Purification Of Enzyme Research Articles

Use of pure recombinant human enzymes to assess the disease-causing potential of missense mutations in urea cycle disorders, applied to N-acetylglutamate synthase deficiency.

N-acetylglutamate synthase (NAGS) makes acetylglutamate, the essential activator of the first, regulatory enzyme of the urea cycle, carbamoyl phosphate synthetase 1 (CPS1). NAGS deficiency (NAGSD) and CPS1 deficiency (CPS1D) present identical phenotypes. However, they must be distinguished, because NAGSD is cured by substitutive therapy with the N-acetyl-L-glutamate analogue N-carbamyl-L-glutamate, while curative therapy of CPS1D requires liver transplantation. Since their differentiation is done genetically, it is important to ascertain the disease-causing potential of CPS1 and NAGS genetic variants. With this goal, we previously carried out site-directed mutagenesis studies with pure recombinant human CPS1. We could not do the same with human NAGS (HuNAGS) because of enzyme instability, leading to our prior utilization of a bacterial NAGS as an imperfect surrogate of HuNAGS. We now use genuine HuNAGS, stabilized as a chimera of its conserved domain (cHuNAGS) with the maltose binding protein (MBP), and produced in Escherichia coli. MBP-cHuNAGS linker cleavage allowed assessment of the enzymatic properties and thermal stability of cHuNAGS, either wild-type or hosting each one of 23 nonsynonymous single-base changes found in NAGSD patients. For all but one change, disease causation was accounted by the enzymatic alterations identified, including, depending on the variant, loss of arginine activation, increased Km Glutamate, active site inactivation, decreased thermal stability, and protein misfolding. Our present approach outperforms experimental in vitro use of bacterial NAGS or in silico utilization of prediction servers (including AlphaMissense), illustrating with HuNAGS the value for UCDs of using recombinant enzymes for assessing disease-causation and molecular pathogenesis, and for therapeutic guidance.

Development of a glutamate-functionalized macroporous cation-exchange matrix for partial purification of lysozyme from chicken egg white

The polymeric monoliths, called cryogels, arouse interest due to their versatility and to be a good alternative for enzyme purification processes. Lysozyme is an enzyme that stands out for its antimicrobial activity and is being used as a preservative in the food industry. This work aimed to produce a new cationic exchange matrix functionalized with monosodium glutamate for partial lysozyme purification of egg white. The structural characteristics of the support delivered were evaluated, presenting an ion exchange capacity (IEC) of 362×10−3 mol Na+. kg−1dry cryogel, zero charge point (PCZ) around pH 6.0, and porosity more significant than 90 %. The FTIR and TGA analyses proved that the modification of the XRD matrix indicated the material's amorphous nature. The hydrodynamic studies showed asymmetry, indicating the pores' non-uniformity and low resistance to flow. The lysozyme was purified from egg white, and a purification factor of 15.11 was obtained. SDS-PAGE electrophoresis analysis indicated high purity of lysozyme, with no other protein bands appearing in the eluted solution, indicating that the adsorbent has high potential for application in lysozyme purification processes.

Coupling protein scaffold and biosilicification: A sustainable and recyclable approach for d-mannitol production via one-step purification and immobilization of multienzymes

Enzymatic synthesis of biochemicals in vitro is vital in synthetic biology for its efficiency, minimal by-products, and easy product separation. However, challenges like enzyme preparation, stability, and reusability persist. Here, we introduced a protein scaffold and biosilicification coupled system, providing a singular process for the purification and immobilization of multiple enzymes. Using d-mannitol as a model, we initially constructed a self-assembling EE/KK protein scaffold for the co-immobilization of glucose dehydrogenase and mannitol dehydrogenase. Under an enzyme-to-scaffold ratio of 1:8, a d-mannitol yield of 0.692 mol/mol was achieved within 4 h, 2.16-fold higher than the free enzymes. The immobilized enzymes retained 70.9 % of the initial joint activity while the free ones diminished nearly to inactivity after 8 h. Furthermore, we incorporated the biosilicification peptide CotB into the EE/KK scaffold, inducing silica deposition, which enabled the one-step purification and immobilization process assisted by Spy/Snoop protein-peptide pairs. The coupled system demonstrated a comparable d-mannitol yield to that of EE/KK scaffold and 1.34-fold higher remaining activities after 36 h. Following 6 cycles of reaction, the immobilized system retained the capability to synthesize 56.4 % of the initial d-mannitol titer. The self-assembly co-immobilization platform offers an effective approach for enzymatic synthesis of d-mannitol and other biochemicals.

Thermophilic lipases in industrial applications: Stutzerimonas stutzeri as a Case Study in Nigeria and the US

Thermophilic lipases derived from Stutzerimonas stutzeri have garnered significant attention in recent years due to their robustness and efficiency in various industrial applications. This review delves into the research, development, and application of these enzymes, with a comparative analysis between Nigeria and the United States. In both nations, researchers have explored the enzymatic properties of thermophilic lipases from Stutzerimonas stutzeri, aiming to optimize their performance for diverse industrial processes. The research encompasses studies on enzyme purification, characterization, and genetic engineering to enhance catalytic activity and stability under extreme conditions. Additionally, efforts have been directed towards understanding the enzyme's structure-function relationship to tailor it for specific applications. In Nigeria, the focus on thermophilic lipases from Stutzerimonas stutzeri has been primarily driven by the need for sustainable biotechnological solutions in various industries, including food, pharmaceuticals, and biofuel production. The enzymes' thermostability makes them particularly suitable for high-temperature processes prevalent in Nigeria's tropical climate, offering potential for cost-effective and eco-friendly alternatives to conventional chemical catalysts. In contrast, the United States has witnessed extensive research and development in thermophilic lipases for industrial applications, with a strong emphasis on biocatalysis in the pharmaceutical, detergent, and biofuel sectors. The advanced infrastructure and investment in biotechnology have facilitated the commercialization of Stutzerimonas stutzeri lipases, leading to their integration into various industrial processes to improve efficiency and sustainability. Despite the similarities in research objectives, disparities exist in the technological advancements and industrial utilization of thermophilic lipases from Stutzerimonas stutzeri between Nigeria and the US. While Nigeria faces challenges related to limited resources and infrastructure, the US benefits from well-established research institutions and industrial networks, enabling accelerated innovation and commercialization. Bridging these gaps through international collaborations and knowledge exchange could facilitate the widespread adoption of thermophilic lipases for sustainable industrial development globally.

Protective effects of oyster polypeptide on cyclophosphamide-induced immunosuppressed rats.

Oyster polypeptide (OP) is a mixture of oligopeptides extracted from oysters through enzyme lysis, separation, and purification. It is associated with immunomodulatory effects, but the underlying mechanisms are not known. This study therefore combined proton nuclear magnetic resonance (1H-NMR) urinary metabolomics and 16S rRNA gene sequencing of the gut microbiome to determine the immunoprotective mechanisms of OP in rats subjected to cyclophosphamide-induced immunosuppression. Oyster polypeptide restored the body weight and the structure of spleen and thymus in rats with cyclophosphamide-induced immunosuppression. It upregulated the levels of white blood cells (WBCs), hemoglobin (HGB), platelets (PLT), red blood cells (RBCs), immunoglobulin G (IgG), immunoglobulin M (IgM), cytokines such as interleukin‑6 (IL-6) and tumor necrosis factor-α (TNF-α), and increased the numbers of CD3+ and CD4+ T cells in the immunosuppressed rats. The 1H-NMR metabolomics results showed that OP significantly reversed the levels of ten metabolites in urine, including 2-oxoglutarate, citrate, dimethylamine, taurine, N-phenylacetylglycine, alanine, betaine, creatinine, uracil, and benzoate. The 16S rRNA gene sequencing results showed that OP restored the gut microbiome homeostasis by increasing the abundance of beneficial bacteria and reducing the abundance of pathogenic bacteria. Finally, a combination of metabolomics and microbiomics found that the metabolism of taurine and hypotaurine, and the metabolism of alanine, aspartate, and glutamate were disturbed, but these metabolic pathways were restored by OP. This study demonstrated that OP had immunoprotective effects in rats with cyclophosphamide-induced immunosuppression by restoring key metabolic pathways and the gut microbiome homeostasis. Our findings provide a framework for further research into the immunoregulatory mechanisms of OP and its potential use in drugs and nutritional supplements. © 2024 Society of Chemical Industry.

Purification and Biochemical Characterization of a Novel Fibrinolytic Enzyme from Culture Supernatant of Coprinus comatus.

A novel fibrinolytic enzyme was produced by the liquid fermentation of Coprinus comatus. The enzyme was purified from the culture supernatant by hydrophobic interactions, gel filtration, and ion exchange chromatographies. It was purified by 241.02-fold, with a specific activity of 3619 U/mg and a final yield of 10.02%. SDS-PAGE analysis confirmed the purity of the enzyme, showing a single band with a molecular weight of 19.5 kDa. The first nine amino acids of the N-terminal of the purified enzyme were A-T-Y-T-G-G-S-Q-T. The enzyme exhibited optimal activity at a temperature of 42 °C and pH 7.6. Its activity was significantly improved by Zn2+, K+, Ca2+, Mn2+, and Mg2+ while being inhibited by Fe2+, Fe3+, Al2+, and Ba2+. The activity of the enzyme was completely inhibited by ethylenediamine tetraacetic acid (EDTA), and it was also dose-dependently inhibited by phenylmethylsulfonyl fluoride (PMSF) and soy trypsin inhibitor (SBTI). However, inhibitors such as N-α-tosyl-L-phenylalanine chloromethyl ketone (TPCK), aprotinin, and pepstatin did not significantly affect its activity, suggesting that the enzyme was a serine-like metalloproteinase. The enzyme acted as both a plasmin-like fibrinolytic enzyme and a plasminogen activator, and it also exhibited the capability to hydrolyze fibrinogen and fibrin. In vitro, it demonstrated the ability to dissolve blood clots and exhibit anticoagulant properties. Furthermore, it was found that the enzyme prolonged activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT), and reduced the levels of fibrinogen (FIB) and prothrombin activity (PA). Based on these studies, the enzyme has great potential to be developed as a natural agent for the prevention and treatment of thrombotic diseases.

Precision Engineering of the Co-immobilization of Enzymes for Cascade Biocatalysis.

The design and orderly layered co-immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N-terminus of an alcohol dehydrogenase (ADH) and an aldo-keto reductase (AKR), respectively. A non-canonical amino acid (ncAA), p-azido-L-phenylalanine (p-AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide-alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual-enzyme coating on porous microspheres. The ordered dual-enzyme reactor was subsequently used to synthesize (S)-1-(2-chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double-layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single-layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Precision Engineering of the Co‐immobilization of Enzymes for Cascade Biocatalysis

AbstractThe design and orderly layered co‐immobilization of multiple enzymes on resin particles remain challenging. In this study, the SpyTag/SpyCatcher binding pair was fused to the N‐terminus of an alcohol dehydrogenase (ADH) and an aldo‐keto reductase (AKR), respectively. A non‐canonical amino acid (ncAA), p‐azido‐L‐phenylalanine (p‐AzF), as the anchor for covalent bonding enzymes, was genetically inserted into preselected sites in the AKR and ADH. Employing the two bioorthogonal counterparts of SpyTag/SpyCatcher and azide–alkyne cycloaddition for the immobilization of AKR and ADH enabled sequential dual‐enzyme coating on porous microspheres. The ordered dual‐enzyme reactor was subsequently used to synthesize (S)‐1‐(2‐chlorophenyl)ethanol asymmetrically from the corresponding prochiral ketone, enabling the in situ regeneration of NADPH. The reactor exhibited a high catalytic conversion of 74 % and good reproducibility, retaining 80 % of its initial activity after six cycles. The product had 99.9 % ee, which that was maintained in each cycle. Additionally, the double‐layer immobilization method significantly increased the enzyme loading capacity, which was approximately 1.7 times greater than that of traditional single‐layer immobilization. More importantly, it simultaneously enabled both the purification and immobilization of multiple enzymes on carriers, thus providing a convenient approach to facilitate cascade biocatalysis.

Surface Engineering of Escherichia coli to Display Its Phytase (AppA) and Functional Analysis of Enzyme Activities.

Escherichia coli phytase (AppA) is widely used as an exogenous enzyme in monogastric animal feed mainly because of its ability to degrade phytic acid or its salt (phytate), a natural source of phosphorus. Currently, successful recombinant production of soluble AppA has been achieved by gene overexpression using both bacterial and yeast systems. However, some methods for the biomembrane immobilization of phytases (including AppA), such as surface display on yeast cells and bacterial spores, have been investigated to avoid expensive enzyme purification processes. This study explored a homologous protein production approach for displaying AppA on the cell surface of E. coli by engineering its outer membrane (OM) for extracellular expression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of total bacterial lysates and immunofluorescence microscopy of non-permeabilized cells revealed protein expression, whereas activity assays using whole cells or OM fractions indicated functional enzyme display, as evidenced by consistent hydrolytic rates on typical substrates (i.e., p-nitrophenyl phosphate and phytic acid). Furthermore, the in vitro results obtained using a simple method to simulate the gastrointestinal tract of poultry suggest that the whole-cell biocatalyst has potential as a feed additive. Overall, our findings support the notion that biomembrane-immobilized enzymes are reliable for the hydrolysis of poorly digestible substrates relevant to animal nutrition.

Enzymatic Modification of Cow, Buffalo and Goat Milk Proteins and their Effect on Anti-Oxidant Activity and Digestibility

Aim: Buffalo milk and goat milk were enzymatically hydrolyzed to have 5, 10 and 15 per cent DH to improve the techno-functional properties of milk proteins in order to utilize in production of channa based dairy products. Study Design: A significant contribution to total milk production of India comes from buffalo milk and followed by cow and goat milk. Inspite of that, Buffalo milk and goat milk are not being utilized for many products in view of their inherent problems associated in production of Chhana based dairy products. Buffalo milk and goat milk were enzymatically hydrolyzed to have 5, 10 and 15 per cent DH to improve the techno-functional properties of milk proteins in order to utilize in production of products. Place and Duration of Study: Karnataka Veterinary Animal and Fisheries Sciences University (KVAFSU), Dairy Science College, Hebbal, Bangalore Karnataka, India Methodology: The Goat milk samples were collected from Sinchana Goat and sheep farm, Marenahalli village (Bengaluru Rural Dist) and Buffalo milk was obtained from country Delight Pvt. Ltd., J. P. Nagar, Bengaluru, Karnataka. Cow milk used in this investigation was collected from SEDP, Dairy Science College, Hebbal, Bangalore. Commercially available pure Neutrase enzyme was purchased from DSM Nutritional Products India Pvt. Ltd, Bangalore. All the glassware used were soaked in chromic acid solution, repeatedly washed with water, rinsed with distilled water and dried before use. For microbiological analysis dried test tubes, conical flask, pipettes were cotton plugged and sterilized in hot air oven. The chemicals and reagents used in this study were mainly of analytical grade procured from Prince Laboratory Company Pvt limited, Bangalore. The protein molecular weight markers were used for the electrophoretic study were procured from Bangalore Genei Pvt Ltd. Results: Highest protein content was noted in buffalo casein hydrolysate (83.0 %) than cow (81.0 %) and goat (81.16 %) and lowest nitrogen and ash content was observed in goat casein hydrolysate (13.65 %) and (15.20 %) respectively. (Akshya pl include few lines of enzymatic modification on anti-oxidant property and digestibility copy from discussions) Conclusion: Though buffalo milk contributes nearly half of the total milk production of India and goat milk carries both nutritional and therapeutic benefits, the potential utilization of both these milks are very much limited due to the inherent problems associated with them. This investigation has shown that by enzymatically hydrolyzing buffalo milk and goat milk to 5 per cent DH results significant improvement in physico-chemical and functional attributes Rasagulla which is on par with cow’s milk protein hydrolysates. Recommendation: Scavenging activity or anti-oxidant activity and Digestibility of hydrolysates has been studied as influenced by the extent of enzymatic modification in this study.

Use of Microbial L-asparaginase in Cancer Treatment -A Detail Review

Abstract The exceptional anticancer effects of l-asparaginases, which hydrolyze plasma l-asparagine and l-glutamine, have drew a lot of interest in recent years. This enzyme is effective against acute lymphoblastic leukemia (ALL) and lymphosarcoma, as well as ALL in children, neoplasia, and various other cancer .The l-asparaginase anticancer application technique has achieved amazing advances in modern oncology by depleting plasma l-asparagine to suppress cancer cell development, notably in youngsters. As bacterial alternative enzyme sources for anticancer therapy, high levels of l-asparaginase enzyme production by Escherichia coli, Erwinia species, Streptomyces, and Bacillus subtilis species are very desirable. Purification of L-asparaginase by Ammonium Sulphate precipitation and various chromatographic technique methods, and improvement of enzyme purity by dialysis confirmation of molecular weight with SDS page.

CHEMICAL COMPOSITION, AMINO ACIDS PROFILE AND ANTIOXIDANT ACTIVITY OF ENZYMATIC PROTEIN HYDROLYSATE FROM Bohadschia marmorata SEA CUCUMBER

The sea cucumber is one of the potential sub-exports of fisheries. Sea cucumbers contain highly nutritious and bioactive compounds with antioxidant, antibacterial, anticoagulant, anti-inflammatory, and antiviral activities. Protein hydrolysates from sea cucumbers have the potential as antioxidants with the ability to reduce free radicals. The objective of this research is to examine the chemical characteristics and antioxidant activity of protein hydrolysates from Bohadschia marmorata sea cucumber. The research method used in this study was experimental and laboratory analysis. The results showed that the chemical composition of fresh Bohadschia marmorata sea cucumber had high moisture content (80.45 %), ash content (4.12 %), low fat content (6.17 %), and low protein content (8.96 %). This could be due to the environmental conditions and nutrient absorption by Bohadschia marmorata sea cucumbers. Fifteen types of amino acids with a total of 53.81 % were obtained from this study. The yield obtained for crude papain enzyme concentration was 16.26 %, and for pure papain enzyme concentration, it was 11.23 %. The antioxidant activity with IC50 > 200 indicates very weak antioxidant activity, and the resulting molecular weight reached 73.93 kDa

Purification and Biochemical Characterization of Phytase Enzyme from Lactobacillus brevis Isolated from Fresh Kashar Cheese

In the last 20 years, phytase enzyme has attracted the attention of scientists in the fields of environmental protection, nutrition and biotechnology. Myo-inositol hexaphosphate phosphohydrolase (phytase), which is a type of phosphatase enzyme, catalyzes the hydrolysis of phytate into less phosphorylated inorganic phosphates and phytate. Phytases of microbial origin are widely used in biotechnological applications (paper industry, feed industry, food industry and soil improvement).
 In the present study, phytase enzyme was partially purified from Lactobacillus brevis NM-34 strain isolated from fresh kashar cheese and the pH and temperature values at which the enzyme showed optimum activity were determined. L. brevis NM-34 showed a phytase activity of 243.80 U/mL as a result of ammonium sulfate precipitation. In the ammonium sulfate range (40-60%), where the highest phytase activity was observed, the protein concentration was measured as 0.989 mg/mL. Km and Vmax values of phytase enzyme were determined as 0.0146 mM and 1.6 µmol/min, respectively. The pH and temperature values at which the partially purified phytase showed optimum activity were found to be pH 5 and 50 °C, respectively. Based on the findings from our research, the enzyme purified from this bacterium was found to have unique properties that make it suitable for use in industrial applications.

Affinity gel synthesis from the p-aminobenzoic acid derivative 4-amino-2-methylbenzoic acid and purification of polyphenol oxidase from various plant sources

The polyphenol oxidase (PPO) enzyme, which causes enzymatic browning, has been repeatedly purified from fruit and vegetables by affinity chromatography. In the present research, Sepharose 4B-l-tyrosine-4-amino-2-methylbenzoic acid, a novel affinity gel for the purification of the PPO enzyme with high efficiency, was synthesized. Additionally, Sepharose 4B-l-tyrosine-p-aminobenzoic acid affinity gel, known in the literature, was also synthesized, and 9.02, 16.57, and 28.13 purification folds were obtained for the PPO enzymes of potato, mushroom, and eggplant by the reference gel. The PPO enzymes of potato, mushroom, and eggplant were purified 41.17, 64.47, and 56.78-fold from the new 4-amino-2-methylbenzoic acid gel. Following their isolation from the new affinity column, the assessment of PPO enzyme purity involved the utilization of SDS-PAGE. According to the results from SDS-PAGE and native PAGE, the molecular weight of each enzyme was 50 kDa. Then, the inhibition effects of naringin, morin hydrate, esculin hydrate, homovanillic acid, vanillic acid, phloridzin dihydrate, and p-coumaric acid phenolic compounds on purified potato, mushroom, and eggplant PPO enzyme were investigated. Among the tested phenolic compounds, morin hydrate was determined to be the most potent inhibitor on the potato (Ki: 0.07 ± 0.03 μM), mushroom (Ki: 0.7 ± 0.3 μM), and eggplant (Ki: 4.8 ± 1.2 μM) PPO enzymes. The studies found that the weakest inhibitor was homovanillic acid for the potato (Ki: 1112 ± 324 μM), mushroom (Ki: 567 ± 81 μM), and eggplant (Ki: 2016.7 ± 805.6 μM) PPO enzymes. Kinetic assays indicated that morin hydrate was a remarkable inhibitor on PPO.

One-Step Purification and Immobilization of Glycosyltransferase with Zn-Ni MOF for the Synthesis of Rare Ginsenoside Rh2.

Uridine diphosphate (UDP)-glucosyltransferases (UGTs) have received increasing attention in the field of ginsenoside Rh2 conversion. By harnessing the metal chelation between transition metal ions and imidazole groups present on His-tagged enzymes, a specific immobilization of the enzyme within metal-organic frameworks (MOFs) is achieved. This innovative approach not only enhances the stability and reusability of the enzyme but also enables one-step purification and immobilization. Consequently, the need for purifying crude enzyme solutions is effectively circumvented, resulting in significant cost savings during experimentation. The use of immobilized enzymes in catalytic reactions has shown great potential for achieving higher conversion rates of ginsenoside Rh2. In this study, highly stable mesoporous Zn-Ni MOF materials were synthesized at 150 °C by a solvothermal method. The UGT immobilized on the Zn-Ni MOF (referred to as UGT@Zn-Ni MOF) exhibited superior pH adaptability and thermal stability, retaining approximately 76% of its initial activity even after undergoing 7 cycles. Furthermore, the relative activity of the immobilized enzyme remained at an impressive 80.22% even after 45 days of storage. The strong specific adsorption property of Zn-Ni MOF on His-tagged UGT was confirmed through analysis using polyacrylamide gel electrophoresis. UGT@Zn-Ni MOF was used to catalyze the conversion reaction, and the concentration of rare ginsenoside Rh2 was generated at 3.15 μg/mL. The results showed that Zn-Ni MOF is a material that can efficiently purify and immobilize His-tagged enzyme in one step and has great potential for industrial applications in enzyme purification and ginsenoside synthesis.

Bioproduction, purification, partial characterization and phenol removal efficacy of tyrosinase enzyme from Streptomyces sp. strain MR28

The study focused on the production of the tyrosinase enzyme from Streptomyces sp. MR28 and its potency in removal of phenol content from water using free and immobilized tyrosinase enzyme. The tyrosinase was produced by Streptomyces sp. MR28 in liquid tyrosine broth medium, and it was further purified to near its homogeneity by employing, precipitation, dialysis, and column chromatography. After the purification, 44.49% yield with a 4 fold purification was achieved. The characterization of the purified enzyme showed a single major peak on HPLC and a solitary band on SDS-PAGE. The purified tyrosinase enzyme was active at a pH of 7.0 and a temperature of 30 °C. Further immobilization of purified tyrosinase was performed using the sodium alginate entrapment method. The capacity of the purified tyrosinase to remove phenol in water was evaluated by spectrophotometric method. The free tyrosinase enzyme-treated solutions showed a gradual decrease in the concentration of phenol with increased incubation time at 30 °C and 40 °C, at 90 min of the incubation time, it showed maximum efficacy in removing phenol from the solution. At 50 °C and 60 °C, the free tyrosinase enzyme exhibited very less capacity to remove the phenol. The immobilized enzyme showed good capacity for the removal of phenol from the solutions; the concentration of phenol in the solution decreased with an increase in the incubation time. At temperatures of 40 °C and 50 °C, the immobilized tyrosinase enzyme beads showed significant removal of phenol from the solution, and at temperatures of 30 °C and 60 °C, they also exhibited good capacity for the removal of phenol. At the end of the 90 min incubation period, it exhibited good capability. The current study suggests using immobilized microbial tyrosinase enzyme can be used for the removal of phenol from the contaminated water in a greener manner.

Production, characterization, and applications of a novel thermo-acidophilic L-asparaginase of Pseudomonas aeruginosa CSPS4

In present investigation, a potential L-asparaginase-producing bacterial isolate, Pseudomonas aeruginosa CSPS4, has been explored to enhance the production and purification of the asparaginase enzyme. Production of L-asparaginase is enhanced using the 'one variable at a time approach (OVAT)'. In Placket Burman (PB) analysis, pH, sucrose, and temperature significantly influence L-asparaginase production. Thereafter, L-asparaginase enzyme was recovered from culture broth using fractional precipitation with chilled acetone. The partially purified L-asparaginase showed a molecular weight of ~35 KDa on SDS-PAGE. L-asparaginase was characterized as a thermo-acidophilic enzyme exhibiting optimum pH and temperature of 6.0 and 60 °C, respectively. These characteristics render this enzyme novel from other available asparaginases of Pseudomonas spp. L-asparaginase activity remained unaffected by different modulators. L-asparaginase of this investigation was successfully employed for acrylamide degradation in commercial fried potato chips, establishing its applicability in food industries.

Extraction and purification techniques of the bio-catalyst cabbage peroxidase enzyme to remove reactive dyes and bisphenol-A pollutants

The industrial dyes and phenols resulting from various industries have a devastating impact on human health and the ecosystem when they enter the food chain, making it necessary to research effective and sustainable treatments. This study informs a novel resource of peroxidase, extracted from discarded cabbage stems purchased from restaurant garbage to use as a biocatalyst treatment. The research focused on comparing the effectiveness of crude and purified enzymes for biodegrading dyes and bisphenol-A. The purification fold for Cabbage legs peroxidase (CLP) was 2.14 with a 39 % yield. At pH 6 and 40 °C the optimum activity of the enzyme was observed, and from its initial activity, the retention is 65% at 60 °C. The (CLP) has been assessed by determining the Michaelis-Menten constant Km value for pyrogallol and H2O2, which were found to be 0.78 mM and 0.047 mM, respectively. For RB49 and RG19 the percentage decolorization was 86% and 78%, respectively after 10-h incubation. The pure enzyme concurrently displayed 79% and 75% decolorization percentages for RB49 and RG19 at the same time and conditions. The crude enzyme demonstrated outstanding effectiveness in removing pollutants. The results indicate that (CLP) holds a promising potential for environmental remediation and bioengineering problems.

Cellulolytic and Ethanologenic Evaluation of Heterotermes indicola's Gut-Associated Bacterial Isolates.

Cellulose is the basic component of lignocellulosic biomass (LCB) making it a suitable substrate for bioethanol fermentation. Cellulolytic and ethanologenic bacteria possess cellulases that convert cellulose to glucose, which in turn yields ethanol subsequently. Heterotermes indicola is a subterranean termite that causes destructive damage by consuming wooden structures of infrastructure, LCB products, etc. Prospectively, the study envisioned the screening of cellulolytic and ethanologenic bacteria from the termite gut. Twenty six bacterial strains (H1-H26) based on varied colonial morphologies were isolated. Bacterial cellulolytic activity was tested biochemically. Marked gas production in the form of bubbles (0.1-4 cm) in Durham tubes was observed in H3, H7, H13, H15, H17, H21, and H22. Sugar degradation of all isolates was indicated by pink to maroon color development with the tetrazolium salt. Hallow zones (0.42-11 mm) by Congo red staining was exhibited by all strains except H2, H7, H8, and H19. Among the 26 bacterial isolates, 12 strains were identified as efficient cellulolytic bacteria. CMCase activity and ethanol titer of all isolates varied from 1.30 ± 0.03 (H13) to 1.83 ± 0.01 (H21) umol/mL/min and 2.36 ± 0.01 (H25) to 7.00 ± 0.01 (H21) g/L, respectively. Likewise, isolate H21 exhibited an ethanol yield of 0.40 ± 0.10 g/g with 78.38 ± 2.05% fermentation efficiency. Molecular characterization of four strains, Staphylococcus sp. H13, Acinetobacter baumanni H17, Acinetobacter sp. H21, and Acinetobacter nosocomialis H22, were based on the maximum cellulolytic index and the ethanol yield. H. indicola harbor promising and novel bacteria with a natural cellulolytic tendency for efficient bioconversion of LCB to value-added products. Hence, the selected cellulolytic bacteria can become an excellent addition for use in enzyme purification, composting, and production of biofuel at large.

Purification, biochemical characterization, and biotechnological applications of a multifunctional enzyme from the Thermoascus aurantiacus PI3S3 strain

The filamentous Thermoascus aurantiacus fungus characterized by its thermophilic nature, is recognized as an exceptional producer of various enzymes with biotechnological applications. This study aimed to explore biotechnological applications using polygalacturonase (PG) derived from the Thermoascus aurantiacus PI3S3 strain. PG production was achieved through submerged fermentation and subsequent purification via ion-exchange chromatography and gel filtration methods. The crude extract exhibited a diverse spectrum of enzymatic activities including amylase, cellulase, invertase, pectinase, and xylanase. Notably, it demonstrated the ability to hydrolyze sugarcane bagasse biomass, corn residue, and animal feed. The purified PG had a molecular mass of 36 kDa, with optimal activity observed at pH 4.5 and 70 °C. The activation energy (Ea) was calculated as 0.513 kJ mol−1, highlighting activation in the presence of Ca2+. Additionally, it displayed apparent Km, Vmax, and Kcat values of at 0.19 mg mL−1, 273.10 U mL−1, and 168.52 s−1, respectively, for hydrolyzing polygalacturonic acid. This multifunctional PG exhibited activities such as denim biopolishing, apple juice clarification, and demonstrated both endo- and exo-polygalacturonase activities. Furthermore, it displayed versatility by hydrolyzing polygalacturonic acid, carboxymethylcellulose, and xylan. The T. aurantiacus PI3S3 multifunctional polygalacturonase showed heightened activity under acidic pH, elevated temperatures, and in the presence of calcium. Its multifunctional nature distinguished it from other PGs, significantly expanding its potential for diverse biotechnological applications.