proximate and compositional assessment of pretreatment methods on selected lignocellulose biomass for biogas production

&#x0D; The efficient conversion of lignocellulosic biomass into biogas is a critical avenue in sustainable bioenergy production. Pretreatment enhances the accessibility of lignocellulose components for subsequent enzymatic hydrolysis and biogas production. This study evaluated the proximate and compositional changes induced by various pretreatment techniques on lemon grass and fluted pumpkin stalk, with a focus on optimizing biogas yield. Alkaline, hydrothermal, and combined hydrothermal pretreatment processes were applied on the feedstocks. The biomass surface characteristics were determined through proximate and compositional analysis.The characterization of the untreated biomass showed that lemon grass and fluted pumpkin stalk recorded; dry matter, moisture content, fixed carbon, total kjeldahl nitrogen and ash content in the ranges of 94.49%-94.56%, 5.44%-5.51%, 26.35%-28.95%, 1.33%-1.932% and 6.00%-12.00% respectively. The cellulose values of the untreated biomass ranged from 34.04% for fluted pumpkin stalk to 38.66% for lemon grass. However, the content of the cellulose increased with pretreatment to values ranging from 44.20% to 51.06%. The results of this analysis showed that the selected lignocellulose biomass has properties that are consistent with past literature stating them as suitable substrates for biogas production. Nonetheless, the potential for increased biogas yield from anaerobic digestion is greater when combined hydrothermal and alkaline pretreatment is used.

Valorization of meat and bone residue by ultrasound and high voltage electrostatic field assisted two-stage enzymatic hydrolysis: Nutritional characteristics and flavor analysis

Enzymatic hydrolysis has been used to extract protein and bioactive compounds. Choosing appropriate enzymes can improve the hydrolysis of meat and bone proteins and enhance the flavor of the final product. In this study, the by-products of meat and bone residue after cooking in food processing plants were hydrolyzed by a two-stage method of complex protease (neutral protease, flavor protease and alkaline protease), and the effects of ultrasound (US) and high voltage electrostatic field (HVEF) pretreatment before enzymatic hydrolysis on the nutritional characteristics and flavor of the enzymolysis solution were investigated. The results showed that pretreatment by US alone and combined with US and HVEF could promote proteolysis and increase the content of free amino acids. HVEF pretreatment alone had no significant effect on protein hydrolysis. The enzymatic hydrolysate pretreated by US-HVEF had the strongest umami flavor, the highest flavor nucleotide content and the strongest antioxidant capacity. And electronic nose and GC-MS analysis showed that the combination of US-HVEF pretreatment could enhance the flavor characteristic intensity of enzymatic hydrolysate and increase the volatile compound content, but the compound types decreased slightly. Therefore, physical field pretreatment before enzymatic hydrolysis can help to produce high value-added enzymatic hydrolysis products.

Qualitative and quantitative analysis of the component variations of Raphani Semen during the stir-frying process and elucidation of transformation pathways of multiple components

Raphani Semen (RS) encompasses two distinct application forms in Chinese clinical practice: raw RS (RRS) and stir-fried RS (SRS). They exhibit divergent drug properties and effects, as described in traditional Chinese medicine theory known as “Sheng shu yi zhi, sheng sheng shu jiang”. The dissimilarity in RS's drug properties is intrinsically linked to alterations in its internal components during the stir-frying process. Previous studies have demonstrated that stir-frying renders myrosinase inactive, thereby preventing the enzymatic hydrolysis of glucosinolates in RS. However, the precise enzymatic hydrolysis pathway and products of glucosinolates remain unclear. Furthermore, it remains uncertain whether other components undergo changes influenced by endogenous enzymes. The objective of this study is to systematically analyze the chemical components disparities between RRS and SRS using high-performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-TOF-MS). Additionally, it seeks to elucidate the potential transformation pathways of multiple components from an enzymatic hydrolysis perspective. We have developed a sensitive and efficient high-performance liquid chromatography coupled with triple quadrupole tandem mass spectrometry (HPLC-QQQ-MS) method for quantifying the content of 5 characteristic components, including glucoraphenin, sinapine thiocyanate, sulforaphene, sinapic acid, and 3′,6-disinapoylsucrose. Based on retention time and MS spectra, we have identified 19 characteristic components in both SRS and RRS, encompassing glucosinolates and sulfur-containing derivatives, oligosaccharide esters, and small-molecule phenolic acids. Notably, 18 of these components undergo changes during the enzymatic hydrolysis process, leading to the identification of 4 transformation pathways: glucoraphenin, 6-sinapoylglucoraphenin, 3′,6-disinapoylsucrose and β-D-(3,4-disinapoyl) furanofructosyl-α-D-(6-sinapisoyl) glucoside, along with 3′-O-sinapoyl-6-O-feruloylsucrose. Quantitative analysis reveals significant differences, including lower levels of glucoraphenin in RRS compared to SRS, higher sulforaphene and sinapic acid levels in RRS, while sinapine thiocyanate and 3′,6-disinapoylsucrose remain unchanged before and after stir-frying. The results of this study highlight distinct chemical compositions between RRS and SRS. Additionally, the method of characterization and content determination constructed in this paper has strong practical value and provides a useful approach for comprehensively evaluating the chemical composition and quality of RS.

Effect of deep eutectic solvent pretreatment on biohydrogen production from corncob: pretreatment temperature and duration

ABSTRACTDeep eutectic solvent pretreatment with different temperatures and durations was applied to corncob to increase hydrogen yield via photo-fermentation. The correlation of composition, enzymatic hydrolysis, and hydrogen production in pretreated corncobs, as well as energy conversion was evaluated. Deep eutectic solvent pretreatment effectively dissolved lignin, retained cellulose, and enhanced both enzymatic hydrolysis and hydrogen production. The maximum cumulative hydrogen yield obtained under a pretreatment condition of 50°C and 12 h was 677.45 mL; this was 2.72 times higher than that of untreated corncob, and the corresponding lignin removal and enzymatic reduction of sugar concentration were 79.15% and 49.83 g/L, respectively; the highest energy conversion efficiency was 12.08%. The hydrogen production delay period was shortened, and the maximum shortening time was 18.9 h. Moreover, the cellulose content in pretreated corncob was positively correlated with both reducing sugar concentration and hydrogen yield and had the strongest influence on hydrogen production.

Synthesis and characterization of the N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) alternate substrate analog N,N-dimethyl-l,l-SDAP

Growing antibiotic resistance by pathogenic bacteria has led to a global crisis. The bacterial enzyme N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) provides a very attractive target for the discovery of a new class of antibiotics, as it resides exclusively in many pathogenic bacterial strains and is a key enzyme in the lysine biosynthetic pathway. This pathway is responsible for the production of lysine as well as meso-diaminopimelate (m-DAP), both of which are required for peptidoglycan cell-wall synthesis, and lysine for peptide synthesis. The enzyme DapE catalyzes the hydrolysis of N-succinyl-l,l-diaminopimelic acid (l,l-SDAP) to succinate and l,l-diaminopimelic acid (l,l-DAP), and due to its absence in humans, inhibition of DapE avoids mechanism-based side effects. We have executed the asymmetric synthesis of N,N-dimethyl-SDAP, an l,l-SDAP substrate analog and an analog of the synthetic substrate of our previously described DapE assay. Previous modeling studies advocated that N,N-dimethyl-SDAP might function as an inhibitor, however the compound behaves as a substrate, and we have demonstrated the use of N,N-dimethyl-SDAP as the substrate in a modified ninhydrin-based DapE assay. Thermal shift experiments of DapE in the presence of N,N-dimethyl-SDAP are consistent with a melt temperature (Tm) shifted by succinate, the product of enzymatic hydrolysis.

Integrated laccase delignification with improved lignocellulose recalcitrance for enhancing enzymatic saccharification of ensiled rice straw

The recalcitrant of straw biomass due to its severe lignification hinders its efficient use as a biofuel of feedstock. Therefore, this study was aimed to develop an integrated approach for treatment of rice straw to improve enzymatic saccharification and increase bioethanol production. We evaluated the effect of Lactobacillus plantarum (LP) and cellulases (CL), along with the ligninolytic enzyme laccase (Lac), on the quality of silage, enzymatic hydrolysis, and ethanol production from rice straw ensiled for 60 days. The rice straw was either ensiled without additive (control) or treated with five combinations of the additives, i.e., LP, CL, LP+CL, LP+Lac, or LP+CL+Lac (LPCL+Lac). The results indicated that adding laccase improved the fermentation and silage preservation quality of rice straw biomass by increasing lactic acid production and decreasing pH (<4) (P < 0.05). It also maintained high water-soluble carbohydrate and mono- and disaccharides. The LPCL+Lac treatment managed to reduce the lignin content of rice straw up to 3.82%. Fourier transform infrared spectroscopic analysis and scanning electron microscopic images confirmed that laccase degraded lignin and altered the physico-chemical structure of rice straw. The delignification and reducing the recalcitrance of lignocellulose of rice straw biomass with integrated laccase pretreatment (LPCL+Lac) resulted in release of high content of reducing sugar (52.42 mg/mL), and produced the highest quantity of ethanol (24.45 mg/mL). These results demonstrate that laccase effectively degraded lignin content of rice straw, and its integrated use with LP and CL provides a feasible approach for improving the preservation and bioconversion efficiency of highly lignified and recalcitrant straw biomass.

Multi-Scale Structural Insights into Enzymatically Hydrolyzed Lentil Starch Concentrates Prepared by In Vitro Method Using Different Types of Enzymes.

This study was undertaken to investigate the enzymatic hydrolysis of lentil starch concentrates from conventional cooked seeds (CCLSC) by the action of different types of enzymes, including pancreatin (PC-EHSC), heat-stable α-amylase (HS-EHSC), β-amylase (βA-EHSC), amyloglucosidase (AMG-EHSC), and multi-enzymes (βA-HS-AMG-EHSC); their multi-scale structural characteristics of the enzymatic hydrolysis products of lentil starch concentrates were compared. The morphological features distinguished among different samples. The Fourier-transform infrared spectroscopy and solid-state 13C CP/MAS NMR spectral features indicated the possible formation of a binary and ternary complex among amylose, protein and lipids. The X-ray diffraction results revealed that the V-type characteristic diffraction peaks were more obvious for samples including PC-EHSC and βA-EHSC, which was in line with their lowest polydispersity index (DPn). PC-EHSC and βA-EHSC also showed an increased peak intensity of the scattering maximum on the small-angle X-ray scattering spectra, whereas CCLSC exhibited an overall lower peak intensity within the studied q range of scattering. The highest XRD crystallinity and the lowest DPn value obtained for PC-EHSC indicated that the starch polymers modified by pancreatin could produce glucan chains with a comparatively homogenous Mw distribution that are readily recrystallized by hydrogen bonding through chain aggregation. Comparatively, the lowest relative crystallinity for HS-EHSC obtained from XRD suggested that thermostable α-amylolysis was unfavorable for the formation of starch structure with a higher degree of molecular order. This study could provide useful information for the needed research to obtain a deeper understanding of the impact of different amylolysis actions on the structural organization of starch hydrolysates and to provide a theoretical foundation for the development of fermentable enzymatically hydrolyzed starch with well-tailored physiological properties.