Ammonium Sulfate Precipitation Research Articles

Production of Fibrinolytic Enzyme by Soil Actinobacteria

Thrombotic diseases are increasingly becoming among the prominent causes of death across the globe. Researchers are now turning attention towards fibrinolytic enzymes as potential alternative treatment for thrombolytic diseases. This present study focused on the production of extracellular fibrinolytic enzyme from soil Actinobacteria and evaluation of its hydrolytic activity on blood clot. The Actinobacteria was isolated from compost soil of semi-arid climate in Nigeria. Bacterial isolation was achieved using starch casein agar. Potent fibrinolytic enzyme producing Actinobacteria were identified and subjected to enzyme production using submerged fermentation method. The interactive effects of incubation time, temperature, pH and media components on enzyme production were analysed. Extracellular fibrinolytic enzyme produced by the selected Actinobacteria was partially purified by ammonium sulphate precipitation and subsequently assayed for blood clot lysis activity. Results of these studies indicated that fibrinolytic enzyme was produced optimally at pH 8 and temperature of 40 oC after 72 hour of fermentation. Partially purified fibrinolytic enzyme was able to degrade blood clot comparable to the positive control. These results shows that soil Actinobacteria of unexplored semi-arid climate of Nigeria present a prospect in search of novel microorganisms with potentials in the production of fibrinolytic enzyme that can serve as an alternative blood clot buster in treating thrombolytic diseases.

Identification of a cytotoxic factor from a non-pigmented entomopathogenic Serratia marcescens isolate toxic towards human carcinoma cell lines.

It has been reported that cell-free culture broths and some proteins from pigmented and non-pigmented Serratia spp. are cytotoxic towards cancerous and non-cancerous human cell lines. Looking for new molecules toxic against human cancerous cells but harmless towards normal human cells, the aim of this work was (a) to determine whether cell-free broths from the entomopathogenic non-pigmented S. marcescens 81 (Sm81), S. marcescens 89 (Sm89) and S. entomophila (SeMor4.1) presented cytotoxic activity towards human carcinoma cell lines; (b) to identify and purify the associated cytotoxic factor(s) and (c) to evaluate whether the cytotoxic factor(s) was cytotoxic towards non-cancerous human cells. This research was focussed on the observed morphology changes and the proportion of remaining viable cells after incubation in the presence of cell-free culture broths from the Serratia spp isolates to evaluate cytotoxic activity. The results showed that broths from both S. marcescens isolates presented cytotoxic activity and induced cytopathic-like effects on the human neuroblastoma CHP-212 and the breast cancer MDA-MB-231 cells. Slight cytotoxicity was observed in the SeMor4.1 broth. A serralysin-like protein of 50 kDa was identified in Sm81 broth as responsible for cytotoxic activity after purification by ammonium sulphate precipitation and ion-exchange chromatography followed by tandem-mass spectrometry (LC-MS/MS). The serralysin-like protein was toxic against CHP-212 (neuroblastoma), SiHa (human cervical carcinoma) and D-54 (human glioblastoma) cell lines in a dose-dependent manner and showed no cytotoxic activity in primary cultures of normal non-cancerous human keratinocytes and fibroblasts. Therefore, this protein should be evaluated for a potential use as an anticancer agent.

Purification and Production Optimization of Alpha Amylases from Bacillus Species

Amylases are the digestive enzymes belong to the family of glycoside hydrolase capable of hydrolysing starch and polysaccharides. These are the recent breakthroughs in food, feed, pulp and pharma industries. Our aim of this study is to purify amylases from Bacillus subtilis and Bacillus megaterium and optimize the growth conditions to achieve the cost-effective production of amylases from bacillus species. Extensive research on amylases from different sources is essential in producing enzymes in large scale and in optimizing the growth conditions and minimize the production cost to meet the increased demand of these industrially important enzymes. Amylases were produced in the current study using submerged fermentation and purified the extracellular enzymes from bacillus species using ultracentrifugation, Ammonium sulphate precipitation and gel filtration chromatography. There was no effect on amylase production from bacillus species when the initial substrate concentration is increased from 1% to 5%. Increase in inoculum size from 1% to 5% and the addition of yeast extract to broth had significantly increased the amylase production. The study observed significantly higher levels of amylase production using rice starch when compared with wheat starch also higher levels of amylase production observed with B. megaterium when compared with B. subtilis. The study concludes 1% rice starch and 5% B. megaterium inoculum can be used for cost effective production of amylases. In the current study, cost effective amylase production was evaluated and able to optimize the yield of extracellular amylases from Bacillus megaterium to a concentration level of 120 IU/ml of fermentation broth.

Partial Purification of Catalase Enzyme from Muscle Tissue of Dusky Spinefoot (Siganus luridus)

Catalase, one of the antioxidant enzymes, decomposes hydrogen peroxide into water and oxygen. From the discovery of catalase, several studies have been carried out to reveal its importance in health, food and cosmetics industries etc., and these studies are still ongoing. In this study, catalase enzyme was partially purified from muscle tissue of Dusky spinefoot (Siganus luridus). Purification procedure consisted of homogenate preparation, ammonium sulfate precipitation and dialysis. The enzyme was precipitated in the range of 40-60 % Ammonium Sulfate concentration. The optimum buffer was determined as 200 mM phosphate buffer, optimum pH 8.0 and optimum substrate concentration 24mM for H₂ and O₂, respectively.

Rhizopus oryzae Inulinase Production and Characterization with Application in Chicory Root Saccharification

The objective of this study was to create a fermentation process for the production of inulinase, an important enzyme with numerous applications in the food and pharmaceutical industries, using low-cost agricultural waste as substrates for Rhizopus oryzae NRRL 3563. High titer inulinase production in chicory roots by Rhizopus oryzae in a submerged culture was accomplished using a statistical experimental design. A two-level Plackett–Burman design followed by a three-level Box–Behnken design producing a high inulinase titer of 1085.11 U/mL, 2.83-fold the maximum level, was obtained in the screening experiment. The optimal levels were as follows: chicory root, 10 g/L; NaNO3, 5 g/L; and KCl, 0.2 g/L. The produced inulinase enzyme was purified using 70% ammonium sulfate precipitation and ultra-filtration causing 3.63-fold purification with 60% activity recovery. The enzyme had a molecular weight of approximately 130 KDa. The purified enzyme showed optimum activity at 50 °C and pH 6.0. The pH stability range was three to six and the temperature stability was up 70 °C. The purified inulinase could hydrolyze inulin and sucrose, but not cellobiose or soluble starch. Km and Vmax for inulin were determined to be 0.8 mg/mL and 50,000 U/mg, respectively. The two-level Plackett–Burman design was applied followed by a Box–Behnken model for optimization of fermentation conditions. Accordingly, the optimal combination of fermentation was a reaction time of seven hours, a temperature of 60 °C, and an enzyme concentration of 40,000 U/mL, which resulted in a 58.07% saccharification yield. The characteristics of the enzyme and its kinetic parameters suggested that it was highly effective in the fermentation of inulin and inulin-containing substrates. Additionally, it raises the potential of using inulinase enzymes in pharmaceutical and food industries.

Laccase Production from Local Biomass Using Solid State Fermentation

The large family of enzymes, known as polyphenols oxidases, includes laccase. Due to the inclusion of a copper atom in their catalytic core, laccases are frequently referred to as multi-copper oxidases. Laccases are versatile enzymes that can catalyze the oxidation of a wide range of phenolic and non-phenolic substances. In the current study, a local strain of Aspergillus niger was used for solid-state fermentation to produce fungal laccase, as well as purify and optimize laccase. The enzyme profile, which was acquired using guaiacol to measure enzyme activity, showed that after five days of incubation, wheat straw provided the highest level of laccase activity, or 2.551 U/mL. A technique called response surface methodology (RSM) was used to examine the effects of various conditions on the production of enzymes. The RSM results demonstrated that after five days of incubation, the enzyme activity was at its highest at 45 °C, pH 5.5, and 30% moisture level, inoculated with 2 mL mycelium. Through ammonium sulphate precipitation and dialysis, the enzyme was purified. Additionally, column chromatography was used to further purify laccase. The next step was enzyme characterization to evaluate how temperature and pH affected enzyme activity. At 45 °C and pH 5.5, the isolated enzyme produced its highest level of activity. The findings of the current study showed that A. niger is capable of producing laccase in an economical and environmentally friendly way. Due to its unique oxidative and catalytic features, this enzyme has received a lot of attention recently.

Enhanced uricase production using novel Escherichia marmotae strain (DJDSS001): Characterization and optimization

Uricase is a diagnostic and therapeutic enzyme used to detect the levels of purine end product namely uric acid in the biological samples and it also promotes the conversion of uric acid to a soluble allantoin. In this study, a potential extracellular uricase-producing Escherichia sp. was isolated from canal water near dairy and molecular characterization using16 S rRNA sequence was performed and the data showed 98.95% similarity with Escherichia marmotae. Morphological and biochemical characteristics of the isolated bacterium were determined. The effect of medium components on uricase production was studied and optimized medium enhanced the uricase production to 27.47 U/ml ± 0.44 which is 1.37 fold greater than initial uricase production. The enzyme was purified using ammonium sulphate precipitation followed by ion exchange chromatography and the resulted enzyme is stable over wide pH range from 6.0 to 9.0 and temperatures ranging from 30 °C to 37 °C retaining more than 94% activity. The enzyme activity and specific activity of the purified enzyme were 40.02 U/mL and 87.76 U/mg respectively with an overall purification fold of 4.04 and the yield obtained is 14.57%.

Study of Thioredoxin Isolated from Human Blood Serum

The research included the separation of thioredoxin (Trx) from human blood serum using different biochemical techniques. One proteinous peak had been isolated by gel filtration using sephadex )G-50) that produced by ammonium sulphate precipitation(65%) after dialysis. It was noted that there are two peaks of Trx when using ion exchange-type DEAE-cellulose for the product of gel filtration column.
 The approximately molecular weight of the isolated Trx using gel filtration chromatography (G-50) and electrophoresis technique type SDS-PAGE were (12305 + 190) Dalton and 12461 Dalton respectively .
 The results showed that inhibited and activated effects of drugs on Trx working. The results showed that inhibited to 44.59% for paracetamol, dexathamazone, diphenhydramine-HCl, theophlline anhydrous, pseudoephedrine, ceramide and caffeine anhydrous, but metronidazole and chlorpheniramine malate have activated effect to 110.8% and 21.62 respectively.

Production, optimization, and purification of alkaline thermotolerant protease from newly isolated Phalaris minor seeds

The present work aims to purify and perform a preliminary analysis on a thermostable serine alkaline protease from a recently identified P. minor. The enzyme was purified 2.7-fold with a 12.4 % recovery using Sephadex G-100 chromatography, DEAE-cellulose, and ammonium sulphate precipitation. The isolated enzyme has a specific activity of 473 U/mg. The purified protease had a molecular mass of 29 kDa, and just one band was seen, which matched the band obtained using SDS-PAGE. High thermostability was demonstrated by the enzymes, which had half-lives of 31.79 and 6.0 min (a 5.3-fold improvement), enthalpies of denaturation (ΔH°) of 119.53 and 119.35 KJ mol−1, entropies of denaturation (ΔS°) of 32.96 and 41.11 J/mol·K, and free energies of denaturation (ΔG°) of 108.87 and 105.58 KJ mol−1 for the protease enzyme. Studies on the folding and stability of alkaline proteases are important since their use in biotechnology requires that they operate in settings of extreme pH and temperature. According to the kinetic and thermodynamic properties, the protease produced by P. minor is superior to that produced by other sources and previously described plants, and it might find utility in a variety of industrial fields.

Engineered Alcaligenes sp. by chemical mutagen produces thermostable and acido-alkalophilic endo-1,4-β-mannanases for improved industrial biocatalyst

This study reported physicochemical properties of purified endo-1,4-β-mannanase from the wild type, Alcaligenes sp. and its most promising chemical mutant. The crude enzymes from fermentation of wild and mutant bacteria were purified by ammonium sulfate precipitation, ion exchange and gel-filtration chromatography followed by an investigation of the physicochemical properties of purified wild and mutant enzymes. β-mannanase from wild and mutant Alcaligenes sp. exhibited 1.75 and 1.6 purification-folds with percentage recoveries of 2.6 and 2.5% and molecular weights of 61.6 and 80 kDa respectively. The wild and mutant β-mannanase were most active at 40 and 50 °C with optimum pH 6.0 for both and were thermostable with very high percentage activity but the wild-type β-mannanase showed better stability over a broad pH activity. The β-mannanase activity from the parent strain was stimulated in the presence of Mn2+, Co2+, Zn2+, Mg2+ and Na+. Vmax and Km for the wild type and its mutant were found to be 0.747 U//mL/min and 5.2 × 10−4 mg/mL, and 0.247 U/mL/min and 2.47 × 10−4 mg/mL, respectively. Changes that occurred in the nucleotide sequences of the most improved mutant may be attributed to its thermo-stability, thermo-tolerant and high substrate affinity- desired properties for improved bioprocesses.

Purification and biochemical characterization of a novel ene- reductase from Kazachstania exigua HSC6 for dihydro-β-ionone from β-ionone.

We purified and characterized a novel ene-reductase (KaDBR1) from Kazachstania exigua HSC6 for the synthesis of dihydro-β-ionone from β-ionone. KaDBR1 was purified to homogeneity by ammonium sulfate precipitation and phenyl-Sepharose Fast Flow and Q-Sepharose chromatography. The purified enzyme was characterized by measuring the amount of dihydro-β-ionone from β-ionone with LC-MS analysis method. The molecular mass of KaDBR1 was estimated to be 45kDa by SDS-PAGE. The purified KaDBR1 enzyme had optimal activity at 60°C and pH 6.0. The addition of 5mM Mg2+, Ca2+, Al3+, Na+, and dithiothreitol increased the activity of KaDBR1 by 25%, 18%, 34%, 20%, and 23%, respectively. KaDBR1 favored NADH over NADPH as a cofactor, and its catalytic efficiency (kcat/Km) toward β-ionone using NADH was 8.1-fold greater than when using NADPH. Owing to its unique properties, KaDBR1 is a potential candidate for the enzymatic biotransformation of β-ionone to dihydro-β-ionone in biotechnology applications.

Biochemical and Kinetic Study for the Partial Purified Thioredoxin Reductase from Healthy Human Serum

The research included the separation of thioredoxin reductase (TrxR) from human blood serum using different biochemical techniques. Two proteinous peaks had been isolated by gel filtration using sephadex)G-50) and three proteinous peaks isolated from sephadex (G-100) that produced by ammonium sulphate precipitation (65%). The approximately molecular weight of the isolated protein as a source of enzyme using gel filtration chromatography (G-100) was (118059 + 925) Dalton.
 The results showed two peaks of enzyme when using ion exchange-type DEAE-cellulose and when using electrophoresis technique type SDS-PAGE indicated the presence of two bands from peak of separation column type sephadex G-100 and one band of each peak, resulting from the ion exchange process with molecular weights of 58231 +490 Dalton.
 The results showed that the optimum conditions of purified enzyme from human serum was at (200 µg/ml) of protein as a source of the enzyme using (0.4 mol/l) citric acid-Na2HPO4 buffer solution at pH (7) act for (25) minutes at (40°C). Using Line Weaver-Burk plot, the values of maximum velocity (Vmax) and Michaelis constant (Km) were found to be (0.182 µmol/ min) and (0.013 mol/l) respectively using 5,5-- dithio bis (2-nitrobenzoic acid) (DNTB) as a substrate. Finally, this was, also, involved the study of the effect of some chemicals and drugs on the enzyme activity. The results showed that gundinium - HCl is uncompetitive inhibitor, while bromoacetic acid is noncompetitive inhibitor and zinc sulphate is a competitive inhibitor for the enzyme at different concentrations of inhibitors, but dexathamazone was an activator to the enzyme.

LEGUME INDIAN BEAN (PHASEOLUS VULGARIS) PARTIALLY PURIFIED ANTI-NUTRITIONAL PROTEIN FACTOR EXHIBIT ANTI-OXIDANT, IMMUNOMODULATORY AND ANTI-CANCER PROPERTIES

Objective: This study was aimed to access one of the hidden ANF in Indian bean and assessment of its antioxidant, immunomodulatory, antiangiogenic, and anti-cancer properties. Methods: Indian beans were processed and evaluated for the presence of ANF through HA activity. Bean-ANF was partially purified by ammonium sulfate precipitation (ASP) and dialysis. Obtained Partially Purified bean-ANF (PPb-ANF) resolved on 12% SDS-PAGE, analyzed protease digestibility. The immunogenicity of PPb-ANF was characterized by producing polyclonal antibodies and checked for the presence of natural antibodies in healthy human sera. Pharmacological studies of PPb-ANF were assessed through anti-oxidant, immunogenic, anti-angiogenic and anti-cancer assays. Results: Partially purified Bean-ANF (PPb-ANF) showed significant specific HA activity up to 150 Units/mg compared to crude extract 9.3 of Units/mg. The PPb-ANF was identified to have more than 90% homogeneity in purity with an observed M W of 30-32 kDa. PPb-ANF showed significant pepsin digestive stability up to 2 h at 37 ᵒC and appears digestive resistant. Rabbit produced anti-PPb-ANF IgG polyclonal antibody on immunization and exhibited immunogenic properties. Natural human IgG and IgE antibodies were found in the non-atopic human sera against PPb-ANF, indicative of its immunogenicity. Pharmacological studies of PPb-ANF confirmed its potent antioxidant activity. At 400 µg of PPb-ANF showed the highest around 90% DPPH activity and at 150 µg of PPb-ANF showed 89% inhibitory lipid peroxidation activity. The PPb-ANF showed 70% (3 μg) and 40% (1.5 μg) inhibitory in blood vessel density compared to control and exhibited significant anti-angiogenic activity. The PPb-ANF showed a significant (*p<0.05.) reduction in solid tumor weight upon dose-2 treatment (50 mg/kg body weight) and also increased lifespan up to 70 d compared with the control tumor model. Conclusion: PPb-ANF was identified as digestive-resistant, hemagglutinin with a molecular weight of 30-32 kDa. The protein is immunogenic and has potential antioxidant, antiangiogenic and anti-cancer properties. Further purification and mechanistic-based pharmacological characterization might explore this protein as a potential anti-cancer molecule.

Purification and enzymatic properties of a new thermostable endoglucanase from Aspergillus oryzae HML366.

Aspergillus oryzae HML366 is a newly screened cellulase-producing strain. The endoglucanase HML ED1 from A. oryzae HML366 was quickly purified by a two-step method that combines ammonium sulfate precipitation and strong anion exchange column. SDS-PAGE electrophoresis indicated that the molecular weight of the enzyme was 68kDa. The optimum temperature of the purified endoglucanase was 60 ℃ and the enzyme activity was stable below 70 ℃. The optimum pH was 6.5, and the enzyme activity was stable at pH between 4.5 and 9.0. The analysis indicated that additional Na+, K+, Ca2+, and Zn2+ reduced the catalytic ability of enzyme to the substrate, but Mn2+ enhanced its catalytic ability to the substrate.The Km and Vmax of the purified endoglucanase were 8.75mg/mL and 60.24μmol/min·mg, respectively. In this study, we report for the first time that A. oryzae HML366 can produce a heat-resistant and wide pH tolerant endoglucanase HML ED1, which has potential industrial application value in bioethanol, paper, food, textile, detergent, and pharmaceutical industries.

Characterization of Catalase Enzyme from Leaf Tissue of Aronia (Aronia melanocarpa) Plant

Aronia is among the most antioxidant containing plants which is found commonly around the world. Aronia cultivation started in Turkey for the first time in 2012 at the Atatürk Central Research Institute of Garden Cultures, and a plantation was constructed in the experimental area. Since antioxidants help to preserve food by blocking oxidation processes and contributing to the health promotion provided by numerous dietary supplements, nutraceutical and functional food additives, antioxidant capacity of these plants should be well characterized. To assess and evaluate the antioxidant content of foods and plant products, many approaches are utilized. In this study, catalase (CAT) enzyme was partially purified from aronia plant leaf tissue and characterization was carried out. Purification process consisted of homogenate preparation, ammonium sulfate precipitation and dialysis. The optimal ionic strength, pH, substrate concentration and enzyme quantity were examined. These values were found to be 300 mM Tris, pH:8.0, 12 mM H2O2 and 75 μl, respectively, for the catalase enzyme of the Aronia plant leaf tissue. This study is the first in the literature dealing with the characterization of antioxidant enzyme from Aronia plant.

Purification, Characterization and Bactericidal Action of Lysozyme, Isolated from Bacillus subtillis BSN314: A Disintegrating Effect of Lysozyme on Gram-Positive and Gram-Negative Bacteria.

In the present study, lysozyme was purified by the following multi-step methodology: salt (ammonium sulfate) precipitation, dialysis, and ultrafiltration. The lysozyme potential was measured by enzymatic activity after each purification step. However, after ultrafiltration, the resulting material was considered extra purified. It was concentrated in an ultrafiltration centrifuge tube, and the resulting protein/lysozyme was used to determine its bactericidal potential against five bacterial strains, including three gram-positive (Bacillus subtilis 168, Micrococcus luteus, and Bacillus cereus) and two gram-negative (Salmonella typhimurium and Pseudomonas aeruginosa) strains. The results of ZOI and MIC/MBC showed that lysozyme had a higher antimicrobial activity against gram-positive than gram-negative bacterial strains. The results of the antibacterial activity of lysozyme were compared with those of ciprofloxacin (antibiotic). For this purpose, two indices were applied in the present study: antimicrobial index (AMI) and percent activity index (PAI). It was found that the purified lysozyme had a higher antibacterial activity against Bacillus cereus (AMI/PAI; 1.01/101) and Bacillus subtilis 168 (AMI/PAI; 1.03/103), compared to the antibiotic (ciprofloxacin) used in this study. Atomic force microscopy (AFM) was used to determine the bactericidal action of the lysozyme on the bacterial cell. The purified protein was further processed by gel column chromatography and the eluate was collected, its enzymatic activity was 21.93 U/mL, while the eluate was processed by native-PAGE. By this analysis, the un-denatured protein with enzymatic activity of 40.9 U/mL was obtained. This step shows that the protein (lysozyme) has an even higher enzymatic potential. To determine the specific peptides (in lysozyme) that may cause the bactericidal potential and cell lytic/enzymatic activity, the isolated protein (lysozyme) was further processed by the SDS-PAGE technique. SDS-PAGE analysis revealed different bands with sizes of 34 kDa, 24 kDa, and 10 kDa, respectively. To determine the chemical composition of the peptides, the bands (from SDS-PAGE) were cut, enzymatically digested, desalted, and analyzed by LC-MS (liquid chromatography-mass spectrometry). LC-MS analysis showed that the purified lysozyme had the following composition: the number of proteins in the sample was 56, the number of peptides was 124, and the number of PSMs (peptide spectrum matches) was 309. Among them, two peptides related to lysozyme and bactericidal activities were identified as: A0A1Q9G213 (N-acetylmuramoyl-L-alanine amidase) and A0A1Q9FRD3 (D-alanyl-D-alanine carboxypeptidase). The corresponding protein sequence and nucleic acid sequence were determined by comparison with the database.

Honey proteins regulate oxidative stress, inflammation and ameliorates hyperglycemia in streptozotocin induced diabetic rats

BackgroundDiabetes Mellitus (DM) poses a serious health problem worldwide and several inflammatory mediators are involved in the pathogenesis of this disease. Honey composed of various constituents which have been proven to have immunomodulatory and anti-inflammatory properties. The aim of this study is to investigate the in vitro and in vivo effects of Ziziphus honey and its isolated crude proteins in modulation of immune system and inflammation involved in the pathogenesis of diabetes.MethodologyThe proteins from Ziziphus honey were isolated by ammonium sulfate precipitation and estimated by Bradford method. In vitro anti-inflammatory activities were evaluated by inhibition of reactive oxygen species (ROS) from phagocytes via chemiluminescence immunoassay and nitric oxide (NO) by Griess method. Cytotoxicity was evaluated by MTT Assay. The comparative effect of oral and IP routes of honey and isolated proteins was observed in streptozotocin (STZ) induced diabetic male Wistar rats. qRT-PCR technique was utilized for gene expression studies.ResultsThe honey proteins suppressed phagocyte oxidative burst and nitric oxide (NO) at significantly lower concentrations as compared to crude honey. The isolated proteins showed promising anti-inflammatory and hypoglycemic effects along with maintenance of body weight of rodents via both oral and IP routes, with significant down-regulation of inflammatory markers TNF-α, IL-1β, IFN-γ, iNOS, caspase 1, Calgranulin A (S100A8) and NF-κB expression in diabetic rats.ConclusionThe isolated honey proteins showed better immunomodulatory and therapeutic potential at significantly lower doses as compared to crude honey.

Ochratoxin A degrading enzymes of Stenotrophomonassp. 043-1a.

Ochratoxin A is a secondary metabolite that acts as a mycotoxin and is produced by Aspergillus, Penicillium, and other fungal species. It is a threat to animal and human health due to nephrotoxic, carcinogenic, and genotoxic properties and its widespread incidence in agricultural products. To reduce this threat, biological remediation processes are of growing interest. The aerobic gram-negative bacterium Stenotrophomonassp. 043-1a, isolated from soil, was previously shown to degrade ochratoxin A into the non-toxic ochratoxin α and l-phenylalanine (Schatzmayr et al. 2002). However, the enzyme or enzymes catalyzing this reaction in this strain remained elusive. Here, we report the targeted purification of Stenotrophomonassp. 043-1a lysate via ammonium sulfate precipitation, size-exclusion chromatography, and hydrophobic interaction chromatography to identify the ochratoxin A degrading enzymes by subsequent peptide fragment fingerprinting. The metallo-dependent hydrolase Chr1_3858681_3267 and a member of the peptidase S9 family, Chr1_3858681_771, were shown to degrade ochratoxin A. This was, to our knowledge, the first report of an ochratoxin A degrading enzyme from the peptidase S9 family.

An alkaline protease from Bacillus cereus NJSZ-13 can act as a pathogenicity factor in infection of pinewood nematode

Endophytic bacteria are an important biological control for nematodes. We isolated the nematicidal Bacillus cereus NJSZ-13 from healthy Pinus elliottii trunks. Bioassay experiments showed killing of all tested nematodes by proteins from the NJSZ-13 culture filtrate within 72 h. Degradation of the nematode cuticles was observed, suggesting the action of extracellular bacterial enzymes. The responsible protease was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, ion-exchange chromatography, and SDS-PAGE. The protease had a molecular weight of 28 kDa and optimal activity at 55 °C and pH 9, indicating an alkaline protease. The study suggests the potential for using this B. cereus NJSZ-13 strain protease to prevent pinewood nematode infection.

Purification and biochemical characterization of two laccase isoenzymes isolated from Trichoderma harzianum S7113 and its application for bisphenol A degradation

Two laccase isoenzymes (LacA and LacB) were isolated from a novel Trichoderma harzianum S7113 isolate employing ammonium sulfate precipitation, Sephadex G100, and DEAE Sepharose ion exchange chromatography. The molecular weights of the purified LacA and LacB laccases were estimated to be 63 and 48 kDa, respectively. The two isoenzymes had their optimum activities at the same temperature (50 °C), but at slightly different pH values (pH 3.0 for LacA and pH 2.5 for LacB). LacA and LacB had the same thermal stability at 40 °C and pH stability at pH 9.0. The two isoenzymes also showed a high level of specific activity toward ABTS, where the Km values of LacA and LacB were 0.100 and 0.065 mM, whereas their Vmax values were 0.603 and 0.182 µmol min−1, respectively. LacA and LacB catalytic activity was stimulated by Mg2+, Zn2+, K+, and Ni2+, whereas it was inhibited by Hg2+ and Pb2+, β-mercaptoethanol, EDTA, and SDS, and completely inhibited by sodium azide. Our findings indicate that purified laccase has a promising capacity for bisphenol A (BPA) bioremediation across a broad pH range. This finding opens up new opportunities for the commercialization of this technique in a variety of biotechnology-based applications, particularly for removing endocrine chemicals from the environment.