Purification Of Enzyme Research Articles

Purification of Glutathione Reductase Enzyme from Whiting Fish Gill Tissue and Investigation of Metal Inhibition

Pollution in the seas basically accumulates in marine organisms. Heavy metal residues in aquatic ecosystems can pass through food and cause toxic effects and accumulations in human health. Glutathione reductase (GR), which is among the basic enzymes, has an important place in the suppression of stress in the cell. In this study, glutathione reductase enzyme from whiting fish gill tissue was partially purified for the first time in the literature and the effects of heavy metal compounds on enzyme activity were determined. The purification process was carried out in three stages as homogenate preparation, ammonium sulfate precipitation and also dialysis. In conclusion the study, optimum level pH 7.0, optimal substrate concentration 2 mM NADPH and optimum buffer 150 mM KH2PO4 were determined. After partial purification, the inhibition effects of Cd2+, Ni2+, Zn2+ as heavy metal ions were investigated. The IC50 levels of heavy metals were calculated as 20.17 µM, 33.7 µM and 59.31 µM, respectively.

Food wastes clean water wastes: melon peel peroxidase isolation and immobilization onto magnetite nanoparticles for phenol removal

In this study, melon peel waste was utilized to isolate peroxidase enzyme through three-phase portioning (TPP) and subsequently immobilized onto magnetite nanoparticles for effective bioremediation of phenol pollutants from water. The optimization of TPP parameters ensured maximum activity recovery and enzyme purity. Magnetite nanoparticles were synthesized and used as a substrate for immobilizing the isolated peroxidase, achieving an activity recovery of 157% and a purification fold of 5.2. Protein homogeneity testing confirmed the purity of the peroxidase enzyme. The magnetite nanoparticles had an average diameter of 62 nm, and the immobilization efficiency reached 93% at pH 8 with an enzyme/nanoparticles v/v ratio of 1:9. The immobilized peroxidase demonstrated the ability to degrade 57% of phenol within 3 h and retained 30% relative activity even after five catalytic cycles. This immobilized melon peel peroxidase on magnetite nanoparticles proves to be a robust, enduring, and reusable biocatalyst with potential for various applications, especially in bioremediation processes.Graphical

Development of a new affinity chromatography method for purification of horseradish peroxidase enzyme.

In this study, benzohydroxamic acid molecules were synthesized from methyl 4-amino-2-methoxy, methyl 4-amino-3-nitro, methyl 4-amino-3-methyl, and methyl 4-amino-3-chloro benzoate molecules, and the horseradish peroxidase (HRP) enzyme was purified in one step using the affinity chromatography technique for the first time. The IC50 and Ki values for the 4-amino 3-methyl benzohydroxamic acid molecule were 0.136 and 0.132 ± 0.054μM, respectively, while the IC50 and Ki values for the 4-amino-3-nitro benzohydroxamic acid molecule were 56.00 and 51.90 ± 9.90μM, respectively. It was found that the IC50 and Ki values for the 4-amino-3-chloro benzohydroxamic acid molecule were 218.33 and 175.67 ± 43.78μM, respectively, whereas the IC50 and Ki values for the 4-amino-2-methoxy benzohydroxamic acid molecule were 306.00 and 218.00 ± 68.80μM, respectively. The HRP enzyme was synthesized from 4-amino-2-methoxy hydroxamic acid column with a 35.97% yield 601.13 times, 4-amino-3-nitro hydroxamic acid column, with a 14.00% yield 404.11 times, 4-amino-3-methyl hydroxamic acid column with an 8.70% yield 394.88 times, and 4-amino-3-chloro hydroxamic acid column with a 4.48% yield 284.85 times. Thus, the HRP enzyme was purified in a single step with hydroxamic acids, and its molecular weight was found to be 44kDa. The optimum pH was 8.0, the optimum temperature was 15°C, and the optimum ionic strength was 0.4M for the purified HRP enzyme.

Whole-cell and Pure Enzyme-catalyzed Degradation of Microcystins: A Comparative Study of the Efficacy of Heterologously Expressed mlrA Gene

The increasing occurrence of cyanobacterial blooms, driven by factors such as water eutrophication and climate warming, has raised concerns due to associated environmental pollution and health risks. Among the toxins produced by these blooms, microcystins, particularly Microcystin MC-LR, pose significant threats to ecosystems and human health. This study investigates the efficiency of degrading microcystins, particularly Microcystin MC-LR, using two catalytic methods: whole-cell and pure enzyme catalysis, both relying on the mlrA gene. The research successfully engineered an Escherichia coli strain with the capability to degrade MC-LR, achieving remarkable results with the whole-cell catalysis approach. Within a 24-hour period, the concentration of MC-LR dropped from 5 μg/mL to 0.681 μg/mL, demonstrating an efficiency of 84%. In contrast, enzymatic degradation yielded less satisfactory outcomes, degrading only 100 ng/mL within the same timeframe. The whole-cell catalysis approach exhibits clear advantages, including cost-effectiveness, making it a promising option for industrial applications. This technology holds potential for safeguarding water resources and public health by addressing algal toxin contamination.

Purification and characterization of thioredoxin reductase enzyme from commercial Spirulina platensis tablets by affinity chromatography and investigation of the effects of some chemicals and drugs on enzyme activity.

Thioredoxin reductase (TrxR, enzyme code [E.C.] 1.6.4.5) is a widely distributed flavoenzyme that catalyzes nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of thioredoxin and many other physiologically important substrates. Spirulina platensis is a blue-green algae that is often used as a dietary supplement. S. platensis is rich in protein, lipid, polysaccharide, pigment, carotenoid, enzyme, vitamins and many other chemicals and exhibits a variety of pharmacological functions. In the present study, a simple and efficient method to purify TrxR from S. platensis tablets is reported. The extractions were carried out using two different methods: heat denaturation and 2',5'-adenosine diphosphate Sepharose 4B affinity chromatography. The enzyme was purified by 415.04-fold over the crude extract, with a 19% yield, and specific activity of 0.7640U/mg protein. Optimum pH, temperature and ionic strength of the enzyme activity, as well as the Michaelis constant (Km ) and maximum velocity of enzyme (Vmax ) values for NADPH and 5,5'-dithiobis(2-nitrobenzoic acid) were determined. Tested metal ions, vitamins, and drugs showed inhibition effects, except Se4+ ion, cefazolin sodium, teicoplanin, and tobramycin that increased the enzyme activity in vitro. Ag+ , Cu2+ , Mg2+ , Ni2+ , Pb2+ , Zn2+ , Al3+ , Cr3+ , Fe3+ , and V4+ ions; vitamin B3 , vitamin B6 , vitamin C, and vitamin U and aciclovir, azithromycin, benzyladenine, ceftriaxone sodium, clarithromycin, diclofenac, gibberellic acid, glurenorm, indole-3-butyric acid, ketorolac, metformin, mupirocin, mupirocin calcium, paracetamol, and tenofovir had inhibitory effects on TrxR. Ag+ exhibited stronger inhibition than 1-chloro-2,4-dinitrobenzene (a positive control).

The Synthesis of Ginsenoside Compound K Using a Surface-Displayed β-Glycosidase Whole-Cell Catalyst

Ginsenoside compound K (CK) has garnered considerable attention due to its versatile pharmacological properties, including anti-inflammatory, anti-allergic, anti-aging, anti-diabetic, and hepatoprotective effects, along with neuroprotection. The conventional approach to synthesizing ginsenoside CK involves enzymatic conversion. However, the purification of enzymes necessitates effort and expense, and enzymes are prone to inactivation. Additionally, whole-cell catalysis suffers from inefficiency due to limited cell permeability. To address these challenges, we harnessed the YiaT protein as an anchoring motif, establishing a surface display system for β-glycosidase Bgp3. This innovative system served as a whole-cell catalyst for the efficient synthesis of ginsenoside CK. We further optimized the YiaT-Bgp3 system, enhancing display levels and significantly increasing ginsenoside CK production. Optimal conditions were achieved at an IPTG concentration of 0.5 mM, an induction temperature of 16 °C, a ginsenoside substrate concentration of 15 mg/mL, and a catalytic temperature of 30 °C. Ultimately, the YiaT-Bgp3 system synthesized 5.18 ± 0.08 mg/mL ginsenoside CK within 24 h, with a conversion of 81.83 ± 1.34%. Furthermore, the YiaT-Bgp3 system exhibited good reusability, adding to its practicality and value. This study has successfully developed an efficient whole-cell Bgp3 biocatalyst, offering a convenient, highly productive, and economically viable solution for the industrial production of ginsenoside CK.

The Influence of Enzymatic Hydrolysis on Bee Pollen Antioxidant and Antibacterial Activities.

Bee pollen is one of the most valuable apitherapeutic products with high nutritional value. To obtain a higher diversity of compounds, higher bioactivity, and improve the release of nutrients from bee pollen, additional processing of the raw material may be applied: fermentation using microorganisms or hydrolysis using selective enzymes. This research aimed to determine the impact of enzymatic hydrolysis on the antioxidant and antibacterial activities of bee pollen. Bee pollen samples from Sweden, Spain, Netherlands, Italy, Poland, Denmark, Slovakia, Malta, and Lithuania were hydrolyzed using pure enzymes, including lipase, cellulase, protease, and amyloglucosidase, as well as enzyme mixtures such as Viscozyme® L and Clara-diastase. Total phenolic content, total flavonoid content, and antioxidant activity were analyzed spectrophotometrically. Antibacterial activity against Staphylococcus aureus, Listeria monocytogenes, Staphylococcus enteritidis, and Salmonella typhimurium was evaluated using the agar well diffusion assay. Obtained results revealed a positive effect of enzymatic hydrolysis on biologically active compound content and activity: total phenolic content increased by 1.1 to 2.5 times, total flavonoid content by 1.1 to 3.0 times, radical scavenging activity by 1.1-3.5 times, and antibacterial activity by 1.1 to 3.3 times. K-means clustering analysis grouped samples into 5-9 clusters and was dependent on the measured characteristic used as an input-total phenolic compounds content, total flavonoid content, antioxidant activity, and antibacterial activity against four different bacteria. Chemometrics showed, that the enzyme used for the hydrolysis had a higher impact on clustering results than the geographical origin of the samples.

Purification of fibrinolytic enzyme from Bacillus amyloliquefaciens GUTU06 and properties of the enzyme

A producing-fibrinolytic enzyme strain was isolated with high yield. The strain was identified as Bacillus amyloliquefaciens. B. amyloliquefaciens GUTU06 fibrinolytic enzyme was purified by acetone precipitation and reverse micelle. Acetone precipitation condition and reverse micelle condition were examined. Results showed that the total reverse micelle extraction efficiency was 64.49 % ± 1.6 %. The purification fold of the entire process reached 13.38. The optimum pH of purified enzyme is 5, and the optimum temperature is 45 °C. Fe3+ and K+ can enhance the fibrinolytic activity of the enzyme. Compared to commercial fibrinolytic enzymes such as urokinase and lumbrukinase, GUTU06 fibrinolytic enzymes have a lower pH optimal range and higher temperature stability. The molecular weight of the enzyme was approximately 28 kDa. Reverse micelle extraction with cetyl trimethylammonium bromide as a surfactant combined with acetone precipitation is suitable for separating and purifying fibrinolytic enzymes and a promising technique for obtaining active proteins.

Влияние рН и состава питательной среды на дегидрогеназную активность экстрактов, полученных из Escherichia coli

“Crude” extracts are a new perspective type of bioelectrocatalysts for biofuel cells. The method of obtaining a “crude” extract by disintegrating bacterial cell membranes (for example, by ultrasound) is simpler and more economical in comparison with pure enzymes. From a scientific point of view, the study of the biocatalytic properties of “crude” extracts is very interesting, since the disintegrated biomass contains a cascade of enzymes involved in the metabolism of microorganisms and coenzymes necessary for oxidative/reductive transformation. This set of bioactive substances makes it possible to simulate the processes occurring during the operation of a “natural” fuel cell in an artificial environment. The effect of the pH and composition of the LB nutrient medium used in the cultivation of E.coli on the dehydrogenase activity of protein extracts obtained from this culture was studied. It is shown that by selecting the pH and modifying the composition of the LB nutrient medium, it is possible to increase the value of the specific dehydrogenase activity of extracts by almost 9 times (from 0.25 to 2.2 mg/mg).

Profile of Amino Acids and Proximate on the Extraction of Red Ginger Protease Enzymes in Commercial Feed for Aquaculture

Quality feed in aquaculture is very important to increase the growth and health of fish. Low feed efficiency can be overcome by feed supplementation using catalytic enzymes. One of the enzymes that can stimulate digestion is protease in red ginger plants. The protease enzyme in red ginger plants plays a role in breaking peptide bonds in place of carboxyl groups so that peptides are formed from complex compounds into simpler compounds. This study aims to determine the profile of amino acids and proximate in feed given red ginger enzymes. This research uses descriptive method. The feed sample used was commercial feed with 4 treatments namely Control (without red ginger enzyme), treatment A (1%), treatment B (3%), and treatment C (5%). The analyzes carried out included red ginger enzyme extraction, protease enzyme purification, proximate and amino acid analysis of each type of feed. The results showed that the results of the SDS-Page analysis on the red ginger protease sample contained at 67.1 kDa. The results of the proximate analysis showed the highest value in feed containing 3% red ginger protease (B) with a percentage of 42.69%. The resulting amino acid content consists of 8 non-essential amino acids namely, L-serine, L-glutamic acid, L-alanine, L-arginine, glycine, L-aspartic acid, L-tyrosine and L-proline. While the essential amino acids consist of 7 types of amino acids namely, L-Phenylalani, L-Isoleucine, L-Valine, L-Lysine, L-Leucine, L-Threonine and L-Histidine. The best type of feed is feed B, which is feed with the addition of 3% red ginger protease.

Purification And Characterization of Mannanase Enzyme from Lentinula edodes Isolate and Evaluation of Its Activity in Removing Blood Stains from Cotton Fabrics.

Shiitake mushroom is the Japanese common name for the genus Lentinula edodes, and Shiitake is a fungus of medicinal and industrial importance. The enzyme Mannanase was purified from the fungal culture filtrate. It included a series of steps, including precipitation with ammonium sulfate and a saturation rate of 80%, in which the specific activity reached 2 units/mg, and the enzymatic accumulation was 80%. It was followed by enzyme dialysis for a whole day, which caused an increase in the specific activity to 3 units/mg, the number of purification times reached 3 times, and the enzymatic accumulation reached 71%. It is noticed from the ion exchange step using DEAE-cellulose that the specific activity increased to 10 units/mg, the number of purification times reached 10 times, and the enzymatic accumulation reached 53%. Subsequently, the last purification step was carried out using gel filtration chromatography Sephadex G-150, in which the specific activity of the enzyme reached 24 units/mg and the number of purification times reached 24 times, with an enzymatic accumulation of 48%. The molecular weight was determined using electrophoresis in a polyacrylamide gel, and its molecular weight was 58 kilodaltons. The optimum pH for its activity was 6.5, while the pH ranged between (5.5-7) to prove its activity. The optimum temperature for enzyme activity was 60°C, while the thermal stability was between (20-80) °C. Keywords: Lentinula edodes, β-mannanase activity, purifi-cation, DEAE-Sephadex A-150, Characterization, removing and ability of detergents to clean clothes.

Purification and characterization of catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) using the local isolate of phenol-degrading Pseudomonas putida.

The purpose of the present study was to purify and characterize the catechol 1,2-dioxygenase (EC 1.13.11.1; catechol-oxygen 1,2-oxidoreductase; C12O) enzyme from the local isolate of Pseudomonas putida. This enzyme catalyzes the initial reaction in the ortho-pathway for phenol degradation in various gram-negative bacteria, including the genus of Pseudomonas. Pseudomonads are commonly used in the biodegradation of xenobiotics due to their versatility in degrading a wide range of chemical compounds. Eighty-nine soil samples were taken from the contaminated soil of the Midland Refineries Company (MRC) of Al-Daura refinery area at Baghdad from April to August 2021. The samples were grown in a mineral salt medium containing 250mg per L of phenol to test their ability to biodegrade phenol. The pH was adjusted to 8.0 at 30°C using a shaking incubator for 24-48h. A number of 62 (69.6%) isolates of the total number were able to degrade phenol efficiently. The findings of the VITEK system and the housekeeping gene 16S rDNA confirmed that out of the positive isolates for phenol degradation, 36 from 62 (58.06%) were identified as Pseudomonas spp. isolates. Those isolates were distributed as P. aeruginosa 30 (83.3%) and P. putida 6 (16.6%). The enzyme production capabilities of the isolates were evaluated, and the highest activity was 2.39 U per mg for the isolate No. 15 which it was identified as P. putida. The previous isolate was selected for enzyme production, purification, and characterization. The enzyme was purified using ion exchange and gel filtration chromatography, with a combined yield of 36.12% and purification fold of 15.42 folds. Using a gel filtration column, the enzyme's molar mass was calculated to be 69kDa after purification. The purified enzyme was stable at 35°C and a pH of 6.0.

Production of laccase enzyme from Curvularia lunata MY3: purification and characterization

Laccase-producing fungus (MY3) was successfully isolated from soil samples collected from Mansoura Governorate, Egypt. This fungal isolate has shown a high laccase production level over other isolated fungi. The identity of this isolate was determined by the molecular technique 18SrRNA as Curvularia lunata MY3. The enzyme purification was performed using ammonium sulfate precipitation followed by Sephacryl S-200 and DEAE-Sepharose column chromatography. The denatured enzyme using SDS-PAGE had a molar mass of 65 kDa. The purified laccase had an optimum temperature at 40 °C for enzyme activity with 57.3 kJ/mol activation energy for 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) oxidation. The enzyme had an optimum pH of 5.0, and it has shown a high stability at the acidic range (4.5 to 5.5). Mn2+ and Mg2+ ions enhanced the enzyme activity, while most of the enzyme activity was inhibited by Hg2+. Some compounds such as 2-mercaptoethanol, L-cysteine, and sodium azide at a concentration of 10 mmol/L had shown a high suppression effect on the enzyme activity. The enzyme strongly oxidized ABTS and syringaldazine and moderately oxidized DMP and guaiacol. The antimicrobial activity of the purified enzyme towards three pathogenic strains (Escherichia coli ATCC-25922, Staphylococcus aureus NRRLB-767, and Candida albicans ATCC-10231) was evaluated for the potential use as an antimicrobial therapeutic enzyme.

Aqueous two-phase extraction and characterization of thermotolerant alkaliphilic Cladophora hutchinsiae xylanase: biochemical properties and potential applications in fruit juice clarification and fish feed supplementation

Xylanase finds extensive applications in diverse biotechnological fields such as biofuel production, pulp and paper industry, baking and brewing industry, food and feed industry, and deinking of waste paper. Here, polyethylene glycol (PEG)-phosphate aqueous two-phase system (ATPS) was applied for the purification of an alkaline active and thermotolerant xylanase from a marine source, Cladophora hutchinsiae (C. hutchinsiae). In the purification process, the effects of some experimental factors such as PEG concentration and PEG molar mass, potassium phosphate(K2HP04) concentration, and pH on xylanase distribution were systematically investigated. Relative enzymatic activity and purification factor obtained were 93.21% and 7.18, respectively. A single protein band of 28 kDa was observed on SDS-PAGE. The optimum temperature and pH of xylanase with beechwood xylan were 30 °C and 9.0, respectively. The Lineweaver-Burk graph was utilized to determine the K m (4.5 ± 0.8 mg/mL), V max (0.04 ± 0.01 U) and k cat (0.001 s−1) values of the enzyme. It was observed that the purified xylanase maintained 70% of its activity at 4 °C and was found stable at pH 4.0 by retaining almost all of its activity. Enzymatic activity was slightly enhanced with Na+, K+, Ca2+ and acetone. The highest increase in the reducing sugar amount was 53.6 ± 3.8, for orange juice at 50 U/mL enzyme concentration.

Purification and characterization of extracellular PHB depolymerase enzyme from Aeromonas caviae Kuk1-(34) and their biodegradation studies with polymer films.

PHB depolymerase enzymes are able to breakdown the PHB polymers and thereby get significant economic value in the bioplastics industry and for bioremediation as well. This study shows the purification of novel extracellular PHB depolymerase enzyme from Aeromonas caviae Kuk1-(34) using dialysis followed by gel filtration and HPLC. The purification fold and yield after HPLC were 45.92 and 27.04%, respectively. HPLC data showed a single peak with a retention time of 1.937min. GC-MS analysis reveals the presence of three compounds, of which 1-Dodecanol was found to be most significant with 54.48% area and 8.623-min retention time (RT). The molecular weight of the purified enzyme was obtained as 35kDa with Km and apparent Vmax values of 0.769mg/mL and 1.89 U/mL, respectively. The enzyme was moderately active at an optimum temperature of 35 °C and at pH 8.0. The stability was detected at pH 7.0-9.0 and 35-45 °C. Complete activity loss was observed with EDTA, SDS, Tween-20 at 5 mM and with 0.1% Triton X 100. A biodegradation study of commercially available biodegradable polymer films was carried out in a liquid medium and in soil separately with pure microbial culture and with purified enzyme for 7, 14, 28, and 49 consecutive days. In a liquid medium, with a pure strain of Aeromonas caviae Kuk1-(34), the maximum degradation (89%) was achieved on the PHB film, while no changes were observed with other polymer films. With purified enzyme in the soil, 71% degradation of the PHB film was noticed, and it was only 18% in the liquid medium. All such weight analysis were confirmed by SEM images where several holes, pits, grooves, crest, and surface roughness are clearly observed. Our results demonstrated the potential utility of Aeromonas caviae Kuk1-(34) as a source of extracellular PHB depolymerase capable of degrading PHB under a wide range of natural/ lab conditions.

Production of Industrial Applicable Enzymes from Rhizopus Oryzae through Response Surface Methodology

The agricultural wastes are used for production of vital enzymes like cellulase, protease and lipase through Solid State Fermentation (SSF), which reveal their industrial potential aspect. This study was done to find out the ability of Rhizopus oryzae to produce lipase, protease and cellulase through SSF. Wheat bran showed maximum enzyme activities as related to others agricultural wastes. The enzymes showed maximum activities (21.4±0.04) (4.6±0.02 IU/mL) and (1.1 ± 0.01 IU/mL) for cellulase, lipase and protease respectively at temperature 25ºC and incubation period 72 hours. Response surface methodology (RSM) used to optimize conditions likes temperature, pH, moisture contents, inoculum size and substrate concentration. RSM revealed maximum enzyme activities using 100% moisture contents, temperature 25ºC, pH 5, substrate concentrations 12g and inoculum size of 6 mL. The method of ammonia sulphate precipitation used for the purification and crystallization of enzymes like lipase, protease and cellulase. The precipitation of cellulase, lipase and protease performed with 80%, 70 % and 90% ammonium sulphate concentrations to increase yield. The characterization of lipase, protease, cellulase revealed that it worked best below acidic conditions (pH 5) and at moderate temperature (25ºC). The results revealed that the mold oryzae had potential to produce industrially important enzymes at acidic ph.

Structure-Guided Discovery of N5-CAIR Mutase Inhibitors.

Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N5-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N5-CAIR mutase of the human pathogens Legionella pneumophila (LpPurE) and Burkholderia cenocepacia (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds in silico and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC50 values. Several of these compounds, including the α1-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.

Research progress on the fibrinolytic enzymes produced from traditional fermented foods.

Cardiovascular diseases (CVDs) are a global health problem and leading cause of death worldwide. Thrombus formation, one of the CVDs, is essentially the formation of fibrin clots. The existing thrombolytic agents have the disadvantages of high price, short half-life, and high bleeding risk; hence, there is an urgent need to find the alternative thrombolytic agents. In recent years, traditional fermented foods have been widely investigated for their outstanding effects in the prevention and treatment of thrombus formation. In this review, we have focused on fibrinolytic enzymes produced by microorganisms during the fermentation of traditional fermented foods and their potential use for treating CVDs. First, we discussed about the sources of fibrinolytic enzymes and microbial strains that produce those enzymes followed by the optimization of fermentation process, purification, and physicochemical properties of fibrinolytic enzymes. Finally, we have summarized the thrombolytic effects of fibrinolytic enzymes in humans and mice. Fibrinolytic enzymes produced by microorganisms during the fermentation of traditional fermented foods not only lyse thrombi but also acts as anti-atherosclerotic, anti-hyperlipidemia, and neuroprotection agents. Therefore, fibrinolytic enzymes from traditional fermented foods have great potential for the prevention and treatment of CVDs.

Purification and Activity of the Second Recombinant Enzyme for Biodegrading Linearized Microcystins by Sphingopyxis sp. USTB-05.

Hepatotoxic microcystins (MCs) are produced and released by the harmful bloom-forming cyanobacteria, which severely threaten drinking water safety and human health due to their high toxicity, widespread distribution, and structural stability. The linearized microcystinase (MlrB) further hydrolyses the poisonous linearized MCs produced by the microcystinase-catalysed MCs to form tetrapeptides. Here, the purification and activity of MlrB were investigated. The results showed that the linearized products generated by 12.5 mg/L MC-LR and MC-RR were removed by purified recombinant MlrB at a protein concentration of 1 mg/L within 30 min. The high catalytic activity of MlrB can be obtained via heterologous expression and affinity purification, which lays the foundation for further studies on the properties and mechanism of MCs biodegradation enzymes.

Enhanced invertase binding from baker's yeast via cryogels included boronic acids.

Invertase, an industrially significant glycoenzyme, was purified from baker's yeast using poly (2-Hydroxyethyl methacrylate) [PHema-Pba] cryogels functionalized with boronic acid. At subzero temperatures, PHema-Pba cryogels were synthesized and characterized using swelling tests, scanning electron microscopy, and Fourier-transform infrared spectroscopy. The surface area of the PHema-Pba cryogels was 14 m2/g with a swelling ratio of 88.3% and macroporosity of 72%. The interconnected macropores of PHema-Pba cryogels were shown via scanning electron microscopy. Invertase binding capacity of PHema-Pba cryogel was evaluated by binding studies in different pH, temperature, and interaction time conditions and the maximum Invertase binding of PHema-Pba cryogel was found as 15.2mg/g. and 23.7 fold Invertase purification was achieved from baker's yeast using PHema-Pba cryogels. The results show that PHema-Pba cryogels have high Invertase binding capacity and may be used as an alternative method for enzyme purification via boronate affinity systems.