Purification Of Enzyme Research Articles

Synergistic Effect of Purified Protease Enzyme with Antibiotics on Pathogenic S. Aureus In-Vitro and In-Vivo

Objective: The goal of this study is the detection of the P. aeruginosa isolate for production the protease, the optimal conditions for produce, extraction, purification and characterization of the enzyme and study the synergistic effect of protease with antibiotics on S. aureus. Method: Among (110) isolates from clinical sources, only (74) isolates identified as P. aeruginosa isolated from Baghdad hospitals. Detection of the optimal isolate for production the protease enzyme and the optimal conditions its produce, purification of enzyme by using ammonium sulfate, ion exchange chromatography and gel filtration, enzyme characterization of PH and temperature activity and stability, the study of synergistic effect of protease with antibiotics on S. aureus. Results: The optimal conditions to produce protease is pH (8), (37) ºC, (BHI) broth, and during (48 hrs.). The precipitation saturation ratio (80%). Ion- exchanges (DEAE and CM) Cellulose and Gel filtration have specific activity ((121.25), (161.6), (660.53)) units/ mg)) respectively. The characterization done by pH and temperature activity purified protease was active in (138.51units/ ml) at pH (8), and stable in pH (8), the temperature for protease activity is (158.21 units/ ml) at (37) ºC. The synergistic effect of purified protease with antibiotics on S.aureus in- vitro,was detected using the agar diffusion method, the effectiveness of the prepared hydrogel types, the results showed S.aureus was more sensitive isolate to prepare (Gel & Protease & Ceftriaxone) the diameter of the inhibition zone reached (44 mm) ,the synergistic effect we noticed when using a (Gel & Ceftriaxone & Protease) healing was observed in time (five days) with wound healing without effect. Conclusion: In this study, it was discovered that mixing (protease + hydrogel + Ceftriaxone) accelerates the wound healing without leaving traces.

Transforming cancer detection and treatment with nanoflowers.

Nanoflowers, an innovative class of nanoparticles with a distinctive flower-like structure, have garnered significant interest for their straightforward synthesis, remarkable stability, and heightened efficiency. Nanoflowers demonstrate versatile applications, serving as highly sensitive biosensors for rapidly and accurately detecting conditions such as diabetes, Parkinson's, Alzheimer's, and foodborne infections. Nanoflowers, with their intricate structure, show significant potential for targeted drug delivery and site-specific action, while also exhibiting versatility in applications such as enzyme purification, water purification from dyes and heavy metals, and gas sensing through materials like nickel oxide. This review also addresses the structural characteristics, surface modification, and operational mechanisms of nanoflowers. The nanoflowers play a crucial role in preventing premature drug leakage from nanocarriers. Additionally, the nanoflowers contribute to averting systemic toxicity and suboptimal therapy efficiency caused by hypoxia in the tumor microenvironment during chemotherapy and photodynamic therapy. This review entails the role of nanoflowers in cancer diagnosis and treatment. In the imminent future, the nanoflowers system is poised to revolutionize as a smart material, leveraging its exceptional surface-to-volume ratio to significantly augment adsorption efficiency across its intricate petals. This review delves into the merits and drawbacks of nanoflowers, exploring synthesis techniques, types, and their evolving applications in cancer.

Characterization of high Fischer ratio oligopeptides produced from casein

High Fischer ratio oligopeptides mixture is a mixed peptide system which possess many beneficial effects to the human body. Casein, a milk protein, was used as feedstock to prepare high Fischer ratio oligopeptides via enzymatic hydrolysis, adsorption and purification. Gel electrophoresis, densitometric analysis, amino acid assay and molecular weight measurement were used to visualize and quantify the prepared peptides. Casein, with Fischer ratio of 2.17, was subjected to hydrolysis by alcalase® (2.4 AU/g, pH 8.5, temperature 55 °C, enzymolysis time 2 h) and flavorzyme® (3.4 AU/g, temperature 50 °C, enzyme amount 120 μL, enzymolysis time 3 h), followed via AK-220 activated carbon adsorption (carbon-hydrolysate ratio: 1:10, adsorption time 3 h, pH 8.0, temperature 55 °C) and 3 KDa membrane ultrafiltration. In conclusion, the processing parameters for alcalase® / flavorzyme® and AK-220 activated carbon helped to obtain oligopeptides with Fischer ratio 9.87 which is significantly higher than feedstock casein F value (p < 0.05) and could potentially be used as food supplement to enhance product nutritional property.

Enhancing the Purification and Stability of Superoxide Dismutase by Fusion with Thermoresponsive Self‐Assembly of Elastin Like Polypeptide

AbstractElastin‐like polypeptide (ELP) is a thermo‐sensitive biosynthetic polymer composed of a Val‐Pro‐Gly‐Xaa‐Gly repeating unit possessing a sharp reversible phase transition property at specific temperatures. Here, ELP was fused to human superoxide dismutase 1 (hSOD1) modified with His tag to produce recombinant hSOD1‐Linker‐ELP‐His (hSODLEH) which was expressed in E. coli and purified via inverse thermal cycling (ITC) and Ni‐NTA resin. The results showed that ELP tag did not affect the soluble expression of SOD1. The purification by ITC was superior to Ni‐NTA resin due to its convenient purification process, improved recovery rate and purification fold, thus indicating industrial suitability for large scale protein purification in a cost‐ and time‐efficient manner. Moreover, one round of ITC was sufficient for the recovery of highly purified recombinant SOD. The results further showed that ELP improved the stability of the SOD, and did not affect its secondary structure nor its ability to bind divalent metal ions. Overall, ELP‐protein fusion system could be a promising tool for large scale enzyme purification.

The Wood-rot Basidiomycete Candolleomyces eurysporus VAST02.52 Produces a Versatile Peroxidase that Catalyses the Biotransformation of α-Pinene to Release Verbenone

Background Versatile peroxidase (VP; EC 1.11.1.16) has recently emerged as a novel peroxidase highly valued in biotechnology due to its exceptional ability to oxidize a diverse range of substrates into value-added products. Objectives To gain insight into the versatile peroxidase system from fungi, this study was designed to purify and characterize a CeuVP from the newly isolated fungus Candolleomyces eurysporus and to investigate its involvement in the biotransformation of plant α-pinene. Materials and methods The fungal isolate was identified by analysis of the internal transcribed spacer region. VP-active protein was purified from liquid culture of C. eurysporus VAST02.52 by using fast protein liquid chromatography. The catalytic properties and stability of enzyme were studied for various substrates, different pH- and temperatures. Moreover, its involvement in the biotransformation of plant α-pinene was assessed by HPLC chromatography. Results CeuVP was successfully purified with 27.3-fold purity and a 20.9% yield by using three steps of anion exchange and size exclusion chromatography. The molecular weight of CeuVP was determined to be 40 kDa by SDS-PAGE electrophoresis. This pure enzyme exhibited optimal activity at pH 4.0 and 35 °C. CeuVP showed the highest specific activity of 231.6 U/mg against ABTS, followed by Mn2+ (205.8 U/mg), veratryl alcohol (112.7 U/mg), cathecol (77.8 U/mg), p-aminophenol (68.9 U/mg), RBBR (56.2 U/mg), 4-methoxyphenol (31.1 U/mg), guaiacol (18.3 U/mg), and Reactive Black 5 (12.9 U/mg). Moreover, CeuVP catalyzed the oxidation of α-pinene derived from the essential oil of Eucalyptus robusta to release verbenone as the sole metabolite after a 7 h incubation. Conclusions Versatile peroxidase ( CeuVP) from C. eurysporus VAST02.52 exhibits the highest specific activity against ABTS while also catalyzing oxidation of various substrates tested. Moreover, this study indicated that CeuVP could be effective in the bioconversion of α-pinene into verbenone, a promising compound with numerous applications in the flavors and fragrances industry.

Evaluation of blue textile dye decolorization by immobilized polyphenol oxidase using pumice stone under optimum conditions

Industrial dyes are major pollutants in wastewater and river water with an initial visible concentration of 1 mg/L. Recent studies have shown the possibility of using polyphenol oxidase in catalytic biological treatment due to its ability to oxidize a large number of dyes and pollutants in wastewater and the flexibility to work in wide ranges of temperature, pH and salinity. It is easy availability as well as the low economic cost resulting from its use in biological treatments, this enzyme polyphenol oxidase was used. The findings in this study showed that the extraction of polyphenol oxidase (PPO) from potato peel was homogenized with potassium phosphate buffer (0.1 M, pH 7) at a ratio of 1:10 (weight: volume) for two min. The result of enzyme purification by ion exchange chromatography showed that the yield of this step was 64 % and the specific activity was 6541.67 U/mg. The polyphenol oxidase was immobilized covalently on the functionalized pumice stone (25.2 U/gm) compared with other methods. The characterization results demonstrated that the optimum pH for immobilized and free enzyme activity was 6.0 while the pH range of free and immobilized polyphenol oxidase stability was from 4.5 -7.0 and 3.5 - 8.5 respectively. The better temperature for free and immobilized polyphenol oxidase activity was 25 ºC, whereas the free and immobilized polyphenol oxidase was stable at 15-35 and 15-50 °C respectively. The outcomes showed that the decolorization efficiency of blue textile dye employing immobilized polyphenol oxidase reached 99.85 % after 24 hr. for 100 mg/L concentration while for other concentrations 200, 300, 400, 500 and 1000 mg/L the decolorization efficiencies were 85.96, 76.15, 72.54, 66.94 and 63.5 % respectively. Based on the results, the immobilized polyphenol oxidase on pumice stone is highly efficient in removing textile dyes at large concentrations and in different environmental conditions.

A novel diglycosidase for the transformation of naringin to naringenin and neohesperidose

A novel fungal diglycosidase that transforms naringin into naringenin and neohesperidose, a rare biotransformation, has been purified to homogeneity using a simple procedure involving precipitation of the enzyme from the culture filtrate of the fungal strain using 80 % saturation of ammonium sulphate, dissolving the precipitate in minimum volume of the buffer and dialysing that against the buffer. The purified enzyme gives single protein bands of molecular mass 64.6 kDa in SDS-PAGE analysis. The purity of the enzyme has been further confirmed by the appearance of single protein band in native page analysis. Using naringin as the substrate, the diglycosidase has Km and kcat values of 0.20 m mol L−1 and 0.66 s−1, respectively, at pH 4.0 and 313 K. The specific activity of the purified enzyme using naringin as the natural substrate is 1.018 katal/kg. The diglycosidase also transforms rutin into quercetin and rutinose but has no effect on hesperidin. The feasibilities of preparing neohesperidose from naringin and rutinose from rutin on milligram scales using the pure enzyme have been demonstrated. These results open the way for developing an enzymatic process for preparation of neohesperidose from naringin. The reported diglycosidase has immense future applications in food and pharmaceutical industries.

In Vitro Activation of Paraoxonase 1 by Steroids: An Experimental, Molecular Docking, and Molecular Modelling Study

AbstractParaoxonase 1 (PON1) is an antioxidant enzyme that prevents lipid oxidation by hydrolysing lipid peroxides in the oxidised low‐density lipoprotein (LDL) structure, bound to high‐density lipoprotein (HDL) in serum, and exhibits esterase and lactonase activity. In this work, the hPON1 enzyme was purified from human serum using a hydrophobic gel with Sepharose 4B‐L‐tyrosine‐naphthylamine, and the affinity of some steroid derivatives previously isolated from fungal steroid biotransformations was examined on the pure hPON1 enzyme. The results indicate that all these derivaties activate the hPON1 enzyme to a different extent. Additionally, the binding potential of the most active five steroid derivatives to the PON1 enzyme to form a stable complex was explored through molecular docking and molecular dynamics (MD) simulation. The compounds with the highest potency in the enzymatic assay, S‐8 and S‐20, had the highest binding potential to the enzyme. The stability of the complexes formed by the two compounds was assessed and compared to the stability of the unbound enzyme structure. The enzyme‐compound complexes generally had similar stability to the unbound enzyme structure. Together with this, the MD simulation revealed that compound S‐8 would remain inside the enzyme's binding site during the simulation period, unlike compound S‐20. This situation varies according to the respective derivatives’ functional groups and hydrophobic characteristics.

A Self-Assembling γPFD-SpyCatcher Hydrogel Scaffold for the Coimmobilization of SpyTag-Enzymes to Facilitate the Catalysis of Regulated Enzymes.

In this study, a γPFD-SpyCatcher hydrogel scaffold with the capacity for spontaneous assembly was established. With a maximum loading capacity of a 1:1 molar ratio with SpyTag-enzymes, the immobilized proteins can not only rapidly provide pure enzymes but also exhibit improved thermal and pH stability. The results of the transmission electron microscopic analysis and the traits they present indicated that SpyCatcher promotes the aggregation of γPFD and the formation of hydrogels. In the cell-free pyruvate synthesis system, the γPFD-SpyCatcher coimmobilized SpyTag-hexokinase (HK), SpyTag-phosphofructokinase (PFK) and SpyTag-pyruvate kinase (PK) were employed, and the production of pyruvate increased by 43, 78 and 47% respectively. In in vitro experiments, the oxidative deamination activity of glutamate dehydrogenase (GDH) coimmobilized with γPFD-SpyCatcher was 38% higher than that of purified enzymes. These findings indicate that the γPFD-SpyCatcher-based hydrogels play an important role in breaking the barrier of regulatory enzymes and will provide more strategies for the development of synthetic biology.

Prospects of crude enzymes in replacing pure enzymes for dissolving pulp production.

High-purity cellulose from paper pulp can be obtained after appropriate treatments involving pure xylanases and cellulases/endoglucanases. This study investigated the efficacy of using crude xylanase and cellulase instead of commercial ones to improve process economics. Kraft paper grade pulp produced from veneer waste, hardwood, and non-wood sources was utilized as a more sustainable option. Crude xylanase and cellulase from isolated soil bacteria Bacillus pumilus 3GAH and Bacillus subtilis PJK6 were used for process optimization. The correlation between Fock reactivity, chain scission, and crystallinity after crude-cellulase treatment was established through chemical, FTIR, and XRD analyses. Pentosans in kraft pulp were reduced from an initial 18.7% to 4.9% through sequential treatments with crude xylanase and alkali. Subsequent crude-cellulase treatment, even at 8U/g o.d. pulp, improved Fock reactivity from 28.2% to 61.2%, fulfilling a major criterion for viscose. Thus, crude enzymes can be effectively used for the efficient and economical upgrading of paper pulp to dissolving pulp.

Enzymatic Preparation, Identification by Transmembrane Channel-like 4 (TMC4) Protein, and Bioinformatics Analysis of New Salty Peptides from Soybean Protein Isolate.

To address the public health challenges posed by high-salt diets, this study utilized pepsin and flavourzyme for the continuous enzymatic hydrolysis of a soy protein isolate (SPI). The separation, purification, and identification of salt-containing peptides in SPI hydrolysate were conducted using ultrafiltration (UF), gel filtration chromatography (GFC), and Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS). Subsequently, a molecular docking model was constructed between salt receptor protein transmembrane channel 4 (TMC4) and the identified peptides. Basic bioinformatics screening was performed to obtain non-toxic, non-allergenic, and stable salt peptides. After the enzymatic hydrolysis, separation, and purification of SPI, a component with a sensory evaluation score of 7 and an electronic tongue score of 10.36 was obtained. LC-MS/MS sequencing identified a total of 1697 peptides in the above component, including 84 potential salt-containing peptides. A molecular docking analysis identified seven peptides (FPPP, GGPW, IPHF, IPKF, IPRR, LPRR, and LPHF) with a strong theoretical salty taste. Furthermore, residues Glu531, Asp491, Val495, Ala401, and Phe405 of the peptides bound to the TMC4 receptor through hydrogen bonds, hydrophobic interactions, and electrostatic interactions, thereby imparting a significant salty taste. A basic bioinformatics analysis further revealed that IPHF, LPHF, GGPW, and IPKF were non-toxic, non-allergenic, and stable salt-containing peptides. This study not only provides a new sodium reduction strategy for the food industry, but also opens up new avenues for improving the public's healthy eating habits.

Purification of Glycolate Oxidase Enzymes from Peasm (Pisum sativum l.) and Sorghum (Sorghum sudanense J.) Using Ion-Exchange Chromatography and their Physicochemical Characteristics

Purification of Glycolate Oxidase Enzymes from Peasm (Pisum sativum l.) and Sorghum (Sorghum sudanense J.) Using Ion-Exchange Chromatography and their Physicochemical Characteristics

Insight into protein cross-linking and casein polymerization in pre- and post-hydrolyzed lactose-free UHT milk during long-term storage

During processing and storage of both conventional and lactose-hydrolyzed UHT milk (LHM), aggregation of milk proteins occurs. Protein aggregation can inter alia occur via non-reducible covalent cross-links derived from either Maillard or dehydroalanine (DHA) pathways. To study this further in relation to processing method and lactase enzyme purity, LHM was produced using 3 different lactase preparations, with lactase enzymes added in a dairy setting either before (pre-hydrolysis) or after (post-hydrolysis) UHT treatment. The prepared LHM types were subsequently stored at either 25°C or 35°C for up to one year. Mass spectrometry was used to absolutely quantify the level of furosine, N-ɛ-(carboxymethyl)lysine (CML) and N-ɛ-(carboxyethyl)lysine (CEL), lanthionine (LAN) and lysinoalanine (LAL) in these products using a newly developed method on Triple Q for these processing-induced markers. The results showed higher levels of Maillard related processing markers in pre-hydrolyzed LHM compared with post-hydrolyzed LHM and conventional UHT milk which, on the other hand, contained higher concentrations of DHA-derived cross-links. Proteomics of collected particles from asymmetrical flow field-flow fractionation (AsFlFFF) in combination with gel electrophoresis indicated presence of intra-micellar cross-links during storage, for especially larger particles involving αS1- and αS2-caseins.

Optimization and purification of L-methioninase enzyme purified from clinical samples of Pseudomonas spp.

Background: This study focused on Pseudomonas aeruginosa, which secrete important enzymes in medical and pharmaceutical applications and are isolated from clinical samples. One of these enzymes is L-methioninase, the most desirable enzyme in the medical and industrial fields today since it converted L-methionine into methanethiol, ammonia, and alpha-ketobutyrate. Objectives: Evaluated the optimum conditions of L-methioninase production and then purified it. Methods: Pseudomonas samples were identified by microscopic and biochemical tests and confirmed with the VITEK2 system. After the L-methioninase production was examined, the optimal conditions for the production of L-methioninase (MGL) were established using clinical samples of P. aeruginosa. Additionally, MGL was purified from the supernatant of P.aeruginosa using ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G150 accordingly. Results: There were 33 isolates of P.aeruginosa according to microscopic characteristics and biochemical tests, followed by the VITEK2 system to ensure identification. The optimization conditions were achieved when supplement media with sucrose 1% w\v, casein 1% w\v, pH 7, temperature 37oC, and incubation for 48hrs within the specific activity of 1.6, 3.3, 3.4, 3.4, and 3.5U\mg protein, respectively, and 4.6 fold the amount of the purified enzyme as compared to the crude enzyme. A single 55 kDa band on the SDS-PAGE indicates the enzyme is purified. Conclusions: P.aeruginosa can be an efficient source of L-methioninase, and this enzyme had better and higher specific activity after applying optimum conditions for it and purifying it.

Functionalized ZnFe2O4@Mesoporous silica nano-support for lipase enzyme immobilization: Enhanced biocatalysis and antibacterial activity for food industry applications

Enzymes, as highly efficient biocatalysts, play a crucial role in diverse biotechnological applications. However, enzyme (re)purification and recovery challenges pose substantial obstacles, rendering them costly for industrial utilization. Addressing this, our study developed mesoporous zinc ferrite nanoparticles coated with amine-functionalized mesoporous silica structure (ZnFe2O4@MS) through the solvothermal method. These nanoparticles are an effective nano-support for immobilizing Candida rugosa Lipase (CRL), ensuring easy separability and recoverability through a magnetic field and providing a significant surface area for high mass transference capacity. Immobilizing the lipase on the nano-support (ZnFe2O4@MS/CRL) through covalent bonds on the surface and within the pores led to a notable increase in enzyme activity from 324 U/mg for free enzyme to 689 U/mg for immobilized enzyme. This indicates unchanged active sites post-immobilization, accompanied by enhanced catalytic activity. The immobilized lipases demonstrated prolonged stability at elevated temperatures, maintaining over 59% of initial catalytic activity through five cycles. Furthermore, ZnFe2O4@MS/CRL exhibited substantial antibacterial activity against Staphylococcus aureus, more than that observed in Gram-negative bacteria. The immobilized lipase was also effective in synthesizing banana flavor (isoamyl acetate) in n-hexane, achieving 64% esterification at 45 °C after 4 h. Overall, the study underscores the industrial promise of this immobilized enzyme system, emphasizing its potential for sustainable and cost-effective biotechnological processes.

Production and partial purification of an innovative heat resistant α-keratinase with some remarkable medical and industrial applications

Background and objectives Keratinase has ultimate practical importance in industry, medicine, food industry and waste management fields. Their applications in wool and silk are as good cleaners and in leather industry as the best ever green dehairing agents providing high leather quality, as well they are used as crucial components of sophisticated detergents. Their medical prospective applications are in prion degradation and human callus removal. They convert keratinaceous wastes to valuable products saving the environment from hard keratin waste pollution. Bacteria, fungi and actinomycetes are effective keratinase producers and they are considered the most suitable sources. This study aims to formulate the production medium and to pinpoint the proper physiological conditions for the potent microorganism producing an efficient α-keratinase enzyme. The partial purification of the crude enzyme was successfully performed. The effect of the reaction temperature on both the crude and the partially purified enzyme (PPE) was duly studied with the thermostability of PPE. Some important applications have been implemented on PPE and these include leather dehairing, cloth stain removal, and topical treatment of human callus. Materials and Methods Thirteen recommended microbial strains were screened for effective and applicable α-keratinase productivity. Optimization of the cultural conditions for extracellular enzyme production and also the partial purification of the crude enzyme by ammonium sulphate salting out or by ethanol or acetone precipitation were carried out. The effect of reaction temperature on the enzyme and its thermostability were studied. Finally, the efficiency of the PPE on leather dehairing, stain removal, and human callus treatment was explored. Results and conclusion Among the 13 organisms screened, the fungal strain Trichoderma polysporum HZ-31 was the most potent producer of an influencial α-keratinase. The maximum α-keratinase activity of 58.2 UmL−1 was obtained by the previous-mentioned strain after 5-days fermentation medium containing (%, w/v): whole chicken feathers 0.5, glucose 0.2, peptone 0.5, yeast extract 0.5, K2HPO4 0.1, KH2PO4 0.3, CaCl2 0.1, MgSO4 0.1, and pH 7.0. Acetone fractionation of the crude keratinase was the most proper and offered the most promising keratinase fraction PPE at 80–90% acetone. This fraction had high thermostability and was kept at 55°C for more than 98% of its original activity after 60 min heating and this temperature (55°C) was also the optimum for 2 h enzymatic reaction. Conclusively, the present study succeeded in the achievement of a constitutive extracellular alkaline α-keratinase, which successfully proceeded to complete leather unhairing after 12–16 h at 37°C, afforded high performance to cloth blood stain removal with Arial detergent after 2 h at 50°C and complete degradation of the human callus after 4 h at 50°C.

Advancements in Aqueous Two-Phase Systems for Enzyme Extraction, Purification, and Biotransformation.

In recent years, the increasing need for energy conservation and environmental protection has driven industries to explore more efficient and sustainable processes. Liquid-liquid extraction (LLE) is a common method used in various sectors for separating components of liquid mixtures. However, the traditional use of toxic solvents poses significant health and environmental risks, prompting the shift toward green solvents. This review deals with the principles, applications, and advantages of aqueous two-phase systems (ATPS) as an alternative to conventional LLE. ATPS, which typically utilize water and nontoxic components, offer significant benefits such as high purity and single-step biomolecule extraction. This paper explores the thermodynamic principles of ATPS, factors influencing enzyme partitioning, and recent advancements in the field. Specific emphasis is placed on the use of ATPS for enzyme extraction, showcasing its potential in improving yields and purity while minimizing environmental impact. The review also highlights the role of ionic liquids and deep eutectic solvents in enhancing the efficiency of ATPS, making them viable for industrial applications. The discussion extends to the challenges of integrating ATPS into biotransformation processes, including enzyme stability and process optimization. Through comprehensive analysis, this paper aims to provide insights into the future prospects of ATPS in sustainable industrial practices and biotechnological applications.

Supported ionic liquids to purify microbial L-Asparaginase

L-Asparaginase (ASNase) is a versatile enzyme that converts L-asparagine into ammonia and aspartic acid. This enzyme has applications in the food industry and health sector. However, high purity ASNase is required, resulting in high production costs. Therefore, in this work, two supported ionic liquids (SILs), specifically silica modified with dimethylbutylpropylammonium chloride ([Si][N3114]Cl) or triethylpropylammonium chloride ([Si][N3222]Cl), were investigated as alternative adsorption materials to purify ASNase. Different conditions were evaluated to improve enzyme purity, including total protein content in the cell extract, contact time, and SIL/cell extract ratio (w/v). Under optimal conditions using [Si][N3114]Cl, a maximum ASNase purification of 6.1-fold is achieved in a single step, resulting from the preferential attachment of other proteins on [Si][N3114]Cl SIL. According to the results, hydrophobic interactions rule the selective adsorption of protein impurities from the cell extract by the SIL, thereby increasing the ASNase purification levels. This approach offers a significant advantage, not requiring the desorption and elution of the target enzyme, while envisioning the application of SILs in a flow-through elution approach. The protonation state of protein surface was calculated by computational analysis, revealing that positively charged amino acids such as arginine and lysine block the effective binding of the enzyme to the SILs. Overall, if properly designed, SILs are promising alternative supports for the downstream processing of ASNase from cell extracts.

Purification and characterization of a thermostable Galium aparine β-galactosidase: A competent agent with enhanced cytotoxic activity against MCF-7 cell line

β-galactosidases (EC 3.2.1.23) are capable of catalysing two distinct types of reactions: transgalactosylation and hydrolysis. It is very important for the senescence of cancer cells. It is a lysosomal exoglycosidase involved in the catabolism of glycoconjugates by sequential release of β-linked terminal galactosyl residues. It has profound significance in cancer cell senescence. It can be derived from various sources including plants, animals, bacteria, yeasts, and filamentous fungi. Purification and thermodynamic characterization of β-galactosidase from Galium aparine seeds, and in vitro efficacy assessment of a breast cancer cell line. The enzyme was purified by using ammonium sulphate precipitation, dialysis, DEAE cellulose, and Sephadex G-100 chromatography. The enzyme was purified to 6.4 fold with a yield of 14 % and specific activity of 11.6 U/mg of protein. SDS-PAGE and MALDI-TOF analysis revealed that β-galactosidase is a monomer with a molecular weight of 35 kDa. The optimum pH and temperature for the purified enzyme were 6.0 and 50 ºC. The enzyme had Km and Vmax values of 0.25 mM and 32 µmol/min, respectively. The thermodynamic parameters (ΔHº, ΔGº, ΔSº) for the irreversible denaturation of the enzyme were also investigated at different temperatures (50–75 ºC). Lactose in milk was successfully hydrolyzed using a purified enzyme at a rate of 0.0121 min−1 and 12.12 min. This is better than other studied β-galactosidases. These results suggest the use of β-galactosidase from G. aparine for producing delactosed milk for the lactose-intolerant population and prebiotic synthesis. pH and optimum temperature and its activation by Ca2+ shows that it is suitable for milk processing. As a result, the enzyme was stable even at higher temperatures and had the potential to undergo catalysis, making it economically viable, primarily in the food and pharmaceutical industries. The present study is aimed to purification and thermodynamic characterization of enzyme to assess in vitro efficacy of β-galactosidase on MCF-7 cell line to delineate its therapeutic efficacy.

Direct Screening of PET Hydrolase Activity in Culture Medium Based on Turbidity Reduction.

The development of an efficient screening method for the activity of PET-degrading enzymes represents a significant technological advance in the field of enzyme research, with the potential to facilitate the advancement of enzymes for PET recycling. By examining the stable conditions of PET suspension and enzyme production conditions, we developed a method to quantify PET-degrading enzyme activity in E. coli culture medium using turbidity reduction as an indicator. High PET concentration or ionic strength caused aggregation of PET, and the best condition for activity detection was 0.5 mg mL-1 PET in 50 mM sodium phosphate pH 7.0. Preculture of E. coli increased the purity of enzyme secreted in medium. To evaluate the screening method, 720 colonies of the PET2-7M-H229X-F233X mutant library were analyzed and three candidates of high-activity mutants were obtained. The thermostability of the mutants could also be easily measured by measuring the residual activity after heat treatment. The H229T-F233M mutant showed 3.4 times higher degradation rate against PET film than the template enzyme at the initial time. The molecular dynamics simulation implied that the F233M mutation makes space for making an α helix and that the H229T mutation resolved the steric hindrance with Trp199. These mutations were speculated to change the angle of the Trp199 side chain of PET2 to an angle similar to that of the Trp185 of IsPETase, making it suitable for PET binding to the active center. Screening of activity using PET suspensions is compatible with robotic automation and is expected to be useful for validating computationally predicted mutations.