Enzymatic Hydrolysis Research Articles

Phage-Derived Endolysins Against Resistant Staphylococcus spp.: A Review of Features, Antibacterial Activities, and Recent Applications.

Antimicrobial resistance is a significant global public health issue, and the dissemination of antibiotic resistance in Gram-positive bacterial pathogens has significantly increased morbidity, mortality rates, and healthcare costs. Among them, Staphylococcus, especially methicillin-resistant Staphylococcus aureus (MRSA), causes a wide range of diseases due to its diverse pathogenic factors and infection strategies. These bacteria also present significant issues in veterinary medicine and food safety. Effectively managing staphylococci-related problems necessitates a concerted effort to implement preventive measures, rapidly detect the pathogen, and develop new and safe antimicrobial therapies. In recent years, there has been growing interest in using endolysins to combat bacterial infections. These enzymes, which are also referred to as lysins, are a unique class of hydrolytic enzymes synthesized by double-stranded DNA bacteriophages. They possess glycosidase, lytic transglycosylase, amidase, and endopeptidase activities, effectively destroying the peptidoglycan layer and resulting in bacterial lysis. This unique property makes endolysins powerful antimicrobial agents, particularly against Gram-positive organisms with more accessible peptidoglycan layers. Therefore, considering the potential benefits of endolysins compared to conventional antibiotics, we have endeavored to gather and review the characteristics and uses of endolysins derived from staphylococcal bacteriophages, as well as their antibacterial effectiveness against Staphylococcus spp. based on conducted experiments and trials.

Deep eutectic solvents assisted laccase pretreatment for improving enzymatic hydrolysis of corn stover.

The efficient and eco-friendly removal of lignin is a critical challenge for bioethanol production from lignocellulosic biomass. Herein, we report the integration of laccase with deep eutectic solvents (DESs) for the pretreatment of corn stover to enhance the production of reducing sugars. Three betaine-based DESs were prepared and tested for their effects on the activity and stability of a bacterial laccase from Bacillus amyloliquefaciens LC02. The aqueous solution of DESs showed no adverse influence on laccase activity, and the laccase thermostability was improved in the presence of DESs. More than 95% of the laccase activity was retained in the DESs solution during the first hour of incubation at 70°C. A red shift in the fluorescence spectra was observed for the laccase in the presence of DESs, indicating conformational changes. The laccase was able to degrade a dimeric lignin model compound by cleaving its β-O-4 bond. The transformation products were identified using LC-MS. The maximal lignin removal from corn stover was achieved by pretreatment using laccase in combination with the betaine-glycerol DES, which also resulted in a yield of fermentable sugar that was 130% higher than the control. This combination strategy provides guidance on the application of laccase and DESs in the pretreatment of lignocellulosic biomass.

Characteristics of deep-sea microbial cellulases: key determinants of the ultimate fate of plant biomass on Earth

AbstractLand plants, especially those with significant woody biomass, represent the largest source of biomass on Earth, making the biodegradation of lignocellulosic materials critical to understanding the global carbon cycle. Cellulose, a major component of lignocellulose, is notoriously resistant to degradation due to its highly crystalline structure. While the degradation of cellulose by terrestrial microbes has been extensively studied, the mechanisms of cellulose degradation in deep-sea environments remain largely unexplored. The deep-sea ecosystem depends on organic matter, such as cellulose, that is synthesized in terrestrial environments and surface waters and descends to the deep sea. Recent studies suggest that a significant amount of cellulose is likely to reach the deep sea. Here, we present an in-depth study of cellulases from a novel deep-sea γ-proteobacterial strain TOYAMA8, isolated from Toyama Bay, Japan, using Surface Pitting Observation Technology (SPOT), a highly sensitive assay for enzymatic cellulose hydrolysis. The cellulases of strain TOYAMA8 show similarities to those of a previously reported deep-sea cellulolytic microbe, Marinagarivorans cellulosilyticus strain GE09. Genomic and transcriptomic analyses of these strains reveal novel cellulase genes and mechanisms that differ from terrestrial counterparts, shedding light on the unique adaptations of deep-sea microbes to recalcitrant biomass. In particular, these strains produce high-molecular-weight cellulases with unique domain architectures, likely optimized for membrane anchoring, which prevents enzyme diffusion and ensures efficient localized activity. Our findings provide critical insights into the microbial cellulose degradation in the deep sea, highlighting its role in the fate of organic carbon and the potential for biotechnological applications in biorefineries.

Effects of Different Pre-Treatments on Local Pineapple Leaf as a Potential Substrate for the Production of Value-Added Products

Lignocellulosic biomass such as pineapple leaves can be applied as substrates in various biochemical processes such as solid-state fermentation to produce industrially important enzymes or other value-added products. However, recalcitrant nature of the biomass hinders the accessibility of cellulose for enzymatic hydrolysis in yielding high amounts of the intended products. In order to overcome this obstacle, pre-treatment is necessary as to loosen the lignocellulose structure to ease accessibility of enzymes and biodegradability of the biomass. In this study, local pineapple leaves were subjected to different methods of pre-treatments which are thermal pre-treatment by autoclaving, and chemical pre-treatments involving treatments with alkaline solutions (1% and 1.5% (w/v) sodium hydroxide) as well as acid solutions (1% and 1.5% (v/v) hydrochloric acid). Scanning Electron Microscopy was conducted to compare and study the effect of different pre-treatments on morphological changes of raw and pre-treated leaves. The obtained results revealed that the biomass was severely affected by 1.5% (v/v) hydrochloric acid solution as the surface structure was vigorously ruptured and more aggregated cracks were clearly visible in most parts, compared to the surface morphologies observed on raw samples and samples with other pre-treatment methods.

Current Strategies to Modify the Functional Properties of Proteins Extracted from Pumpkin Seeds: A Comprehensive Review

The functional properties of pumpkin seed proteins remain unutilized in numerous food and industrial applications. Several current approaches aim to improve the functional properties of pumpkin seed proteins, allowing their innovative potential to develop and modify significantly. Several strategies can be implemented to alter the functional properties of proteins isolated from pumpkin seeds. The first is enzymatic hydrolysis, regardless of whether, proteases may free peptide binding and profoundly impact the protein structure and functionality. Thermal treatment can include heating and cooling to replace protein conformation and increase solubility, emulsification, and gelation properties. Chemical modification techniques, including acylation and glycation, can also be used to improve stability, viscosity, and foaming ability. Functional properties and, where possible, ingredients with many applications may include exceptional possibilities for proteins modified in food preparations, such as dairy replacements, plant-based meat analogues, and free gluten that have an outstanding aspect, satisfactory quality, and nutritional profiles. As multiple different proteins act as precursors of active peptides, they can also be used to generate bio-specific foods. This review briefly provides information about various types of protein extraction techniques and functional properties that are modified by different types of processing technologies.

Gentidelasides A-G: loganin derivatives from Gentiana delavayi with reducing Aβ secretion via suppressing BACE1 expression.

Gentidelasides A-G (1-7) seven unreported loganin derivatives and fourteen known compounds (8-21) were isolated from the flowers of Gentiana delavayi Franch. Their structures including absolute configurations were unambiguously elucidated by analysis of extensive NMR spectroscopy, ECD, and HRESIMS, as well as enzymatic hydrolysis. In vitro bioassay, compound 7 showed obvious inhibitory effects on the production of Aβ40 and Aβ42, with IC50 values of 0.052 ± 0.0023 nM and 1.52 ± 0.95 nM, respectively, which probably exert their prevention of Alzheimer's disease by inhibiting the expression of β-site amyloid precursor protein cleaving enzyme 1. The molecular docking simulation revealed that compound 7 inhibited BACE1 through hydrophilic and hydrophobic interactions in the active site cavities.

Differently Processed Low Doses of β-Glucan from Oat Bran Similarly Attenuate Postprandial Glycemic Response

Incorporating β-glucan-rich oat bran (OB) can attenuate postprandial glycemic response (PPGR) in solid foods, but its effect in liquid matrices is unclear. This study investigated the ability of differently processed low-dose-β-glucan-containing beverages to lower PPGR, and the mechanisms of action. Twenty participants consumed five malt beverages made from cocoa powder: intact OB (Intact), OB treated with enzymatic hydrolysis (EnzymA, EnzymB) or extrusion (Extr), or no OB (Ctrl). Four-hour postprandial incremental areas under the curve (iAUC) and peak incremental concentrations (iCmax) of glucose, insulin, glucagon-like peptide 1 (GLP-1), gastric inhibitory polypeptide (GIP), and paracetamol were evaluated. The molecular weight (MW) and extractability of the β-glucan in all the test products were also assessed. The three-hour glucose iAUC significantly decreased by −26%, −28%, −32%, and −38% in Intact, EnzymA, EnzymB, and Extr, respectively, and the insulin levels of the oat-containing products were also significantly lower compared to Ctrl. Intact and Extr elicited a lower insulin iCmax and GLP-1 3 h iAUC compared to Ctrl. However, the GIP and paracetamol levels were not changed. All the processed OBs improved β-glucan extractability and lowered the MW of β-glucan compared to Intact. In conclusion, low-dose oat β-glucan in a beverage significantly reduced PPGR, with effects maintained across different oat processing methods.

Mechanism of a novel Bacillus subtilis JNF2 in suppressing Fusarium oxysporum f. sp. cucumerium and enhancing cucumber growth

Cucumber Fusarium wilt caused by Fusarium oxysporum f. sp. cucumerium (FOC), is a prevalent soil-borne disease. In this study, Bacillus subtilis JNF2, isolated from the high incidence area of cucumber Fusarium wilt in Luoyang, demonstrated significant inhibitory effects on FOC and promoted cucumber seedling growth. The biocontrol mechanism of strain JNF2 were elucidated through morphological observation, physiological and biochemical experiments, and whole genome sequence analysis. Pot experiments revealed an 81.33 ± 0.21% control efficacy against Fusarium wilt, surpassing the 64.10 ± 0.06% efficacy of hymexazol. Seedlings inoculated with JNF2 exhibited enhanced stem thickness and leaf area compared to control and hymexazol-treated plants. Physiological tests confirmed JNF2’s production of indole-3-acetic acid (IAA), siderophores, and hydrolytic enzymes, such as β-1,3-glucanase, amylase, and protease, which inhibited FOC growth and promoted plant development. Genome analysis identified genes encoding antimicrobial peptides and hydrolases, as well as a novel glycocin synthetic gene cluster. These findings underscore B. subtilis JNF2’s potential as a biocontrol agent for sustainable cucumber cultivation.

Investigating the Core Structure of Reuteran with High-Content Linear (α1 → 6) Linkages Synthesized by Limosilactobacillus mucosae L24-B GtfB-Type Glucanotransferase.

Reuteran is a functional homopolysaccharide comprising major (α1 → 4) and interspersed (α1 → 6) linkages whose properties and functions are directly associated with the structure. In order to enrich the structural variety and application prospects of reuteran, a GtfB-type 4,6-α-glucanotransferase from Limosilactobacillus mucosae L24-B (LmGtfB), which can synthesize reuteran with high-content linear (α1 → 6) linkages, was screened. LmGtfB exhibited 4,6-α-glucanotransferase activity against starch or dextrin and can synthesize both linear and branching (α1 → 6) linkages, which offered advantages in terms of source and cost. The as-produced reuteran had the highest content of linear (α1 → 6) linkages (19% in the methylation result) among the reuterans synthesized by other GtfB-type 4,6-α-glucanotransferases. A purification method was established and applied to the extraction of the core structure of the LmGtfB products. 1H NMR and enzymatic hydrolysis revealed that the core structure contains 46.24% (α1 → 6) linkages and alternating (α1 → 4)/(α1 → 6) linkages, which was formed by enzymatic transfer of maltose.

Comparing the improvement of enzymatic hydrolysis effect of Celtis sinensis leaves residue by hydrophilic or hydrophobic deep eutectic solvent pretreatment

Comparing the improvement of enzymatic hydrolysis effect of Celtis sinensis leaves residue by hydrophilic or hydrophobic deep eutectic solvent pretreatment

Statistical optimisation of enzymatic pre-treatment process of marigold flower waste for enhanced hydrolysis and complete release of lutein ester through Taguchi orthogonal design

ABSTRACT The lutein ester of the marigold flower is entrapped in the matrix of pectin, cellulose, hemicellulose and lignin which cannot be released by moderate acidic, alkaline treatment or enzymatic treatment with a single enzyme. Five different parameters are optimized in the current study for two response factors: lutein ester extraction (LEEP) and floral solid hydrolyzed percentage (FSHP). Three commercially available enzymes, namely pectinase, cellulase, and xylanase, at various temperatures and time intervals were utilised to optimise the enzymatic hydrolysis of flower biomass. An L27 orthogonal Taguchi experimental design was employed, investigating temperature (25–35 °C), time (10–30 days), and enzyme levels (pectinase, cellulase and xylanase at 0–1 mL). The optimal conditions for 100% lutein ester extraction from marigold flower waste were: temperature 35 °C, 20 days, and enzymes 1 mL/200 g each of pectinase (2600 µPG/g), cellulase (20000 CMC U/g), and xylanase (300000 U/g). Thus, it could be concluded that optimisation by the Taguchi method could be used to develop an enzyme cocktail for the hydrolysis of marigold flowers to obtain the best yield of lutein ester. The aim is to enhance the yield of lutein ester through the optimisation of the enzymatic pre-treatment of the marigold flower.

Lactic Acid Production from Distiller’s Dried Grains Dilute Acid Hydrolysates

Lactic acid (LA) is an important chemical with diverse applications in various industries. LA can be produced by the fermentation of different substrates by many microorganisms such as bacteria, fungi, yeasts, and algae. Lactic acid bacteria (LAB) are generally accepted as the main producers of LA. A distinct characteristic of LAB is the complexity of the fermentation media. Distiller’s dried grains with solubles (DDGS), a by-product from bioethanol production, represent a promising substitute for costly sugars in the nutrition media for LA production. In the present paper, the possibility of using dilute acid DDGS hydrolysates as a substrate for LA fermentation was investigated. The influence of different factors (acid concentration, time, pressure, solid-to-liquid ratio) on the reducing sugars (RS) obtained was studied. Additional enzyme hydrolysis was carried out to increase RS content in the hydrolysates. LA production from hydrolysates without and with control of the pH during fermentation was monitored and compared with lactose as a substrate. Inhibition of the process was observed in both substrates in the absence of pH control which was overcome in the case of pH control. A mathematical model based on the Verhulst and Ludeking–Piret equations was proposed and tested, showing very good agreement with experimental data.

Screening, Gene Cloning and Expression of Cellulase-Producing Strain Bacillus subtilis Xh-16.

Cellulase is a complex enzyme system composed of multiple hydrolytic enzymes. It can degrade cellulose into glucose and improve the utilization efficiency of cellulose resources. Cellulase produced by microorganisms is the main method used in industry, offering the advantages of convenience and being environmentally friendly. A strain Xh-16 with high cellulase production was screened from the rotten rice straw. It was identified as Bacillus subtilis by morphological identification, physiological and biochemical identification, and 16S rRNA gene sequence analysis. Strain Xh-16 was used to degrade rice straw. After a 48h cellulase treatment, the complete degradation and structural breakdown of the straw were observed. We cloned the endoglucanase Cel5L gene of Bacillus subtilis Xh-16 and induced expression of the cloned gene in Escherichia coli BL21 (DE3). The results showed that the coding length of Cel5L gene was 1500bp, and the molecular weight of the encoded protein was about 55kDa. The molecular formula is C2456H3811N671O761S10 and it has 7709 atoms and 499 amino acids. Cel5L differs significantly from some the glycoside hydrolase family 5 cellulases because it has only one carbohydrate binding module family 3 at the C-terminus of its catalytic domain. The cellulase gene Cel5L in Xh-16 can encode active cellulase and can be heterologously expressed in Escherichia coli, which makes Escherichia coli have cellulase function. This study serves as a foundation for further research on cellulase diversity in Bacillus subtilis and offers insights for enhancing cellulase production.

Mechanisms of Biocontrolling Action of Yeasts in Post-Harvest Fruit: A Review

Objective: The aim of the research is to address the biological control developed by yeasts in the post-harvest of fruits, based on a brief literature review on the subject. Theoretical Framework: The use of chemical control mechanisms and biocontrol is based on the increasing need for sustainable agricultural practices that ensure human health and environmental preservation. While synthetic fungicides are effective in combating fungal diseases, their excessive use raises concerns about pathogen resistance and public health risks. In this context, the development of biocontrol agents, such as yeasts, emerges as a viable and ecological alternative, promoting food safety and crop protection with a lower environmental impact. Method: A narrative literature review was conducted using major scientific databases to collect articles on the biological control of yeasts in the post-harvest of fruits. Key studies were evaluated to measure effectiveness and understand the mechanisms related to the biocontrol action of these yeasts. Results and Discussion: The predominant chemical control for fungal diseases is being replaced by sustainable alternatives, such as yeast-based biofungicides, which demonstrate effectiveness against pathogens in post-harvest settings. Mechanisms such as killer activity and the production of hydrolytic enzymes stand out in biocontrol. Although promising, further research is needed to deepen the understanding of biocontrol agents and their applications.

Effect of a Thermomechanical Pretreatment Coupled to Sulfuric Acid or Sodium Hydroxide Catalysis on Physicochemical Properties and Enzymatic Hydrolysis of Alfa Fibers (Stipa tenacissima L.)

Effect of a Thermomechanical Pretreatment Coupled to Sulfuric Acid or Sodium Hydroxide Catalysis on Physicochemical Properties and Enzymatic Hydrolysis of Alfa Fibers (Stipa tenacissima L.)

Microbial Life in Playa-Lake Sediments: Adapted Structure, Plastic Function to Extreme Water Activity Variations

Saline shallow lakes in arid and semi-arid regions frequently undergo drying episodes, leading to significant variations in salinity and water availability. Research on the impacts of salinity and drought on the structure and function of biofilms in hypersaline shallow lakes is limited. This study aimed to understand the potential changes of biofilms in playa-lake sediments during the drying process. Sediments were sampled at different depths (surface, subsurface) and hydrological periods (wet, retraction, and dry), which included a decrease in water activity (aw, the availability of water for microbial use) from 0.99 to 0.72. aw reduction caused a greater effect on functional variables compared to structural variables, indicating the high resistance of the studied biofilms to changes in salinity and water availability. Respiration and hydrolytic extracellular enzyme activities exhibited higher values under high aw, while phenol oxidase activity and prokaryote biomass increased at lower aw. This shift occurred at both depths but was more pronounced at the surface, possibly due to the more extreme conditions (up to 0.7 aw). The increased levels of extracellular polymeric substances and carotenoids developed at low aw may help protect microorganisms in high salinity and drought environments. However, these harsh conditions may interfere with the activity of hydrolytic enzymes and their producers, while promoting the growth of resistant prokaryotes and their capacity to obtain C and N sources from recalcitrant compounds. The resilience of biofilms in hypersaline lakes under extreme conditions is given by their resistant biochemichal structure and the adaptability of their microbial functioning.

Enhancing Molecular‐Level Biological Monitoring with a Smart Self‐Assembling 19F‐Labeled Probe

AbstractReal‐time monitoring of molecular transformations is crucial for advancements in biotechnology. In this study, we introduce a novel self‐assembling 19F‐labeled nuclear magnetic resonance (NMR) probe that disassembles upon interaction with various nucleotides. This interaction not only activates the 19F signals but also produces distinct signatures for each specific component, thereby enabling precise identification and quantification of molecules in evolving samples. We demonstrate the capability of this probe for real‐time monitoring of adenosine triphosphate (ATP) hydrolysis and screening potential enzyme inhibitors. These applications highlight the probe's significant potential in enzyme analysis, drug development, and disease diagnostics.

Valuable Ca/P Sources Obtained from Tuna Species’ By-Products Derived from Industrial Processing: Physicochemical and Features of Skeleton Fractions

The global tuna canning industry generates substantial volumes of by-products, comprising 50% to 70% of the total processed material. Traditionally, these by-products have been utilized in low-value products such as fish oils and fishmeal. However, there is significant potential to extract high-value compounds from these by-products, such as calcium phosphates (CaP), which can have pharmaceutical, agricultural and biotechnological applications. This work explores the potential of tuna canning by-products, particularly mineral-rich fractions (central skeleton, head and fish bones) as sources of calcium phosphates (CaP), offering a sustainable alternative to conventional synthetic derivatives within a circular bioeconomy framework. By-products from two of the most exploited species (yellowfin and skipjack) were subjected to enzymatic hydrolysis and chemical extraction, followed by controlled calcination to obtain CaP. The content of organic matter, nitrogen, total proteins, lipids and amino acids in the cleaned bones, as well as the main chemical bonds, structure and elemental composition (FT-Raman, XRD, XRF) were evaluated. Results indicated that the highest recovery yield of wet bones was achieved using the chemical method, particularly from the dorsal and caudal fins of yellowfin tuna. The proximal composition, with ash content ranging from 52% to 66% and protein content varying between 30% and 53%, highlights the potential of tuna skeleton substrates for plant growth formulations. Furthermore, variations in crystalline structures of the substrates revealed significant differences depending on the by-product source and species. XRD and Raman results confirmed a monophase calcium phosphate composition in most samples from both species, primarily based on hydroxyapatite (central skeleton, caudal and dorsal fin) or whitlockite/β-tricalcium phosphate (viscera), whereas the heads exhibited a biphasic composition. Comparing the species, yellowfin tuna (YF) exhibited a hydroxyapatite structure in the branchial arch and scales, while skipjack (SKJ) had a biphasic composition in these same regions.

Exploring newly isolated thermotolerant, halotolerant and antimicrobial resistant Bacillus subtilis ProNTL1 from tannery waste and its alkaline protease production.

A hydrolytic enzyme called protease from microbial sources has expansive applications in leather as well as tannery industries and thus leaves no choice but to discover a potential candidate for efficient yield of protease with eccentric characteristics. In the current study, using skim milk agar medium, three bacterial strains (Pro SS14, Pro NTL1, Pro SM) were identified for protease production out of thirty tannery waste samples. The bacterial isolates were identified through morphological, biochemical and molecular basis and the highest protease producer (134U/mL) having the paramount protein content (8.75%) with the greatest biomass (absorbance 1.7 at 600nm) was further bioinformatically identified as Bacillus subtilis Pro NTL1. Moreover, the cultural conditions-pH, temperature, incubation period and salinity were tested to get the optimized condition for the three isolates where Bacillus subtilis Pro NTL1 gave best growth at pH 9 demonstrating alkaline protease producing capability, at 45°C for 72h proving its thermotolerance characteristics and at 3% NaCl concentration declaring its halotolerance property. Furthermore, following Kirby-Bauer agar disc diffusion technique, the antimicrobialsusceptibility pattern was investigated showing Bacillus subtilis Pro NTL1 was resistant to ampicillin, penicillin, sulfafurazole and sensitive to amoxicillin, gentamycin, cefotaxime, kanamycin, imipenem, tetracycline. Additionally, Bacillus subtilis Pro NTL1 showed antagonistic activity against three pathogens named Bacillus cereus, Bacillus megaterium and Escherichia coli. Thus, these findings suggest that Bacillus subtilis Pro NTL1 is a newly isolated strain with unique features which might help to contribute in several biotechnological purposes with significant industrial benefits.

Influence of the Conditions of Alkaline Pretreatment of Vegetable Raw Material before Hydrolysis in a Rotor Pulsation Device on the Cellulose Conversion

Introduction. The growing global energy demand, limited fossil fuel resources, and increasing greenhouse gasemissions highlight the need for expanded use of renewable energy sources, particularly biomass.Problem Statement. A significant drawback in the production of bioethanol is its high cost, primarily due tothe presence of hemicellulose and lignin.Purpose. This study aims to determine the effect of treating an alkaline suspension of wheat straw in a rotary pulsation device on the degree of cellulose conversion during enzymatic hydrolysis, a critical step in the productionof fuel ethanol.Materials and Methods. The raw material is pre-crushed wheat straw. The experiments have been conductedwith the use of a rotary pulsation device in an experimental plant.Results. It has been found that the combined physical effects of the discrete pulse energy input method andreduced straw particle size significantly increase the lignin extraction, from 40.0% at an average particle size of2—1 mm to 62.0% at an average particle size of 0.4—0.1 mm. Additionally, the treatment of an alkaline suspension of wheat straw at 90 оC for one hour, at alkali concentrations ranging from 1 to 4% (wt./wt.), results in anincrease in the cellulose conversion during enzymatic hydrolysis from 38% to 65.8%. Additionally, a corresponding decrease in the lignin content, from 17.1% to 3.16% has been reported.Conclusions. Increasing alkali concentration during the alkaline pretreatment of wheat straw using the discrete-pulse energy input method in a rotary pulsation device enhances the rate of cellulose conversion duringsubsequent enzymatic hydrolysis.