Purification Of Enzyme Research Articles

Supplementation of citrus pectin with whole-cell pectinase PG5 on Pichia pastoris promotes recovery of colitis and enhances intestinal barrier function in DSS-treated mice

A whole-cell biocatalyst was developed by genetically engineering pectinase PG5 onto the cell surface of Pichia pastoris using Gcw12 as the anchoring protein. Whole-cell PG5 eliminated the need for enzyme extraction and purification, while also exhibiting enhanced thermal stability, pH stability, and resistance to proteases in vitro compared to free PG5. Magnetic resonance mass spectrometry analysis revealed that whole-cell PG5 efficiently degraded citrus pectin, resulting in the production of a mixture of pectin oligosaccharides. The primary components of the mixture were trigalacturonic acid, followed by digalacturonic acid and tetragalacturonic acid. Supplementation of citrus pectin with whole-cell PG5 resulted in a more pronounced protective effect compared to free PG5 in alleviating colitis symptoms and promoting the integrity of the colonic epithelial barrier in a mouse model of dextran sulfate sodium-induced colitis. Hence, this study demonstrates the potential of utilizing whole-cell pectinase as an effective biocatalyst to promote intestinal homeostasis in vivo.

Pengaruh suhu berbeda terhadap aktifitas enzim kolagenase dari usus ikan cunang (Congresox talabon)

Collagenase enzyme is a type of protease enzyme. The activity of an enzyme is affected by substrate, temperature, inhibitor, and pH. One of the sources of enzymes is the internal organs of fish, namely the gut. This study aims to determine the activity of the collagenase enzyme at different temperatures. The experimental design used was randomized and full non-factorial. The treatments used were different incubation temperatures (50, 55, and 60oC) with three replications. Parameter analysis comprised fish proportion, enzyme activity, and dissolved protein. The results showed that the parts of the yellow pike conger fish were meat (64.25%), bones (19.98%), internal organs (including intestines) (4.50%), and skin (6.30%). Collagenase enzyme activity at temperatures 50, 55, and 60oC was 1.154, 1.702, and 0.667 U/mL, respectively. The dissolved proteins content was 12.89 mg/mL. The optimum temperature of the collagenase enzyme from the fish gut was 55oC with an enzyme activity of 1,702 U/mL. The dissolved protein content of the gut yellow pike conger extract was 12.89 mg/mL Purification of the collagenase enzyme from of the gut yellow pike conger was needed to obtain a higher enzyme activity value.

Simple and efficient method for the extraction, isolation, and purification of peroxidase enzyme from wheat bran

AbstractPeroxidase is an industrially important enzyme with a broad range of applications. In this study, the extraction, isolation, and purification of peroxidase was conducted with wheat bran as a feedstock. Several commercially available buffers (K2HPO4, Na3PO4, CH3COONa, and Tris‐base) were explored for the extraction of peroxidase from wheat bran. Of the tested media, sodium acetate buffer gave an excellent performance and displayed maximum protein (0.44 mg mL−1) and enzyme activity (28 U mL−1). A process optimization study was also conducted to obtain the maximum protein concentration and enzyme activity. The intensified process provided 1.39 mg mL−1 protein concentration with 90 U mL−1 enzyme activity with specific enzyme activity of 135.8 U mg−1. Crude enzyme was purified using a three‐step process: (a) precipitation, (b) ultrafiltration, and (c) chromatographic purification. Commercially available strong and weak ion exchange resins, with different surface properties, were tested for purification. The weak anion exchange resin showed an excellent performance for maximum binding of peroxidase. Adsorption study investigated the best fit model of Freundlich isotherm with maximum binding capacity of 54 mg mL−1. Overall, purification provided peroxidase with a protein concentration of 1.24 mg mL−1 and 727.58 U mg −1 specific enzyme activity with purity increased by around ~6.6 fold. The reported process provides a simple and efficient method for the extraction and purification of peroxidase enzyme from wheat bran for its efficient valorization.

Enhancement of α-galactosidase production using novel Actinoplanes utahensis B1 strain: sequential optimization and purification of enzyme.

α-Galactosidase is an important exoglycosidase belonging to the hydrolase class of enzymes, which has therapeutic and industrial potential. It plays a crucial role in hydrolyzing α-1,6 linked terminal galacto-oligosaccharide residues such as melibiose, raffinose, and branched polysaccharides such as galacto-glucomannans and galactomannans. In this study,Actinoplanes utahensis B1 was explored for α-galactosidase production, yield improvement, and activity enhancement by purification. Initially, nine media components were screened usingthe Plackett-Burman design (PBD). Among these components, sucrose, soya bean flour, and sodium glutamate were identified as the best-supporting nutrients for the highest enzyme secretion by A. Utahensis B1. Later, the Central Composite Design (CCD) was implemented to fine-tune the optimization of these components. Based on sequential statistical optimization methodologies, a significant, 3.64-fold increase in α-galactosidase production, from 16 to 58.37 U/mL was achieved. The enzyme was purified by ultrafiltration-I followed by multimode chromatography and ultrafiltration-II. The purity of the enzyme was confirmed by Sodium Dodecyl Sulphate-Polyacrylamide Agarose Gel Electrophoresis (SDS-PAGE) which revealed a single distinctive band with a molecular weight of approximately 72kDa. Additionally, it was determined that this process resulted in a 2.03-fold increase in purity. The purified α-galactosidase showed an activity of 2304 U/mL with a specific activity of 288 U/mg. This study demonstrates the isolation of Actinoplanes utahensis B1 and optimization of the process for the α-galactosidase production as well as single-step purification.

Potential Application of Bacterial Lipase from Bacillus subtilis in the Production of Biodisel

This work aims to evaluate the potential of bacterial lipase of Bacillus subtilis in biodiesel production. Assays were performed to obtain, purify, and immobilize the enzyme and compare chemical and enzymatic transesterification tests. The Tryptic Soy Broth (TSB) culture medium without supplementation showed the highest enzymatic activity and purification yields when using 60% of (NH4)2SO4. Citral was the most efficient agent for lipase immobilization. Even though the free enzymes showed a higher activity rate, the immobilized enzymes are associated with a better-quality product. In conclusion, the lipase enzyme showed positive potential in biodiesel production.

Onion (Allium cepa L.) Skin Waste Valorization: Unveiling the Phenolic Profile and Biological Potential for the Creation of Bioactive Agents through Subcritical Water Extraction.

Onion skin waste (OSW), the primary non-edible byproduct from onion processing, offers a renewable source of bioactive compounds. This study aims to valorize OSW through subcritical water extraction (SWE), aligning with a circular economy and biorefinery principles. SWE was carried out at 145 °C and 50 bar for 50 min in a discontinuous reactor, producing a phenolic-rich extract (32.3 ± 2.6 mg/g) dominated by protocatechuic acid (20.3 ± 2.5 mg/g), quercetin-4'-O-glucoside (7.5 ± 0.2 mg/g), and quercetin (3.2 ± 0.6 mg/g). Additionally, the extract contains sugars (207.1 ± 20.3 mg sucrose-Eq/g), proteins (22.8 ± 1.6 mg BSA-Eq/g), and free amino acids (20.4 ± 1.2 mg arginine-Eq/g). Its phenolic richness determines its scavenging activity against ●NO and O2●- radicals and its α-glucosidase and aldose-reductase inhibition without affecting α-amylase. Notably, the extract demonstrates significant α-glucosidase inhibition (IC50 = 75.6 ± 43.5 µg/mL), surpassing acarbose (IC50 = 129.5 ± 1.0 µg/mL) in both pure enzyme and cell culture tests without showing cytotoxicity to AGS, HepG2, and Caco-2 human cell lines. The extract's bioactivity and nutritional content make it suitable for developing antioxidant and antidiabetic nutraceutical/food components, highlighting SWE's potential for OSW valorization without using organic solvents.

Exploring the use of supercritical carbon dioxide in enzymatic hydrolysis of cellulosic substrate

Enzymes are critical components for production of fermentable sugars in lignocellulosic biorefineries. Their activity could be influenced by various reaction parameters. Herein, a formulated cellulolytic enzyme cocktail was immobilized into mesoporous aerogel material and used under high pressure environment of supercritical CO2 (scCO2). The cocktail was immobilized at different enzyme loads (36.0 to 144.0 mg enzyme/g aerogel) using sol-gel entrapment method and scCO2 drying. The immobilized cocktail at 144.0 mg/g aerogel enzyme load retained a residual yield of >50.0 % after fourth cycle of reuse compared to first cycle under atmospheric pressure. Sugar yield by the cocktail aerogel was below 10 wt% in second cycle under scCO2 due to enzyme leaching. Additionally, surface area and porous volume of reused aerogels was decreased over the reuse cycles. The study suggests that scCO2 could be employed for aerogel preparation after optimizing the immobilization process for pure enzymes rather than crude enzymes.

Recombinant production of a highly efficient photolyase from Thermus thermophilus.

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋𝑚𝑎𝑥 ) of 2.0g L-1 was reached after 5h of cultivation, corresponding to 𝑃X =0.4g L-1 h. The µ𝑚𝑎𝑥 corresponded to 1.0 h-1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25mg L-1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15µg mL-1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase kM of 9.5nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products.

Optimization of Expression and Purification of Recombinant One of Central Nervous System Enzyme (Acetylcholinesterase)

In general, the use of natural enzymes is very limited and they are not economical to use in industry. To solve this problem, the recombinant expression systems approach has been used. Optimization of production processes in heterologous expression systems is one of the most important activities in today's competitive industries. One of the key factors for the commercialization of recombinant proteins is the achievement of a low-cost culture medium available for the production of suitable cell biomass that can be used on an industrial scale. Commercialization of recombinant products requires process economics and cost reduction, and reducing the cost of the product is possible by using a cheaper culture medium, so decided optimization of expression and purification of recombinant acetylcholinesterase (EC.3.1.1.7), because it’s one of the body's vital enzymes, that by hydrolyzing acetylcholine, which is a neurotransmitter, terminates neural messages in the area of cholinergic synapses. Because the enzyme acetylcholinesterase produced from natural sources is not suitable for industrial purposes and semi-mass production due to low production efficiency and high costs of enzyme preparation and purification processes, optimizing the growth conditions of microorganisms capable of the production and secretion of the enzyme acetylcholinesterase are very important, both in terms of yield and economic costs. The present study seeks to increase productivity and reduce the cost of producing this enzyme by optimizing the growth medium and expression of pichia pastoris yeast cells that have genes related to this enzyme and also by optimizing the purification and mass production of this enzyme.

Enhancing the efficiency and functionality of xylanase from Bacillus sp. RTS11: Optimization, purification, characterization, and prospects in kraft pulp bleaching.

Bacillus sp. RTS11, a xylanolytic strain, was isolated from the Algerian desert rocks. Genetic analysis revealed a remarkable 98.69% similarity to Bacillus pumilus. We harnessed optimization techniques, including Plackett-Burman screening and Box-Behnken optimization design, to amplify xylanase production and activity. The outcome of these efforts was an optimized medium that yielded an impressive xylanase production titer of 448.89 U, a threefold increase compared to the non-optimized medium (146 U). The Purification of xylanase was achieved through the three-phase partitioning technique, employing t-butanol and various chromatographic methods. Notably, anion exchange chromatography led to isolating a highly pure enzyme with a molecular weight of 60 kDa. The xylanase exhibited its peak activity at a temperature of 60°C and a pH of 9.0. When applied to pulp pretreatment, 20 U/g of xylanase demonstrated a substantial increase in the release of phenolic and chromophore compounds while reducing sugar content in the pulp. Furthermore, this versatile xylanase shows its ability to efficiently hydrolyze a variety of agro-industrial residues, including wheat bran, corn and grape waste, wheat straw, and sugarcane bagasse. These findings underscore the significant potential of this xylanase enzyme in biobleaching processes and the utilization of agro-industrial waste, opening up exciting avenues for sustainable and environmentally friendly industrial applications.

Comparison Study Between Crude and Purified L-Asparaginase From Bacillus Spp. for Reduction of Acrylamide Level During Fries Processing

Acrylamide is a toxic chemical that is created when foods are heated; it is also available in foods containing different additives. The purpose of the study was to determine whether Bacillus spp. isolates could reduce the concentration of acrylamide in food, as well as to compare the different treatments of crude and pure L-asparaginase produced from the same bacteria in acrylamide reduction in potato slices. Our findings reveal that this bacterium could degrade acrylamide and reduce its concentration. Furthermore, the acrylamide content of potato slices reduced dramatically with increasing enzymatic treatment time, reaching the under detection limit (UDL) after 30 minutes of treatment with 84 U/ml of crude and purified L-asparaginase. In addition, the purified enzyme is more active in removing acrylamide from potato slices than the crude enzyme at all three times. The removal efficiency rises as the enzyme concentration increases. After bleaching the potato slices for 20 minutes at 80 °C with 84 U/ml of pure enzyme, the acrylamide level was totally decreased.

Foam fractionation Tags (F-Tags) enabling surfactant-free, activity-preserving recovery of enzymes

Enzymes have become important tools in many industries. However, the full exploitation of their potential is currently limited by a lack of efficient and cost-effective methods for enzyme purification from microbial production. One technology that could solve this problem is foam fractionation. In this study, we show that diverse natural foam-stabilizing proteins fused as F-Tags to β-lactamase, penicillin G acylase, and formate dehydrogenase, respectively, are able to mediate foaming and recovery of the enzymes by foam fractionation. The catalytic activity of all three candidates is largely preserved. Under appropriate fractionation conditions, especially when a wash buffer is used, some F-Tags also allow nearly complete separation of the target enzyme from a contaminating protein. We found that a larger distance between the F-Tag and the target enzyme has a positive effect on the maintenance of catalytic activity. However, we did not identify any particular sequence motifs or physical parameters that influenced performance as an F-tag. The best results were obtained with a short helical F-Tag, which was originally intended to serve only as a linker sequence. The findings of the study suggest that the development of molecular tags that enable the establishment of surfactant-free foam fractionation for enzyme workup is a promising method.Key points• Foam-stabilizing proteins mediate activity-preserving foam fractionation of enzymes• Performance as an F-Tag is not restricted to particular structural motifs• Separation from untagged protein benefits from low foam stability and foam washingsGraphical

Discovery and biocatalytic characterization of opine dehydrogenases by metagenome mining

Enzymatic processes play an increasing role in synthetic organic chemistry which requires the access to a broad and diverse set of enzymes. Metagenome mining is a valuable and efficient way to discover novel enzymes with unique properties for biotechnological applications. Here, we report the discovery and biocatalytic characterization of six novel metagenomic opine dehydrogenases from a hot spring environment (mODHs) (EC 1.5.1.X). These enzymes catalyze the asymmetric reductive amination between an amino acid and a keto acid resulting in opines which have defined biochemical roles and represent promising building blocks for pharmaceutical applications. The newly identified enzymes exhibit unique substrate specificity and higher thermostability compared to known examples. The feature that they preferably utilize negatively charged polar amino acids is so far unprecedented for opine dehydrogenases. We have identified two spatially correlated positions in their active sites that govern this substrate specificity and demonstrated a switch of substrate preference by site-directed mutagenesis. While they still suffer from a relatively narrow substrate scope, their enhanced thermostability and the orthogonality of their substrate preference make them a valuable addition to the toolbox of enzymes for reductive aminations. Importantly, enzymatic reductive aminations with highly polar amines are very rare in the literature. Thus, the preparative-scale enzymatic production, purification, and characterization of three highly functionalized chiral secondary amines lend a special significance to our work in filling this gap.Key points• Six new opine dehydrogenases have been discovered from a hot spring metagenome• The newly identified enzymes display a unique substrate scope• Substrate specificity is governed by two correlated active-site residues

Engineering an Artificial Pathway to Improve the Bioconversion of Lysine into Chiral Amino Alcohol 2-Hydroxycadaverine Using a Semi-Rational Design

Amino alcohols are important compounds that are widely used in the polymer and pharmaceutical industry, particularly when used as chiral scaffolds in organic synthesis. The hydroxylation of polyamide polymers may allow crosslinking between molecular chains through the esterification reactions of hydroxyl and carboxyl groups. Therefore, this may alter the functional properties of polyamide polymers. 2-hydroxycadaverine (2HyC), as a new type of chiral amino alcohol, has potential applications in the pharmaceutical, chemical, and polymer industries. Currently, 2HyC production has only been realized via pure enzyme catalysis or two-stage whole-cell biocatalysis, which faces great challenges for scale-up production. However, the use of a cell factory is very promising for the production of 2HyC in industrial applications. Here, we designed and constructed a promising artificial pathway in Escherichia coli for producing 2HyC from biomass-derived lysine. This biosynthesis route expands the lysine catabolism pathway and employs two enzymes to sequentially convert lysine into 2HyC. However, the catalytic activity of wild-type pyridoxal phosphate-dependent decarboxylase from Chitinophage pinensis (DCCp) toward 3-hydroxylysine is lower, resulting in the lower production of 2HyC. Thus, the higher catalytic activity of DCCp is desired for low-cost and expanded industrial applications of 2HyC. To improve the catalytic activity of DCCp, a mutant library of DCCp was first built using a semi-rational design. The Kcat/Km of mutant DCCp (R53D/V94I) increased by 63%. A titer of 359 mg/L 2HyC was produced in shake flasks, with a 2HyC titer increase of 54% compared to control strain ML101. The results show that the production of 2HyC was effectively increased through a semi-rational design strategy. These findings lay the foundation for the development and utilization of renewable resources to produce 2HyC in microorganisms via an efficient, green, and sustainable biosynthetic strategy for further industrial application.

Novel recombinant aminoacylase from Paraburkholderia monticola capable of N-acyl-amino acid synthesis

N-Acyl-amino acids can act as mild biobased surfactants, which are used, e.g., in baby shampoos. However, their chemical synthesis needs acyl chlorides and does not meet sustainability criteria. Thus, the identification of biocatalysts to develop greener synthesis routes is desirable. We describe a novel aminoacylase from Paraburkholderia monticola DSM 100849 (PmAcy) which was identified, cloned, and evaluated for its N-acyl-amino acid synthesis potential. Soluble protein was obtained by expression in lactose autoinduction medium and co-expression of molecular chaperones GroEL/S. Strep-tag affinity purification enriched the enzyme 16-fold and yielded 15 mg pure enzyme from 100 mL of culture. Biochemical characterization revealed that PmAcy possesses beneficial traits for industrial application like high temperature and pH-stability. A heat activation of PmAcy was observed upon incubation at temperatures up to 80 °C. Hydrolytic activity of PmAcy was detected with several N-acyl-amino acids as substrates and exhibited the highest conversion rate of 773 U/mg with N-lauroyl-L-alanine at 75 °C. The enzyme preferred long-chain acyl-amino-acids and displayed hardly any activity with acetyl-amino acids. PmAcy was also capable of N-acyl-amino acid synthesis with good conversion rates. The best synthesis results were obtained with the cationic L-amino acids L-arginine and L-lysine as well as with L-leucine and L-phenylalanine. Exemplarily, L-phenylalanine was acylated with fatty acids of chain lengths from C8 to C18 with conversion rates of up to 75%. N-lauroyl-L-phenylalanine was purified by precipitation, and the structure of the reaction product was verified by LC–MS and NMR.Key points• A novel aminoacylase from Paraburkholderia monticola was cloned, expressed in E. coli and purified.• The enzyme PmAcy exhibits exceptional temperature and pH stability and a broad substrate spectrum.• Synthesis of acyl amino acids was achieved in good yields.

Optimization of welan gum extraction and purification using lysozyme and alkaline protease.

Welan gum, a natural polysaccharide produced by Sphingomonas sp. ATCC 31555, has attracted considerable attention in the scientific community due to its desirable properties. However, challenges, such as high viscosity, residual bacterial cells, carotenoids, and protein complexation, hinder the widespread application of welan gum. In this study, we established a method for the extraction and purification of welan gum using a synergistic approach with lysozyme and alkaline protease. Lysozyme hydrolysis conditions were optimized by applying response surface methodology, and the best results for bacterial cell removal were achieved at 11 000 U/g, 44°C, and pH 9 after 3h of treatment. Subsequently, we evaluated protein hydrolysis through computer simulation and identified alkaline protease as the most suitable enzyme. Through experimental investigations, we found that the optimal conditions for alkaline protease hydrolysis were 7500 U/g, 50°C, pH 10, and 600rpm. These conditions resulted in a sugar recovery rate of 76.1%, carotenoid removal rate of 89.5%, bacterial removal rate of 95.2%, and protein removal rate of 87.3% after 3h of hydrolysis. The purified welan gum exhibited high transparency and purity. Structural characterization and antioxidant activity evaluation revealed that enzymatically purified welan gum has potential application prospects. Our study provides valuable insights into the optimal method for the enzymatic extraction and purification of welan gum. Such a method is conducive to the development of the multiple potential applications of welan gum. KEY POINTS: • A novel process for the synergistic purification of welan gum using lysozyme and alkaline protease was established. • In silico virtual digestion was employed to select the purification enzyme. • Welan gum with high transparency and purity was obtained.

Гидрофобная хроматография. Разделение РНКазы и ДНКазы

The article discusses hydrophobic chromatography as a modern method for the isolation and purification of enzymes. Methods for using nucleases in medicine are described. The study is devoted to the separation of the ribonuclease enzyme from the deoxyribonuclease enzyme from their mixture using hydrophobic chromatography.

Partial purification of linoleic acid isomerase enzyme from Lactobacillus paracasei bacteria isolated from milk.

Conjugated Linoleic Acid (CLA) has attracted the attention of many researchers, especially that of microbial origin due to its biological importance to the consumer. The current study aims to extract LA Isomerase enzyme from Lactobacillus paracasei bacteria from milk and to use the enzyme in the production of CLA. Selective media, including MRS and MRS-Dagatose, were used in isolating local strains. The selected bacterial isolates were tested for their ability to produce LA-Isomerase enzyme. The isolate with high enzymatic activity was selected. After extraction and partial purification of the enzyme, the optimal conditions for the production of conjugated fatty acid were studied, and the reaction products were diagnosed using GC-MS technology. It was found that 11 isolates have the ability to produce CLA at different concentrations, H1 isolate showed the highest production of conjugated fatty acid at a concentration of 120.45 g.ml-1, this isolate was selected as the source for enzyme extraction. The enzymatic activity of the crude extract and partially purified with ammonium sulfate was estimated using color methods at wavelength of 233 nm. The effect of the optimum conditions (pH, temperature, linoleic acid concentration and enzyme concentration) on the CLA product was studied using the partially purified LA Isomerase enzyme, the optimum conditions for production were 6.5, 45 °C, 100 μg.ml-1 and 0.7 ml, respectively. The GC-MS technique showed the presence of a number of reaction products that are isomers of conjugated linoleic acid (C9T11, T9T12, T10C12) with different concentrations.

Biochemical Properties and Antithrombotic Effect of a Serine Protease Isolated from the Medicinal Mushroom Pycnoporus coccineus (Agaricomycetes).

The purification of a fibrinolytic enzyme from the fruiting bodies of wild-growing medicinal mushroom, Pycnoporus coccineus was achieved through a two-step procedure, resulting in its homogeneity. This purification process yielded a significant 4.13-fold increase in specific activity and an 8.0% recovery rate. The molecular weight of P. coccineus fibrinolytic enzyme (PCFE) was estimated to be 23 kDa using sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. PCFE demonstrated its optimal activity at a temperature of 40 °C and pH 8. Notably, the enzymatic activity was inhibited by the presence of zinc or copper metal ions, as well as serine protease inhibitors, such as phenylmethylsulfonyl fluoride and 4-amidinophenylmethanesulfonyl fluoride. PCFE exhibited remarkable specificity towards a synthetic chromogenic substrate for thrombin. The enzyme demonstrated the Michaelis-Menten constant (Km), maximal velocity (V ), and catalytic rate constant (Kcat) values of 3.01 mM, 0.33 mM min-1 μg-1, and 764.1 s-1, respectively. In vitro assays showed PCFE's ability to effectively degrade fibrin and blood clots. The enzyme induced alterations in the density and structural characteristics of fibrin clots. PCFE exhibited significant effects on various clotting parameters, including recalcification time, activated partial thromboplastin time, prothrombin time, serotonin secretion from thrombin-activated platelets, and thrombin-induced acute thromboembolism. These findings suggest that P. coccineus holds potential as an antithrombotic biomaterials and resources for cardiovascular research.

Purification and biochemical characterization of methanobactin biosynthetic enzymes.

Methanobactin (Mbn) is a ribosomally synthesized and post-translationally modified peptide (RiPP) natural product that binds Cu(I) with high affinity. The copper-chelating thioamide/oxazolone groups in Mbn are installed on the precursor peptide MbnA by the core enzyme complex, MbnBC, which includes the multinuclear non-heme iron-dependent oxidase (MNIO) MbnB and its RiPP recognition element-containing partner protein MbnC. For the extensively characterized Mbn biosynthetic gene cluster (BGC) from the methanotroph Methylosinus trichosporium OB3b, the tailoring aminotransferase MbnN further modifies MbnA after leader sequence cleavage by an unknown mechanism. Here we detail methods to express and purify M. trichosporium OB3b MbnBC and MbnN along with protocols for assessing MbnA modification by MbnBC and MbnN aminotransferase activity. In addition, we describe crystallization and structure determination of MbnBC. These procedures can be adapted for other MNIOs and partner proteins encoded in Mbn and Mbn-like BGCs. Furthermore, these methods provide a first step toward in vitro biosynthesis of Mbns and related natural products as potential therapeutics.