Enzyme Formulations Research Articles

Effect of Different Enzyme Treatments on Juice Yield, Physicochemical Properties, and Bioactive Compound of Several Hybrid Grape Varieties

This study investigates the effects of four enzymatic treatments on the yield, physicochemical properties, and bioactive compounds of grape juices from two red (Golubok, Regent) and two white (Muscaris, Aurora) hybrid grape varieties. A total of 20 samples were prepared using four commercial enzyme formulations (Pectinex Ultra, Safizym Clear Plus, Safizym Press, and Rapidase color) applied at a concentration of 0.02% (w/w). The juices were evaluated for yield, total phenolic content, antioxidant capacities (ABTS, DPPH, FRAP), titratable acidity, turbidity, total soluble solids, and phenolic profile. The addition of enzymes significantly improved juice yield by 10% to 20%, with the effect varying depending on the type of enzyme and the variety of grapes. Pectinex Ultra was the most effective enzyme in reducing turbidity, while enzyme treatments had minimal impact on Brix levels and sugar concentration, which were primarily determined by the characteristics of each grape variety. The enzyme addition showed a minor influence on the titratable acidity of the juices, with slight increases observed in Muscaris, but the grape variety played a major role in determining the titratable acidity levels. Color parameters revealed that white grape juices (Muscaris and Aurora) were brighter than red varieties (Golubok and Regent). Additionally, enzyme treatments influenced the color, enhancing the red hues in red grape juices. Enzyme treatments also improved the antioxidant capacity of grape juices, especially in Aurora and Muscaris, although the effect on polyphenol content was more dependent on the variety of grapes, with red varieties showing higher levels of polyphenols than white varieties. These findings highlight the significant role of both enzyme treatments and grape variety in determining the quality and health-promoting properties of grape juice.

Effectiveness of Fruit Peels Eco Enzyme in Reducing Total Chromium from Yogyakarta Tanning Industry Waste

. Leather tanning industry is essential in Indonesia's economic sector, specifically in the export of hides and leather goods. The high production of goods is often accompanied by a large amount of waste disposal, containing elevated levels of chromium which has a negative effect on the environment. To address this problem, eco enzyme containing high carboxyl groups is expected to be an effective solution. Therefore, this study aimed to determine the functional groups in fruit peels eco enzyme and its effectiveness in reducing total chromium content in leather tanning waste. The experimental process included the preparation of eco enzyme formulations, testing functional groups with FTIR, and determining total chromium content using AAS. Furthermore, the characteristics of waste were determined, including such as BOD, pH, temperature, ammonia, TDS, and TSS, followed by incubation of eco enzyme in waste, as well as data analysis with ANOVA and Duncan test. The results showed that eco enzyme formulations contained hydroxyl (O-H) and carboxyl (-COOH) functional groups, carbon-carbon single bonds (C-C), carbon-oxygen single bonds (C-O), carbon-carbon double bonds (C=C), and carbon-hydrogen bonds (C-H). All formulations effectively reduced chromium levels in leather tanning waste to value lower than quality standards. Based on the results, the best formulation was found to be eco enzyme A, which comprised one type of fruit, namely mango peels.

Immobilization and characterization of β-galactosidase from Aspergillus oryzae in polyvinyl alcohol hydrogels.

One of the main goals of contemporary biotechnology has been the development of novel immobilized enzyme formulations. In the present study, the industrially important β-galactosidase was trapped in a polyvinyl alcohol (PVA) gel to immobilize it. The optimization of immobilization method and characterization of the immobilized enzyme were studied. The results were compared with free enzymes. The results indicate that the optimal temperature range for the enzyme to be at following immobilization is between 40°C and 50°C. At pH 7, the optimal pH, the activity increased, the Vmax value increased from 1.936 to 2.495 Umg‒1, and the Km value decreased from 4.861 to 0.982mM.Depending on how stable the immobilized enzyme when stored, β-galactosidases immobilized on PVA gels showed 52.87% activity at the end of the seventh week and 58.86% activity at the end of the fifth week. Their initial activity subsided after three reuses. The final result was 66%. Therefore, one may argue that it increases the catalytic effect of the enzyme. As a result, it has been found that immobilized β-galactosidase has more potent enzymatic properties than free β-galactosidase, which may make it more advantageous for industrial processes. Further studies could delve deeper into the mechanistic aspects of the immobilization process in an effort to improve optimization and tailor the immobilized enzyme to specific industrial needs.

Application and characterization of Fusarium solani lipase microemulsion used to degrade fatty

Obesity is a chronic disease with a high prevalence, now recognized as a global epidemic. Studies show a strong association between high body mass index (BMI) and various chronic diseases, including non-alcoholic fatty liver (NAFL), cardiovascular diseases, diabetes, musculoskeletal disorders, chronic kidney disease, and mental health issues. These conditions negatively impact quality of life and elevate healthcare costs (Boutari et al., 2022). Objective: This study explores exogenous lipase produced by the fungus Fusarium solani as a biotechnological alternative for aesthetic and health applications, particularly for women with excess abdominal fat. Methods: F. solani was cultivated on solid medium, followed by submerged and semi-solid fermentation. The lipolytic activity of the extracts was evaluated through pH and temperature tests, surfactant effects, and storage stability. Additionally, we assessed cell viability in normal and cancer cells, antioxidant activity, antimicrobial properties, and fat degradation. Results: Submerged fermentation with olive oil yielded Lipase F2, which exhibited significant lipolytic activity. Lipase F2 demonstrated stability at pH 8.0, maintaining an activity of 135.89 U/mg and a purity of 3.63. It remained active between 35 and 70°C, with optimal stability at 35°C, and showed resistance to various surfactants and ions. The enzyme degraded chicken fat from 10g to 0.89g in 96 hours. Conclusion: An enzymatic formulation with F. solani lipase is viable for intradermal and surface applications, proving to be safe and non-toxic for human use.

Large-scale screening identifies enzyme combinations that remove in situ grown oral biofilm

Bacteria in the oral cavity are responsible for the development of dental diseases such as caries and periodontitis, but it is becoming increasingly clear that the oral microbiome also benefits human health. Many oral care products on the market are antimicrobial, killing a large part of the oral microbiome but without removing the disease-causing biofilm. Instead, non-biocidal matrix-degrading enzymes may be used to selectively remove biofilm without harming the overall microbiome. The challenge of using enzymes to degrade biofilms is to match the narrow specificity of enzymes with the large structural diversity of extracellular polymeric substances that hold the biofilm together. In this study, we therefore perform a large-scale screening of single and multi-enzyme formulations to identify combinations of enzymes that most effectively remove dental biofilm. We tested >400 different treatment modalities using 44 different enzymes in combinations with up to six enzymes in each formulation, on in vitro biofilms inoculated with human saliva. Mutanase was the only enzyme capable of removing biofilm on its own. Multi-enzyme formulations removed up to 69 % of the biofilm volume, and the most effective formulations all contained mutanase. We shortlisted 10 enzyme formulations to investigate their efficacy against biofilms formed on glass slabs on dental splints worn by 9 different test subjects. Three of the ten formulations removed more than 50 % of the biofilm volume. If optimal enzyme concentration and exposure time can be reached in vivo, these enzyme combinations have potential to be used in novel non-biocidal oral care products for dental biofilm control.

Recovery of pure PET from wool/PET/elastane textile waste through step-wise enzymatic and chemical processing

Textile waste is mostly incinerated because few recycling processes are available to recover valuable materials. In this work, a feasible chemo-enzymatic recycling process of wool/polyethylene terephthalate (PET)/elastane blends to recover pure PET is for the first time successfully demonstrated. Two novel enzyme formulations were selected for wool hydrolysis, whereas the recovered amino acids were quantified using high-performance liquid chromatography and two assays (Ninhydrin and Folin–Ciocalteu). Kinetic studies on the amino acid formation alongside reaction observations by scanning electron microscopy proved sufficient removal of wool within 8 hours with the new enzyme formulation, marking an acceleration compared to previous studies. Finally, elastane was separated with a non-hazardous solvent to obtain pure PET. Tensile tests on the recovered PET fibres reveal only slight changes through the enzymatic treatment and no changes induced by the applied solvent. The enzyme formulation was successfully tested on five different post-consumer wool/PET textile waste samples. This valorization approach enhances the circular economy concept for textile waste recycling.

Rapid and accurate quantification of trypsin activity using integrated infrared and ultraviolet spectroscopy with data fusion techniques

Proteases play a crucial role in industrial enzyme formulations, with activity fluctuations significantly impacting product quality and yield. Therefore, developing a method for precise and rapid detection of protease activity is paramount. This study aimed to develop a rapid and accurate method for quantifying trypsin activity using integrated infrared (IR) and ultraviolet (UV) spectroscopy combined with data fusion techniques. The developed method evaluates the enzymatic activity of trypsin under varying conditions, including temperature, pH, and ionic strength. By comparing different data fusion methods, the study identifies the optimal model for accurate enzyme activity prediction. The results demonstrated significant improvements in predictive performance using the feature-level data fusion approach. Additionally, substituting the spectral data of the samples in the validation sets into the best prediction model resulted in a minimal residual difference between predicted and true values, further verifying the model's accuracy and reliability. This innovative approach offers a practical solution for the efficient and precise quantification of enzyme activity, with broad applications in industrial processes.

Avalglucosidase alfa in infantile-onset Pompe disease: A snapshot of real-world experience in Italy

Avalglucosidase alfa in infantile-onset Pompe disease: A snapshot of real-world experience in Italy

Enzymatic hydrolysis of waste streams originating from wastewater treatment plants

BackgroundAchieving climate neutrality is a goal that calls for action in all sectors. The requirements for improving waste management and reducing carbon emissions from the energy sector present an opportunity for wastewater treatment plants (WWTPs) to introduce sustainable waste treatment practices. A common biotechnological approach for waste valorization is the production of sugars from lignocellulosic waste biomass via biological hydrolysis. WWTPs produce waste streams such as sewage sludge and screenings which have not yet been fully explored as feedstocks for sugar production yet are promising because of their carbohydrate content and the lack of lignin structures. This study aims to explore the enzymatic hydrolysis of various waste streams originating from WWTPs by using a laboratory-made and a commercial cellulolytic enzyme cocktail for the production of sugars. Additionally, the impact of lipid and protein recovery from sewage sludge prior to the hydrolysis was assessed.ResultsTreatment with a laboratory-made enzyme cocktail produced by Irpex lacteus (IL) produced 31.2 mg sugar per g dry wastewater screenings. A commercial enzyme formulation released 101 mg sugar per g dry screenings, corresponding to 90% degree of saccharification. There was an increase in sugar levels for all sewage substrates during the hydrolysis with IL enzyme. Lipid and protein recovery from primary and secondary sludge prior to the hydrolysis with IL enzyme was not advantageous in terms of sugar production.ConclusionsThe laboratory-made fungal IL enzyme showed its versatility and possible application beyond the typical lignocellulosic biomass. Wastewater screenings are well suited for valorization through sugar production by enzymatic hydrolysis. Saccharification of screenings represents a viable strategy to divert this waste stream from landfill and achieve the waste treatment and renewable energy targets set by the European Union. The investigation of lipid and protein recovery from sewage sludge showed the challenges of integrating resource recovery and saccharification processes.

Applications of Microbial Organophosphate-Degrading Enzymes to Detoxification of Organophosphorous Compounds for Medical Countermeasures against Poisoning and Environmental Remediation.

Mining of organophosphorous (OPs)-degrading bacterial enzymes in collections of known bacterial strains and in natural biotopes are important research fields that lead to the isolation of novel OP-degrading enzymes. Then, implementation of strategies and methods of protein engineering and nanobiotechnology allow large-scale production of enzymes, displaying improved catalytic properties for medical uses and protection of the environment. For medical applications, the enzyme formulations must be stable in the bloodstream and upon storage and not susceptible to induce iatrogenic effects. This, in particular, includes the nanoencapsulation of bioscavengers of bacterial origin. In the application field of bioremediation, these enzymes play a crucial role in environmental cleanup by initiating the degradation of OPs, such as pesticides, in contaminated environments. In microbial cell configuration, these enzymes can break down chemical bonds of OPs and usually convert them into less toxic metabolites through a biotransformation process or contribute to their complete mineralization. In their purified state, they exhibit higher pollutant degradation efficiencies and the ability to operate under different environmental conditions. Thus, this review provides a clear overview of the current knowledge about applications of OP-reacting enzymes. It presents research works focusing on the use of these enzymes in various bioremediation strategies to mitigate environmental pollution and in medicine as alternative therapeutic means against OP poisoning.

An engineered enzyme embedded into PLA to make self-biodegradable plastic.

Plastic production reached 400 million tons in 2022 (ref. 1), with packaging and single-use plastics accounting for a substantial amount of this2. The resulting waste ends up in landfills, incineration or the environment, contributing to environmental pollution3. Shifting to biodegradable and compostable plastics is increasingly being considered as an efficient waste-management alternative4. Although polylactide (PLA) is the most widely used biosourced polymer5, its biodegradation rate under home-compost and soil conditions remains low6-8. Here we present a PLA-based plastic in which an optimized enzyme is embedded to ensure rapid biodegradation and compostability at room temperature, using a scalable industrial process. First, an 80-fold activity enhancement was achieved through structure-based rational engineering of a new hyperthermostable PLA hydrolase. Second, the enzyme was uniformly dispersed within the PLA matrix by means of a masterbatch-based melt extrusion process. The liquid enzyme formulation was incorporated in polycaprolactone, a low-melting-temperature polymer, through melt extrusion at 70 °C, forming an 'enzymated' polycaprolactone masterbatch. Masterbatch pellets were integrated into PLA by melt extrusion at 160 °C, producing an enzymated PLA film (0.02% w/w enzyme) that fully disintegrated under home-compost conditions within 20-24 weeks, meeting home-composting standards. The mechanical and degradation properties of the enzymated film were compatible with industrial packaging applications, and they remained intact during long-term storage. This innovative material not only opens new avenues for composters and biomethane production but also provides a feasible industrial solution for PLA degradation.

A proof of principle investigation of a novel enzyme formulation on dental calculus deposition: a 4-week randomized human clinical trial

BackgroundThe dissolution of dental calculus, safely and at home, is among the more challenging issues facing the over-the-counter healthcare industry. Pontis Biologics, Inc. has developed novel model of calculus development and structure and has formulated a dentifrice (Tartarase™) using digestive enzymes as active ingredients that is shown to dissolve dental calculus in this Proof of Principle clinical trial.MethodsThis investigation was designed to evaluate the safety and efficacy of a novel enzyme formulation to remove existing calculus deposits in 4 weeks, measured using the Volpe-Manhold Index (V-MI) on lingual surfaces of 6 lower anterior teeth. The test formulation was compared to Crest Cavity Protection, as a control dentifrice. A total of 40 randomized test subjects began the study with 20 assigned to the control dentifrice and 20 assigned to the Tartarase groups (ten each, one brushing with Tartarase twice daily and one brushed with Tartarase and wore a dental tray filled with Tartarase for 30 min then brushed again with Tartarase, once daily).ResultsThe Crest group experienced a 12% increase in calculus, in contrast to the results of both Tartarase groups that experienced a 40% reduction in calculus in 4 weeks of unsupervised at home use of the Tartarase toothpaste formulation.ConclusionsThis proof of principle study demonstrates that a dentifrice, formulated along the lines of the Tartarase material, is capable of combating calculus accumulation using the same oral hygiene habits that are common worldwide.Trial registrationThis trial was registered retrospectively at clinicaltrials.gov and has the Unique Identification Number: NCT06139835, 14/11/2023.

Immobilization of human tyrosine hydroxylase onto magnetic nanoparticles – A novel formulation of a therapeutic enzyme

Human tyrosine hydroxylase (hTH) has key role in the production of catecholamine neurotransmitters. The structure, function and regulation of hTH has been extensively researched area and the possibility of enzyme replacement therapy (ERT) involving hTH through nanocarriers has been raised as well. However, our understanding on how hTH may interact with nanocarriers is still lacking. In this work, we attempted to investigate the immobilization of hTH on magnetic nanoparticles (MNPs) with various surface linkers in quantitative and mechanistic detail. Our results showed that the activity of hTH was retained after immobilization via secondary and covalent interactions as well. The colloidal stability of hTH could be also enhanced proved by Dynamic light scattering and Zeta potential analysis and a homogenous enzyme layer could be achieved, which was investigated by Raman mapping. The covalent attachment of hTH on MNPs via aldehyde or epoxy linkers provide irreversible immobilization and 38.1% and 16.5% recovery (ER). The hTH-MNPs catalyst had 25% ER in average in simulated nasal electrolyte solution (SNES). This outcome highlights the relevance of immobilization applying MNPs as a potential formulation tool of sensitive therapeutic enzymes offering new opportunities for ERT related to neurodegenerative disorders.

Development of a fluorescence-based excipient screening for improved stability and shelf-life of recombinant chitin deacetylase

Chitin deacetylase (CDA) modifies chitin into chitosan by removing acetyl groups, but its inherent instability poses a challenge for successful crystallisation. Despite limited successes in crystallizing CDAs, prior attempts with recombinant chitin deacetylase (BaCDA) failed due to poor stability. To address this, we propose an enzyme buffer formulation as a cost-effective strategy to enhance stability, prolong shelf life, and increase the likelihood of crystallisation. Utilizing the high-throughput screening technique FTSA, we developed a screening method correlating BaCDA stability with its activity. The optimised formulation comprises 50mM Tris-HCl buffer pH 7, 1M NaCl, 20% glycerol, and 1mMMg2+ as excipients. This formulation significantly improves BaCDA's thermostability (140.47% increase) and enzyme activity (2.9-fold enhancement). BaCDA remains stable in the formulated buffer at -20°C and -80°C for 30 days and at 4°C for 15 days. The current study has designed a high-throughput screening method approach to assess the stability of CDA enzyme formulations. The results of this study could contribute to the exploration of formulation elements that enhance the structural stability of CDA, thereby facilitating investigations into the enzyme's structure-function relationships.

Effects of Bio-enzyme on the strength properties of soil

Effects of Bio-enzyme on the strength properties of soil

Polymer Chemistry: A primer to enzymatic polymer degradation

AbstractAs highly efficient biocatalysts, enzymes could be used in technical applications for the degradation of polymers, for chemical recycling of polymers, and for the removal of microplastic debris. The formulation of enzymes with preservation of their enzymatic activity is a major challenge for applications with polymers.

Benefitting multi-enzyme system for the purpose of improving the flow properties of waxy oil

Benefitting multi-enzyme system for the purpose of improving the flow properties of waxy oil

Use of carbohydrases to promote protein extraction from rice bran and soybean meal: A comparative study

Use of carbohydrases to promote protein extraction from rice bran and soybean meal: A comparative study

Enhanced energy savings in enzymatic refining of hardwood and softwood pulp

BackgroundPulp refining is an energy consuming, but integral part of paper production with the aim to increase tensile strength and smoothness of paper sheets. Commercial enzyme formulations are used to lower the energy requirements by pre-treatment of pulp before refining. However, a high number of different commercial enzyme products are available on the market containing enzymes of varying origin and composition, which complicates the prediction of their behavior, especially using different pulp types.ResultsEndoglucanase-rich enzyme formulations were characterized regarding enzyme activity at different temperatures, resulting in a significant decrease of activity above 70 °C. Some enzyme preparations additionally contained arabinosidase, xylanase and β-glucosidase activity consequently resulting in a release of xylose and glucose from pulp as determined by high-performance liquid chromatography. Interestingly, one enzyme formulation even showed lytic polysaccharide monooxygenase (LPMO) activity of 3.05 nkat mg−1. A correlation between enzyme activity using the endoglucanase specific derivatized cellopentaose (CellG5) substrate and enzyme performance in laboratory PFI (Papirindustriens forskningsinstitut) refining trials was observed on softwood pulp resulting in a maximum increase in the degree of refining values from 27.7°SR to 32.7°SR. When added to a purified endoglucanase enzyme (31.6°SR), synergistic effects were found for cellobiohydrolase II (34.7°SR) or β-glucosidase enzymes (35.7°SR) in laboratory refining. Comparison with previously obtained laboratory refining results on hardwood pulp allowed differences in enzyme performance based on varying pulp types to be elucidated.ConclusionsInterestingly, the individual enzymes indeed showed different refining effects on softwood and hardwood pulp. This difference could be predicted after development of an adapted enzyme activity assay by combination of the derivatized cellopentaose CellG5 substrate with either softwood or hardwood sulfate pulp.

Effect of enzymatic and talc treatment on olive oil extraction process at the industrial scale

Technological coadjuvants were applied at the beginning of the malaxation phase of the olive oil mechanical extraction process at the industrial scale. An enzymatic formulation consisting of 35% pectinase, 28% pectinmethylesterase and 7% polygalacturonase % (v/v/v) and talc was added during the kneading of olive paste to evaluate its impact on oil extractability and on olive oil quality characteristics. Quantitative and qualitative evaluation of hydrophilic phenols and volatile compounds involved in the main health and sensory properties of high-quality olive oil was carried out. The addition of a combination of enzymatic complex and talc, for the first time at industrial scale, increased the oil extractability by 5.6% (absolute value), improving the degradation of the cell wall structure of olive paste and breaking down the oil-in-water emulsions with a more efficient separation of the oil during the extraction process. The use of the enzymatic complex and talc leads to a percentage increase in the phenolic content in the range of 12%–16% without altering the legal quality parameters and volatile profile of the final product.