Ammonium Sulfate Precipitation Research Articles

The crude oil biodegradation activity of Candida strains isolated from oil-reservoirs soils in Saudi Arabia

Crude oil (petroleum) is a naturally occurring complex composed of hydrocarbon deposits and other organic materials. Bioremediation of crude oil-polluted sites is restricted by the biodiversity of indigenous microflora. They possess complementary substrates required for degrading the different hydrocarbons. In the current study, four yeast strains were isolated from different oil reservoirs in Riyadh, Saudi Arabia. The oil-biodegradation ability of these isolates showed variable oxidation effects on multiple hydrocarbons. The scanning electron microscopy (SEM) images showed morphological changes in Candida isolates compared to the original structures. The drop-collapse and oil emulsification assays showed that yeast strains affected the physical properties of tested hydrocarbons. The content of biosurfactants produced by isolated strains was quantified in the presence of different hydrocarbons to confirm the oil displacement activity. The recovery assays included acid precipitation, solvent extraction, ammonium sulfate, and zinc sulfate precipitation methods. All these methods revealed that the amount of biosurfactants correlates to the type of tested hydrocarbons, where the highest amount was produced in crude oil contaminated samples. In conclusion, the study highlights the importance of Candida isolated from contaminated soils for bioremediation of petroleum oil pollution. That raises the need for further analyses on the microbes/hydrocarbon degradation dynamics.

Fermentation optimization, purification and biochemical characterization of a porphyran degrading enzyme with funoran side-activity from Zobellia uliginosa

A porphyran degrading enzyme was obtained from the marine bacterial strain Zobellia uliginosa with a secondary funoran degradation activity. The optimal fermentation conditions were determined using one-factor-at-a-time experiments and the bacteria produced highest amount of enzyme after 72 h of incubation at 25 °C. The optimal fermentation medium composition was identified as 3% sea salt, furcellaran as carbon source, and peptone served as both nitrogen and carbon source. The extracellular enzyme was purified using ultrafiltration, ammonium sulfate precipitation, ion-exchange chromatography and High performance size-exclusion chromatography (HP-SEC). As compared to that in the crude form, the enzyme had a 7751-fold and 5736-fold increase in maximum specific activity resulting in 2.29 U/mg and 1.13 U/mg for porphyran and funoran respectively. Based on the high performance size-exclusion chromatography, the molecular weight of the purified enzyme was estimated at around ∼41 kDa. The enzymatic activity was observed against funoran with a maximum value at 40 °C and pH 7.5; it remained fairly stable until 40 °C. Significant activation was observed with the presence of Fe3+ and Mg2+; whereas, the enzyme activity was majorly inhibited by EDTA and NH4+. This study is the first to present evidence of funoran degrading enzymes from marine bacteria.

A hemagglutinin from Pongamia pinnata (L.) Pierre seed

Lectins are diverse proteins that bind to carbohydrates and are involved in many important physiological processes. They are highly specific to the sugar molecules they bind and therefore, have many therapeutic and diagnostic applications. There are several lectins that display antiviral, antibacterial antifungal and antitumour activities. Characterization of new lectins paves way for comprehension of their diverse biological roles and mechanism of action, thus aiding in further exploration of lectins in various domains of biology. Here, we endeavoured to purify and characterize lectin from the seed of the legume,Pongamia. Pongamia pinnata (L.) Pierre (syn. Millettia pinnata), seed lectin (PPSL) was purified conventionally by ammonium-sulfate precipitation followed by size exclusion chromatography. The further lectin was physicochemically characterized by CD, fluorescence spectroscopy, mass spectroscopy and isothermal calorimetry. Hemagglutination studies with various mono and disaccharides showed specificity towards galactose. This specificity was reaffirmed by isothermal studies with appreciable thermodynamic parameters. Lectins have tremendous diagnostic applications. They are used as second-generation drug delivery systems.

ISOLATION AND PURIFICATION OF BROMELAIN ENZYME FROM PINEAPPLE JUICE AND STUDY CYTOTOXIC ACTIVITY AGAINST CANCER CELLS

The work included the isolation of Bromelain enzyme from pineapple juice using different biochemical techniques which included: ammonium sulfate precipitation, diagnosis and gel filtration chromatography on Sephadex G-100. It was found that only the second peak had a high activity for Bromelain, the apparent molecular weight of the isolated Bromelain using gel filtration chromatography and SDS-PAGE was (27000) and (24000) Dalton respectively, this study was led to distinguish the cytotoxic action of purified enzyme Bromelain from pineapple juice remove on human breast cancer (MCF-7) cells in vitro contrasted with human hepatic (WRL-68) cells utilizing MTT measure. Charted 6 crystal program incorporating non-straight relapse with one-way ANOVA followed by Dennett's different comparison test to get information addressing the mean+ SD. The outcomes uncovered high poisonousness of the concentrate against MCF-7 cells with IC50 (194.5µg/ml) and showed high-decreased action toward WRL-68 cells with high IC50 esteem while the least feasibility (26.389 ±3.73022) showed up in MCF. 7 cells under treatment with 400 µg.ml-1 contrasted and high practicality in WRL-68 cells. The review reasoned that has a significant wellspring of anticancer medications with protected and particular action on disease cells in contrast with human typical cells.

In Silico Analysis and Functional Characterization of Antimicrobial and Insecticidal Vicilin from Moth Bean (Vigna aconitifolia (Jacq.) Marechal) Seeds.

Vicilin has nutraceutical potential and different noteworthy medicative health-promoting biotic diversions, and it is remarkable against pathogenic microorganisms and insects. In this study, Vigna aconitifolia vicilin (VacV) has been identified and characterized from the seed of Vigna aconitifolia (Jacq.) Marechal (Moth beans). LC-MS/MS analysis of VacV provided seven random fragmented sequences comprising 238 residues, showing significant homology with already reported Vigna radiata vicilin (VraV). VacV was purified using ammonium sulfate precipitation (60%) followed by size exclusion chromatography on Hi-Load 16/60 Superdex 200 pg column and anion-exchange chromatography (Hi trap Q FF column). Purified VacV showed a major ~50 kDa band and multiple lower bands on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under both reduced and non-reduced conditions. After all, a three-dimensional molecular structure of VacV was predicted, which showed β-sheeted molecular conformation similar to crystallographic structure of VraV. All Vicilins from V. aconitifolia and other plants were divided into six sub-groups by phylogenetic analysis, and VacV shared a high degree of similarity with vicilins of Vigna radiata, Pisum sativum, Lupinus albus, Cicer arietinum and Glycine max. Additionally, VacV (20 μg) has significant growth inhibition against different pathogenic bacteria along strong antifungal activity (50 μg). Likewise, VacV (3.0 mg) produced significant growth reduction in Rice Weevil Sitophilus oryzae larvae after 9 days compared with control. Furthermore, by using MMT assay, the cytotoxicity effect of VacV on the growth of HepG2 liver cancerous cells was tested. VacV showed cytotoxicity against the HepG-2 line and the acquired value was 180 µg after 48 h. Finally, we performed molecular docking against caspase-3 protein (PDB ID: 3DEI) for VacV bioactive receptor interface residues. Hence, our results reveal that VacV, has nutraceutical potential and moth beans can be used as a rich resource of functional foods.

Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

SUMMARYSerine carboxypeptidase‐like acyltransferases (SCPL‐ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan‐3‐ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL‐AT paralogs are involved in galloylation of flavan‐3‐ols: CsSCPL4, which contains the conserved catalytic triad S‐D‐H, and CsSCPL5, which has the alternative triad T‐D‐Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non‐catalytic companion paralog (NCCP). Co‐expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull‐down and co‐immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post‐translational processing. Moreover, phylogenetic analyses revealed that their homologs co‐exist in galloylated flavan‐3‐ol‐ or hydrolyzable tannin‐rich plant species. Enzymatic assays further revealed the necessity of co‐expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co‐expression of SCPL‐ATs and their NCCPs contributes to the acylation of flavan‐3‐ols in the plant kingdom.

Biochemical Insights into the functionality of a novel thermostable β-amylase from Diocleareflexa

β-amylases are exohydrolases that catalyze the hydrolysis of α-1,4 glycosidic linkages in polysaccharides to produce maltose. Despite various applications of plant β-amylases, thermolability of the enzyme has been a major challenge that continuously prompts the search for thermostable β-amylases. In this study, β-amylase was isolated from an underutilized legume, marble vine (Dioclea reflexa) and purified to apparent homogeneity, using a combination of 30–80% ammonium sulfate precipitation, ion exchange and gel filtration chromatography. It was characterized for its physicochemical properties. The enzyme gives a purification fold and specific activity of 12.25 and 9.43 U/mg respectively, it has a molecular weight of 56.23 kDa using 12% SDS-PAGE. The kinetic parameters Km and Kcat of the enzyme for p-nitrophenyl-maltopentaoside were 0.22 mM and 1340 s−1. The enzyme had an optimum pH of 6.0 while the optimum temperature was 60 °C. while the thermal inactivation rate constants (kd) value of the enzyme was 0.002, thermal inactivation (ΔG) was 99.1 kJ/mol, ΔH and ΔS were 27.7 kJ/mol and −305.80 J/mol.K respectively at 333 k respectively. The thermal inactivation of the enzyme followed first-order kinetics at 60 °C. The enzyme was relatively stable for 140 min retaining 70% of its initial activity at 60 °C and 67% of the activity at pH 6.0. The activity of purified D. reflexa β-amylase was enhanced in the presence of Zn2+, Ca2+ and Mg2+ and strongly inhibited by Cu2+ and Hg2+. Remarkably, the enzyme exhibited thermostable properties which increase its prospects as a veritable potential enzyme for various industrial and biotechnological applications.

The role of cathepsin L on structural changes of collagen fibers involved in textural deterioration of chilled grass carp (Ctenopharyngodon idella) fillets.

Textural deterioration is a serious problem in chilled fish flesh. Cysteine proteinases are proposed to participate in disintegration of collagen fibers during this process, while its mechanism remains elusive. In the present study, a cysteine proteinase was purified from grass carp muscle and identified by mass spectrometry, and its effect on structural changes of collagen fibers was investigated. During storage at 4°C, cysteine proteinase activity in fillets increased to 1.53-fold at day 5 and maintained a high level later, and this variable was related to a decline in shear force and an increase in drip loss. A 29 kDa cysteine proteinase was purified through ammonium sulfate precipitation and column chromatography, and identified as cathepsin L. Cathepsin L caused collagen fibers to partly disintegrate into fibril bundles and individual fibrils at 48 h, while the triple helical structure of collagen molecules remained stable. Release of soluble proteins and glycosaminoglycans from cathepsin L-treated collagen fibers was time dependent, coinciding with a release of 4.12 ± 0.13% and 8.57 ± 0.03% at 48 h respectively. However, 0.85 ± 0.02% of hydroxyproline was freed from cathepsin L-treated collagen fibers at 48 h. Furthermore, scanning electron microscopy revealed that the inhibitory effect of cathepsin L could retard the destruction of intramuscular connective tissues (IMCTs). These results indicated that cathepsin L might be involved in collagen fiber breakdown by degrading collagen-associated proteoglycans during textural deterioration of grass carp. © 2022 Society of Chemical Industry.

Developing a recombinant expression system for human serine protease PRSS23

The PRSS23 gene, encoding putative serine protease 23, has been associated with several diseases by correlative gene expression studies and genetic‐based comparative models. Our group has recently developed data implicating the expression of this gene as a potential driver of ovarian cancer malignancy. However, there have been no studies defining PRSS23 protein structure or demonstrating its hypothesized proteolytic function. We aim to elucidate PRSS23 protein structure and enzymatic activity. The first step to achieve this goal is to develop a robust expression system and purification protocol for recombinant PRSS23 protein. We hypothesize that developing a methodology to produce PRSS23 recombinant protein will enable us to determine its crystal structure, evaluate its biological and enzyme function, and ultimately target the enzyme therapeutically. We strategized to maximize chances of producing PRSS23 in its native form and with high yield by testing multiple systems and host species: human, yeast, and bacterial. We anticipate, based on the protein sequence, that PRSS23 is endogenously secreted and may be activated in the secretory pathway by furin, releasing the C‐terminal catalytic domain from a smaller N‐terminal domain. The protein is also predicted to be glycosylated. We generated expression plasmids for full‐length PRSS23, codon‐optimized for expression in human and Pichia pastoris expression systems, incorporating a His‐tag for detection and purification. In the human HEK 293F and HEK 293T cell systems, despite optimizing transfection protocols and testing multiple expression constructs, secretion signals, and promoters, the expression level remained very low and was judged unlikely to produce sufficient protein for structural and functional studies. Integration of codon‐optimized PRSS23 into the genome of P. pastoris produced integrants showing a spectrum of expression levels from very low to moderate. Experiments are ongoing to optimize expression conditions of the best clone and develop a purification workflow involving ammonium sulfate precipitation, immobilized nickel ion chromatography, and additional chromatographic steps as needed. In parallel, we cloned the catalytic domain of PRSS23, appropriately codon‐optimized and also containing a C‐terminal His‐tag, into an expression vector for Escherichia coli. High levels of protein were produced as insoluble inclusion bodies, and a protocol was developed for nickel ion affinity purification under denaturing conditions. Experiments are ongoing to identify suitable conditions for in vitro refolding of the PRSS23 catalytic domain. Additionally, we are evaluating a maltose binding protein fusion with the PRSS23 catalytic domain as an alternative strategy to improve soluble protein production in E. coli. Significant quantities of pure protein will be needed to enable crystal structure determination and enzymatic activity elucidation, to unravel the biological mechanism of action and identify endogenous substrates, and potentially lead to new therapeutic strategies targeting PRSS23. The expression systems and purification workflows developed here will be a step forward toward these long‐term goals.

"Production of Equine Anti-Daboia Siamensis Immunoglobulin F(ab’)2 Fragments with Ammonium Sulphate and Caprylic Acid Precipitation and by Multimode Chromatography"

"Production of Equine Anti-Daboia Siamensis Immunoglobulin F(ab’)2 Fragments with Ammonium Sulphate and Caprylic Acid Precipitation and by Multimode Chromatography"

PURIFICATION, THERMOSTABILITY AND INHIBITORS OF α-GLUCOSIDASE FROM Penicllium chrysogenum

α-glucosidase (α-D-glucoside glucohydrolase, EC:3.2.1.20) was isolated from Penicillium chrysogenum Thom ATCC 10106. The enzyme was purified by ammonium sulphate precipitation (85%), DEAE-cellulose and Sephadex G-200. The final specific activity was 360 units/mg protein. The yield was 18 % and the purification fold was 52.9. Trehalose and different sugars offered appreciable protection of α-glucosidase against denaturation at 60 °C. The aqueous extracts from various plants including Trigonella foenum-grecum, Sinapis arvensis, Cucurbita moschata, Apium crispum and Lepidium sativum inhibited α-glucosidase activity with different rates. IC50 values of the tested plants extracts were 88.5, 65.3, 72.6, 84.7 and 97.9 μg/ml, respectively.

Studies on the Production of Protease by Aspergillus Oryzae Ncim 637 under Solid-state Fermentation using Mixed Substrates of Prawn’s Shell and Fish Meal Powder

Proteases are the enzymes that catalyze the breakdown of protein molecules into peptides and amino acids. Because of the vast variety of applications of the proteases in the current investigation. the production of protease from Aspergillus oryzae NCIM 637 was carried out under solid-state fermentation. The highest yield of the enzyme was screened using two substrate powders (Prawn's shell and fish meal powder), and it was observed that combined substrate powder has a higher potential to serve as a substrate for neutral protease synthesis by the fungal strain Aspergillus oryzae NCIM 637. Fermentation time (5 days), fermentation temperature (35°C), optimum pH (7), initial moisture content (46.40%), inoculum age (4 days), and inoculum volume were all optimized (1.0ml). The influence of additives such as carbon source maltose (2%) and nitrogen source casein (2%) was investigated, with a maximum production of 562.57U/gds. When the enzyme was partially purified using ammonium sulfate precipitation, the activity of the protease enzyme was found to be 570U/gds. Proteases have a wide range of applications in the food, pharmaceutical, and leather industries, as well as brewing.

Screening of mushrooms from the woodlands of Zimbabwe: Occurrence of lectins and partial purification of a mucin specific lectin from Boletus edulis.

Mushrooms are known to possess a diversity of bioactive compounds that include lectins, which are proteins or glycoproteins that bind specifically to cell surface carbohydrates, culminating in cell agglutination. The present study describes the screening of lectin activity from ten local mushrooms, namely, Amanita zambiana, Boletus edulis, Cantharellus heinemannianus, Cantharellus miomboensis, Cantharellus symoensii, Lactarius kabansus, Amanita sp., Coprinus sp., Ganoderma lucidum and Trametes strumosa. The lectin content was detected by the haemagglutination activity of mushrooms against sheep and goat erythrocytes. Among the different mushrooms screened Amanita sp., Boletus edulis and Lactarius kabansus showed high lectin activity (39, 617 and 77 HAU/mg mushroom, respectively). Boletus edulis was used for the haemagglutination inhibition assay. A total of twenty sugars and sugar derivatives, namely, α-lactose, D-glucose, D-mannose, D-raffinose, N-acetyl glucosamine, maltose, melibiose, D-ribose, porcine mucin, D-cellobiose, D-arabinose, α-methyl-D-glucoside, methyl-α-D-mannopyranoside, D-trehalose, L-arabinose, L-sorbose, L-lyxose, β-lactose, DL-xylose, and D-galactose, were used for the haemagglutination inhibition assay. Of the various carbohydrates tested, only porcine mucin was found to be the most potent inhibitor of Boletus lectin. The lectin from Boletus mushroom was partially purified using ammonium sulphate precipitation. The highest lectin activity was observed in the 30%-60% fraction. This study revealed for the first time the occurrence of lectins in the local Zimbabwean mushrooms studied as well as isolation of a novel mucin-specific lectin. The information obtained can be used for further investigation of cell surface sugars, purification and characterisation of glycoproteins and their contribution towards the medicinal properties of local mushrooms.

In vitro effects of thirty-eight cardiac drugs on human serum paraoxonase.

In this study, the effects of 38 commonly used cardiac drugs on the human paraoxonase (PON1) were investigated. PON1 was purified from human serum blood by ammonium sulfate precipitation (60%-80%) and hydrophobic interaction chromatography (Sepharose-4B~L-tyrosine~1-napthylamine gel). All of the cardiac drugs inhibited PON1 at the micro molar level. IC50 and Ki values were determined for each drug. The tested drugs displayed potent PON1 inhibitory activity. It was found that the weakest PON1 inhibitors are Irbesartan (Ki : 421.73µM), Glyceryl Trinitrate (Ki : 351.48µM), and Apixaban (Ki : 333.27µM). Bisoprolol hemifumarate (Ki : 269.31µM) is also other weak PON1 inhibitor. Therefore, these drugs, having weak PON1 inhibitory activity, may be preferred primarily in patients with atheroclerotic heart disease compared to other drugs due to the protective effect of PON1 on atherosclerosis. Conversely, the most potent inhibitors against PON1 were propafenone (Ki : 0.35µM), Lacidipine (Ki : 0.78µM), Lidocaine HCl (Ki : 1.78µM), and Propranolol (Ki : 1.86µM). Molecular docking was also applied to confirm the activity of some cardiac drugs on PON1.

Optimization, purification and characterization of laccase from lignocellulolytic fungi Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535

In the present study, lignocellulolytic fungi were isolated by lignocellulolytic waste (Banana waste) soil from the Bhilai-Durg region of Chhattisgarh, India, which was capable to produce laccase enzyme. The laccase producing potent organisms was identified as Dichotomopilus funicola NFCCI 4534 and Alternaria padwickii NFCCI 4535 using molecular characterization by Internal Transcribed Spacer (ITS). For the better production of laccase enzyme physical (incubation period, temperature and pH) and nutritional (carbon, nitrogen and vitamin source) parameters optimized by one factor-at-a time and response surface methodology approach were used. Laccase enzyme was partially purified by ammonium sulphate precipitation and dialysis. The partially purified laccase was characterized by the effect of substrate concentration, temperature, pH, metal ions and inhibitors. Results showed that out of 5 positive cultures only 2 cultures showed the best laccase production after the quantitative screening. ITS-rDNA gene sequencing and BLAST analysis were used to characterize the isolated fungal species and they were found to be D. funicola NFCCI 4534 and A. padwickii NFCCI 4535. The temperature 24 °C and 32 °C and pH 6.0 and 6.5 were suitable for production of D. funicola and A. padwickii, respectively. It was found that Ferric chloride and ferrous sulphate exhibited maximum laccase activity as compared to other metal ions. However, EDTA and l-arginine inhibited D. funicola and A. padwickii, respectively.

Antidiabetic bio-peptides of soft and hard wheat glutens

The effects of various purification techniques on kiwifruit enzyme characteristics (protease activity, kinetic parameters, and protein patterns) and production of wheat gluten bio-active peptides were investigated. The enzyme extract purified by ammonium sulfate precipitation method exhibited the highest protease activity (26), Km (0.04 ± 0.002 mM), Kcat/Km (40), and yield (96%). Using actinidin, the hard and soft wheat gluten subunit proteins produced antidiabetic inhibitory (α-glucosidase and α-amylase) peptides. The smallest Mw fraction of soft wheat gliadin peptide (<1 kDa) showed the highest inhibitory capacity against α-glucosidase (18.4 ± 0.7%) and α-amylase (53.3 ± 1.9%). The presence of high levels of amino acids with hydroxyl groups and proline in P3 sub-fraction had a critical role on α-glucosidase (47.2%) and α-amylase (71.2%) inhibitory activities. In conclusion, wheat gluten subunit peptides showed significant metabolic effects relevant to glucose and insulin control in vitro.

Purification and characterization of antifungal lipopeptide produced by Bacillus velezensis isolated from raw honey.

Raw honey contains a diverse microbiota originating from honeybees, plants, and soil. Some gram-positive bacteria isolated from raw honey are known for their ability to produce secondary metabolites that have the potential to be exploited as antimicrobial agents. Currently, there is a high demand for natural, broad-spectrum, and eco-friendly bio-fungicides in the food industry. Naturally occurring antifungal products from food-isolated bacteria are ideal candidates for agricultural applications. To obtain novel antifungals from natural sources, we isolated bacteria from raw clover and orange blossom honey to evaluate their antifungal-producing potential. Two Bacillus velezensis isolates showed strong antifungal activity against food-isolated fungal strains. Antifungal compound production was optimized by adjusting the growth conditions of these bacterial isolates. Extracellular proteinaceous compounds were purified via ammonium sulfate precipitation, solid phase extraction, and RP-HPLC. Antifungal activity of purified products was confirmed by deferred overlay inhibition assay. Mass spectrometry (MS) was performed to determine the molecular weight of the isolated compounds. Whole genome sequencing (WGS) was conducted to predict secondary metabolite gene clusters encoded by the two antifungal-producing strains. Using MS and WGS data, we determined that the main antifungal compound produced by these two Bacillus velezensis isolates was iturin A, a lipopeptide exhibiting broad spectrum antifungal activity.

Iron oxide nanoparticles immobilized Aspergillus flavus manganese peroxidase with improved biocatalytic, kinetic, thermodynamic, and dye degradation potentialities

Manganese peroxidase (MnP) has gained significant attention for treating textile waste due to its ability to oxidize and detoxify a wide range of recalcitrant xenobiotics, including synthetic dyes. MnP was isolated from a solid-state culture of Aspergillus flavus using pre-optimized conditions of 5 g wheat bran (66% moisture content), pH 4.5, temperature 30 ℃, carbon source: glucose 2%, nitrogen source: yeast extract 0.02%, 5 ml of homogenous fungal spore suspension and fermentation period of 5 days. The extracted MnP was partially purified using ammonium sulfate precipitation and immobilized on iron oxide nanoparticles. Soluble and immobilized MnP showed the maximum catalytic potential at 35 ℃ and 50 ℃, and optimum pH of 4.0 and 5.0, respectively. Vmax and Km for free MnP were 0.126 U/mg and 0.0886 mM, and 0.140 U/mg and 0.704 mM for the immobilized MnP, respectively. Thermodynamic parameters, including Ea, ΔH*, ΔG*, ΔS* were also investigated for free and immobilized MnP. Finally, the soluble and immobilized MnP were exploited for catalytic degradation of two textile dye pollutants, Direct red 31 and Acid black 234. Free MnP catalyzed 94% decolorization of Direct red 31, while 100% decolorization was achieved with immobilized MnP. On the other hand, Acid black 234 showed 85% and 92% decolorization with free and immobilized MnP, respectively. The magnetically separable immobilized MnP was more thermally stable, can work over a wider pH and temperature range, and be more catalytically vigorous than its free counterpart.

ISOLATION, SCREENING, AND PARTIAL CHARACTERIZATION OF OXALATE OXIDASE ENZYME FROM PSEUDOMONAS STRAIN UNDER INDUCED OXALATE STRESS CONDITION

Oxalate oxidase (OxO), a manganese dependent enzyme, is involved in the catalysis of oxalate oxidation to carbon dioxide with the formation of hydrogen peroxide. OxO is present in the cell wall of plants. It increases the resistivity against diseases and external stress. Oxalate is found as waste metabolites in mammals, excess accumulation of oxalate results in hyperoxaluria and urolithiasis in humans. These disorders can be diagnosed by the help of OxO in many analytical methods. The present study is aimed on isolation of OxO enzyme from Pseudomonas strain under high salt stress. The OxO enzyme was produced in bulk under selected salt stress. It was partially purified by ammonium sulphate precipitation method, dialysis and ion-exchange chromatography. The OxO enzyme on enzymatic analysis showed potent activity concerning oxalic acid substrate. The microtiter plate analysis confirmed the ability of OxO enzyme purified from Pseudomonas strain in the diagnosis of oxalate-related disorders and in the future could be used to prepare the diagnostic biosensors.

Isolation, Purification, and Characterization of a Laccase-Degrading Aflatoxin B1 from Bacillus amyloliquefaciens B10.

Aflatoxins, widely found in feed and foodstuffs, are potentially harmful to human and animal health because of their high toxicity. In this study, a strain of Bacillus amyloliquefaciens B10 with a strong ability to degrade aflatoxin B1 (AFB1) was screened; it could degrade 2.5 μg/mL of AFB1 within 96 h. The active substances of Bacillus amyloliquefaciens B10 for the degradation of AFB1 mainly existed in the culture supernatant. A new laccase with AFB1-degrading activity was separated by ammonium sulfate precipitation, diethylaminoethyl (DEAE) and gel filtration chromatography. The results of molecular docking showed that B10 laccase and aflatoxin had a high docking score. The coding sequence of the laccase was successfully amplified from cDNA by PCR and cloned into E. coli. The purified laccase could degrade 79.3% of AFB1 within 36 h. The optimum temperature for AFB1 degradation was 40 °C, and the optimum pH was 6.0–8.0. Notably, Mg2+ and dimethyl sulfoxide (DMSO) could enhance the AFB1-degrading activity of B10 laccase. Mutation of the three key metal combined sites of B10 laccase resulted in the loss of AFB1-degrading activity, indicating that these three metal combined sites of B10 laccase play an essential role in the catalytic degradation of AFB1.