Ammonium Sulfate Precipitation Research Articles

Production of highly cytotoxic and low immunogenic L-asparaginase from Stenotrophomonas maltophilia EMCC2297

L-asparaginase is an important therapeutic enzyme that is frequently utilized in the chemotherapy regimens of adults as well as pediatric patients with acute lymphoblastic leukemia. However, a high rate of hypersensitivity with prolonged use has limited its utilization. Stenotrophomonas maltophilia (S. maltophilia) EMCC2297 isolate was reported as a novel and promising source for L- asparaginase. The present study aimed at the production, purification, and characterization of L- asparaginase from S. maltophilia EMCC2297 isolate. The microbial production of L-asparaginase by the test isolate could be increased by pre-exposure to chloramphenicol at 200 µg/ml concentration. S. maltophilia EMCC2297 L-asparaginase could be purified to homogeneity by ammonium sulphate precipitation and the purified form obtained by gel exclusion chromatography showed total activity of 96.4375 IU/ml and specific activity of 36.251 IU/mg protein. SDS-PAGE analysis revealed that the purified form of the enzyme is separated at an apparent molecular weight of 17 KDa. Michaelis-Menten constant analysis showed a Km value of 4.16 × 10− 2 M with L-asparagine as substrate and Vmax of 10.67 IU/ml. The antitumor activity of the purified enzyme was evaluated on different cell lines and revealed low IC50 of 2.2 IU/ml and 2.83 IU/ml for Hepatocellular cancer cell line (HepG-2), human leukemia cancer cell line (K-562), respectively whereas no cytotoxic effect could be detected on normal human lung fibroblast cells (MRC-5). However, mice treated with native L-asparaginase showed lower IgG titre compared to commercial L-asparaginase. This study highlights the promising characteristics of this enzyme making it a valuable candidate for further research and development to be an adduct in cancer chemotherapy.

Small ubiquitin-like modifier-tag and modified protein purification significantly increase the quality and quantity of recombinant African swine fever virus p30 protein

Background and Aim: African swine fever (ASF) is a highly virulent and contagious viral disease caused by the ASF virus (ASFV). It has a significant impact on swine production throughout the world, while existing vaccines and specific treatments remain ineffective. ASFV p30 is a potent antigenic protein that induces protective antibodies immediately after infection; however, most recombinant p30 is insoluble. This study aimed to improve the solubility, yield, and purity of recombinant p30 by tagging it with a small ubiquitin-like modifier (SUMO) and modifying the protein purification process. Materials and Methods: SUMO fused with ASFV p30 (SUMO-p30) and p30 alone were cloned and expressed in Escherichia coli. SUMO-p30 and p30 solubility and expression levels were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein purification was modified by combining ammonium sulfate precipitation method with affinity chromatography. In addition, large-scale production of all versions of p30 were compared using SDS-PAGE and western blotting, and the purified p30 was used to develop the indirect enzyme-linked immunosorbent assay (ELISA). Results: The solubility and expression levels of SUMO-p30 were dramatically enhanced compared with that of p30. Modification of the purification process significantly increased purified and soluble SUMO-p30 and p30 yields by 6.59 and 1.02 μg/mL, respectively. Large-scale production confirmed that this procedure increased the quantity of recombinant p30 while maintaining protein purity and immunogenicity. The p30-based indirect ELISA was able to discriminate between positive and negative serum samples with statistically significant differences in mean optical density 450 values (p < 0.001). Conclusion: This study demonstrates the enhancement of solubility, purity, and yield of ASFV p30 expressed in E.coli by SUMO fusion tagging and combining ammonium sulfate precipitation with affinity chromatography for protein purification. These positive effects were sustained in large-scale production. Cleavage and removal of hexahistidine-SUMO tag from the fusion protein by protease may not be suitable when handling a large amount of the protein. However, the SUMO-fused p30 retained strong immunoreactivity to convalescent swine serum, indicating its application in immunization and diagnostic purposes. The expression and purification procedures in this study could be applied to increase solubility, quality, and quantity of other recombinant proteins as well. Keywords: African swine fever, diagnosis, indirect enzyme-linked immunosorbent assay, p30, protein expression, purification.

Production, purification, and determination of the biochemical properties of β-glucosidase in Trichoderma koningii via solid substrate fermentation.

The β-glucosidase enzyme was obtained from Trichoderma koningii Oudem. NRRL 54330 under optimal conditions by solid substrate fermentation (SSF) using corn cobs as substrate. The enzyme was purified by two-step procedures, ammonium sulphate precipitation and cefarose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography, followed by biochemical and kinetic characterisation. The β-glucosidase was obtained from T.koningii using ground corn cob as substrate and Na2HPO4, pH 9, as humidification medium. The optimum conditions for enzyme production by SSF were 30 °C and 6days. The purification efficiency of the obtained β-glucosidase was calculated to be 22.56-fold with a yield of 73.51 %. In the determination of β-glucosidase activity, p-nitrophenyl-β-d-glucopyranoside (pNPG) substrate was used, and the optimum pH and temperature values at which β-glucosidase showed high activity were determined to be pH 3.0 and 75 °C. The purity of the enzyme and the presence/number of subunits were checked using two different electrophoretic methods, SDS-PAGE and NATIVE-PAGE electrophoretic methods. The K m and V max values of the purified enzyme were determined to be 0.16 mM and 2000 EU respectively. Itwas also found that d-(+)-glucose and δ-gluconolactone inhibitors exhibited competitive inhibition of β-glucosidase in the presence of pNPG.

PURIFICATION AND CHARACTERIZATION OF AMYLASE PRODUCED FROM PROBIOTIC LACTOBACILLUS PLANTARUM CS FOR INDUSTRIAL APPLICATIONS

Previous studies have demonstrated that probiotic Lactobacillus plantarum CS was able to generate an appreciable amount of extracellular amylase, hence the need to purify and characterize it. The aim of the study was to purify and characterize crude amylase from probiotic Lactobacillus plantarum CS for its industrial applications Three purification steps including ammonium sulphate precipitation, ion exchange chromatography on carboxymethyl sephadex and gel filtration on Sephadex G-75 were utilized. The homogeneity of the purified enzyme was confirmed using sodium deodocyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The purified amylase was characterized on different parameters including substrates hydrolyses, pH and temperature activity and stability profiles. The general purification elution profile revealed two different peaks of amylase activities with outstanding one having a molecular weight of 59.7kDa. Its purification fold was 4.0 with specific activity of 16.44U/mg protein and enzyme yield of 3%. Temperature optimal activity and stability was at 400C and 7.5 for pH activity and stability. Mangenese (Mn2+) (135.17%), tween 80 (128.30%) and some food condiments garlic, thyme, ginger, and tumeric) significantly (p> 0.05) enhanced amylase activity (≥262.40%). However, selenium (Se4+) and hydrogen peroxide (H2O2) were observed to have greatest inhibiting effect (≥30.9%) on the enzyme. Substrate hydrolysis profiles showed that the amylase hydrolyzed all the test starchy substrates with the highest hydrolytic potential on indigenous sweet potato starch (Km value/ Vmax of 1.33mg/ml/ 7.89ml). The rate of hydrolysis of other test substrates had yam> rice>cassava>corn with km values ≤ 4.0mg/ml and Vmax ≤ 25ml. The obtained results gave an insight that amylase produced from Lactobacillus plantarum CS met with the possessed properties suitable for any industrial application especially in food

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

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

Optimization and Detergent Compatibility of Protease Produced from Aspergillus oryzae by Utilizing Agro Wastes.

The biotechnological process called solid-state fermentation (SSF) was applied for hyper production of protease by using a fungal strain called Aspergillus oryzae. From screening of 9 different local substrates (peanut shell, wheat bran, guava leaves, sugar cane bagasse, rice polish, wheat straw, corn straw, reed grass, and rice straw), peanut shells serve as the best substrates for protease production under optimized cultured conditions. The varying physiochemical parameters such as pH (2-9.5), temperature (30-52 °C), incubation time (1-10 days), inoculum size (1-8 mL), moisture level (20-125%), and substrate concentration (1-7 g) were optimized by response surface methodology (RSM). The highest activity of protease was recorded to be 1101.778 U/mL at 660 nm using peanut shell was optimum at pH 8, temperature 52 °C, incubation time 8 days, inoculum size 2 mL, moisture level 20%, and substrate concentration 2 g. The crude form of enzymes produced were further purified through ammonium sulfate precipitation, dialysis, and gel filtration chromatography. Then, purified enzymes were characterized at different pH, temperature, and incubation time. For characterization of purified protease, pH, temperature, and incubation time were 8, 52 °C, and 8 days for peanut shell and was done by one factor at a time method. Hence, isolated enzymes were alkaline in nature, i.e., alkaline proteases. Then, protease produced from peanut shells was applied to locally available detergents to increase their catalytic activity for strain removal. At last, the final results were interpreted in the form of 3D surface and contour plots using Microsoft Excel 2013 and Minitab 17 software. In conclusion, the utilization of A. oryzae and peanut shell as the substrate in the biotechnological process of SSF demonstrated successful hyper production of alkaline protease. The optimized conditions resulted in high enzyme activity and showcased the potential application of the isolated enzymes in improving the catalytic activity of locally available detergents.

Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Purification and characterization of lipase from a new strain Staphylococcus argenteus MG2 (MTCC 12820)

The lipase enzyme was isolated and purified from Staphylococcus argenteus MG2 (MTCC 12820) to homogeneity using ammonium sulphate precipitation followed by chromatographic techniques. This process resulted in a purification factor of 40.96-fold and a 26.25% recovery with a specific activity of 744.68 U mg-1. The molecular weight of the purified lipase was determined by SDS-PAGE to be 45 kDa. The Km and Vmax values of the purified lipase were calculated to be 4.95 mM and 79.36 µmol/min/mg-1, respectively. The maximum lipase activity was observed at pH 7.0 and 30 ºC with 100% stability, and it was also found to be stable in a broad range of pH (5-12) and temperature (30-90 ºC). The enzymatic activity of this Staphylococcal lipase was increased by Ca2+ to 105.71% at a concentration of 1 mM CaCl2. Additionally, it exhibited marked stability and activity in organic solvents. In the presence of 1% SDS surfactant, it retained 85.16% residual activity, while the metal chelator EDTA (inhibitor) reduced the lipase activity to 83.87% residual activity at a concentration of 1% w/v. This alkali-stable and thermo-stable lipase can be exploited by extending its use in the preparation of detergents and in various industrial and biotechnological applications.

Unveiling the anticancer potential of L-Asparaginase (ASNase) from novel Streptomyces isolate HB2AG: Purification and characterization study

L-asparaginase (ASNase) obtained from microbial origins is a significant enzyme in the commercial sphere, crucial for the treatment of acute lymphoblastic leukemia. In the current investigation, we isolated ASNase through techniques involving ammonium sulfate precipitation and DEAE sepharose anion exchange column chromatography, revealing a molecular weight of 35 kDa. The enzyme exhibited its peak activity at pH 8 and 40°C, maintaining approximately 99% of its enzymatic effectiveness. The Michaelis constant (Km) and maximum velocity (Vmax) were determined to be 0.0021 M and 714.3 µM/min, respectively. ASNase was found to be non-toxic on HEK293 cells and selectively effective against breast cancer, hepatocellular carcinoma cells, colorectal adenocarcinoma cells, Human lung cancer cell line, Neck squamous cells, and Human head carcinoma cells. Hepatocellular carcinoma cells exhibited remarkable responsiveness to ASNase, displaying an IC50 of 23 µg/mL. This led to observable changes in nuclear morphology indicative of apoptosis, as evidenced by DAPI and Annexin V-FITC staining. ASNase also showed cell migration potential on HEK293 cells. Streptomyces HB2AG ASNase’s dynamic functioning across pH and temperature ranges, as well as its stability at 40°C and cytotoxic potential, make it a promising pharmaceutical candidate for various applications.

Streptococcus thermophilus: A Source of Postbiotics Displaying Anti-Inflammatory Effects in THP 1 Macrophages.

In addition to traditional use in fermented dairy products, S. thermophilus also exhibits anti-inflammatory properties both in live and heat-inactivated form. Recent studies have highlighted that some hydrolysates from surface proteins of S. thermophilus could be responsible partially for overall anti-inflammatory activity of this bacterium. It was hypothesized that anti-inflammatory activity could also be attributed to peptides resulting from the digestion of intracellular proteins of S. thermophilus. Therefore, total intracellular proteins (TIP) from two phenotypically different strains, LMD-9 and CNRZ-21N, were recovered by sonication followed by ammonium sulphate precipitation. The molecular masses of the TIP of both strains were very close to each other as observed by SDS-PAGE. The TIP were fractionated by size exclusion fast protein liquid chromatography to obtain a 3-10 kDa intracellular protein (IP) fraction, which was then hydrolysed with pancreatic enzyme preparation, Corolase PP. The hydrolysed IP fraction from each strain exhibited anti-inflammatory activity by modulating pro-inflammatory mediators, particularly IL-1β in LPS-stimulated THP-1 macrophages. However, a decrease in IL-8 secretion was only observed with hydrolysed IP fraction from CNRZ-21N, indicating that strain could be an important parameter in obtaining active hydrolysates. Results showed that peptides from the 3-10 kDa IP fraction of S. thermophilus could therefore be considered as postbiotics with potential beneficial effects on human health. Thus, it can be used as a promising bioactive ingredient for the development of functional foods to prevent low-grade inflammation.

Green and efficient method to acquire high-value phycobiliprotein from microalgal biomass involving deep eutectic solvent-based ultrasound-assisted extraction

Phycoerythrin (PE) is a phycobiliprotein holding great potential as a high-value food colorant and medicine. Deep eutectic solvent (DES)-based ultrasound-assisted extraction (UAE) was applied to extract B-PE by disrupting the resistant polysaccharide cell wall of Porphyridium purpureum. The solubility of cell wall monomers in 31 DESs was predicted using COSMO-RS. Five glycerol-based DESs were tested for extraction, all of which showed significantly higher B-PE yields by up to 13.5 folds than water. The DES-dependent B-PE extraction efficiencies were proposedly associated with different cell disrupting capabilities and protein stabilizing effects of DESs. The DES-based UAE method could be considered green according to a metric assessment tool, AGREEprep. The crude extract containing DES was further subjected to aqueous two-phase system, two-step ammonium sulfate precipitation, and ultrafiltration processes. The final purified B-PE had a PE purity ratio of 3.60 and a PC purity ratio of 0.08, comparable to the purity of commercial products.

Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity

In this study, glutathione reductase (EC 1.8.1.7; GR, Glutathione: NADP+ oxidoreductase), which is the key enzyme of antioxidant metabolism, was purified from sheep spleen using ammonium sulfate precipitation and 2', 5'-ADP Sepharose-4B affinity chromatography. As a result of purification, the GR enzyme was purified, with 20.03 EU/mg.protein of specific activity, 1564.8 times a yield of 40.61%. The purity of the enzyme was checked by SDS-PAGE. In the characterization studies, optimum pH, optimum ionic strength, stable pH, optimum temperature and subunit molecular mass of the enzyme were determined. In addition, KM and Vmax values were found to determine the enzyme's affinity for GSSG and NADPH substrates. It was determined as KM constant 0.0061 mM and Vmax value 0.259 EU/mL, for NADPH, KM constant was determined as 0.351 mM and Vmax value was determined as 0.604 EU/mL for GSSG. In addition, the effects of ampicillin, streptomycin sulfate, gentamicin, cefoperazone sodium and precort-lyo on enzyme activity were investigated. It was determined that these drugs showed an inhibitory effect on GR enzyme activity purified from sheep spleen tissue. The IC50 values for precort-lyo, ampicillin, streptomycin sulfate, cefoperazone sodium, and gentamicin were 1.27 mM, 3.22 mM, 7.95 mM, 16.97 mM, and 17.20 Mm, respectively; and Ki constants were calculated as 0.466±0.387 mM (competitive), 1.057±0.110 mM (non-competitive), 3.386±1.305 mM (competitive), 4.910±0.960 mM (competitive), and 20.770±8.169 mM (non-competitive), respectively.

Response surface methodology as an approach for optimization of alpha amylase production by using bacterial consortium under submerged fermentation

The present study aims to isolate amylolytic bacteria and later the formation of a bacterial consortium for alpha-amylase production. The potent amylolytic strains in a selected consortium were identified as B. subtilis and B. licheniformis on the basis of 16S rRNA sequencing. Initially cultural conditions for alpha amylase were optimized in Plackett–Burman design, followed by the central composite design. Analysis of Variance (ANOVA) of the optimization study indicated pH 7, 0.05 %CaCl2 and 24 h incubation time were the significant variables that mainly effect the alpha amylase production. After the process of statistical optimization, the enzyme was then partially purified by means of ammonium sulfate precipitation at a 60% saturation level, resulting in a 2-fold purification with 1.4 U/mg specific activity. Enzyme kinetics studies showed that the maximum velocity of the reaction (Vmax) and Michaelis constant (Km) were 47.4 mg/ml and 13.2 mM, respectively. Current results suggest the newly isolated bacterial consortium could be considered as a promising candidate for industrial applications.

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

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

Purification and Biochemical Characterization of Polyphenol Oxidase Extracted from Wheat Bran (Wan grano).

Currently, little is known about the characteristics of polyphenol oxidase from wheat bran, which is closely linked to the browning of wheat product. The wheat PPO was purified by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column, and Superdex G-75 chromatography column. Purified wheat PPO activity was 11.05-fold higher, its specific activity was 1365.12 U/mg, and its yield was 8.46%. SDS-PAGE showed that the molecular weight of wheat PPO was approximately 21 kDa. Its optimal pH and temperature were 6.5 and 35 °C for catechol as substrate, respectively. Twelve phenolic substrates from wheat and green tea were used for analyzing the substrate specificity. Wheat PPO showed the highest affinity to catechol due to its maximum Vmax (517.55 U·mL-1·min-1) and low Km (6.36 mM) values. Docking analysis revealed strong affinities between catechol, gallic acid, EGCG, and EC with binding energies of -5.28 kcal/mol, -4.65 kcal/mol, -4.21 kcal/mol, and -5.62 kcal/mol, respectively, for PPO. Sodium sulfite, ascorbic acid, and sodium bisulfite dramatically inhibited wheat PPO activity. Cu2+ and Ca2+ at 10 mM were considered potent activators and inhibitors for wheat PPO, respectively. This report provides a theoretical basis for controlling the enzymatic browning of wheat products fortified with green tea.

Production and Optimization of a Lipase-Producing Bacteria Enterobacter hormachei Isolated from an Oil-Contaminated Soil

Lipase enzymes possess a wide range of industrial applications. Thus, the capacity of lipase-producing bacteria to proliferate on tributyrin agar medium was used to screen for them among various sources. The lipase producing bacteria with highest zone of clearance on the screening media were preserved on the agar slants. The preserved slants were characterized by16SrRNA gene sequencing. The nucleotide sequence so obtained by the 16SrRNA gene sequence was then put through phylogenetic analysis and homology search using the NCBI’s BLAST program. The sequence of lipase producing bacteria showed maximum resemblance with Enterobacter hormaechei bacterial strain. These bacterial strains were produced by inoculating the culture in the inoculum media and allowing it to enrich over night. Subsequently, 3% of the inoculum from the inoculum media was added to the production media, which was then incubated for 48 hours in a rotary shaker. After production the media was centrifuged and supernatant was extracted and used further for optimization, Optimization of the physiochemical parameters of the bacterial strain like inoculum pH, incubation period, inoculum size was found using one factor at a time (OFAT) approach and medium parameters like different carbon source, nitrogen source, substrate, minerals, salts (11 factors) were screened using Plackett-Burman (PB) design which is a full factorial design. The Lipase activity was found by using a titrimetric method using olive oil and Arabic gum mixture as substrate mixture. The maximum lipase activity was found for inoculum pH of 5, 48 hours of incubation time, and 5% inoculum size. The results of the PB design showed the significant parameters to be glucose (carbon source), peptone (nitrogen source), KH2PO4 (salt), and NaCl (mineral). The organism of the soil sample containing bacterial strain showed maximum lipase activity of 70 U/ml and protein concentration of 4.3 μg/mL at the optimized conditions. After centrifuging the culture media that had been improved, the supernatant was collected and partially purified using dialysis and the ammonium sulfate precipitation procedure. Following precipitation, the supernatant’s activity was measured to be 74 U/mL. After being gathered, the pallet was dialyzed in a dialysis bag and added to a buffer. Both the protein content and the lipase activity were estimated. The protein concentration determined by Lowry’s technique was found to be 6.2 μg/mL, and the lipase activity was reported to be 85.22 U/mL.

Purification, kinetic characterization of thermostable multicopper oxidase from the oyster mushroom and its versatility for greener agro-pulp bio bleaching in the paper industry.

Production of a thermostable laccase from Pleurotus florida was reported for the first time, both in submerged and solid-state fermentation using agro-industrial residues. This enzyme was purified using ammonium sulphate precipitation (60-90%), Sephadex G-100 and DEAE column ion exchange chromatography, respectively. The laccase was purified to 21.49 fold with an apparent molecular weight of 66 kDa and had an optimal pH of 5 with temperature stability at 60°C. Metal ions such as Cu2+ (91.26 µmole/mL/min), Mg2+ (68.15 µmole/mL/min), and Fe2+ (1.73 µmole/mL/min) enhanced the laccase activity, but Fe2+ (1.73µmole/mL/min) inhibited the enzyme activity. The purified laccase had Km and Vmax of 16.68 mM and 26.73 µmole/mL/min for guaiacol as a substrate. The isolated enzyme was characterized by FT-IR which revealed bands at 3655.0 cm-1, 2894.7 cm-1, and 1151.7 cm-1 corresponding to primary amines, C-H stretch, and amide -III, respectively. The enzymatic bio bleaching of paddy straw pulp was found to be most effective which resulted in a lowering of kappa number and yellowness by 19.47% & 17.84% whereas an increase in brightness and whiteness by 41.92%. & -19.61%. Thus, this might be stated that the crude laccase from P. florida can be exploited to reduce the toxic waste load for managing environmental pollution and helps in enhancing the yield and quality of the paper.

Extraction, purification, and investigation of the antibacterial potential of lysozyme isolated from the latex of Calotropis procera

Calotropis procera produces a latex used in traditional medicine because of its components which are found to be biologically active. The latex of C. procera has many benefits such as pain reduction, antimicrobial attitude and other precious attributes. Latex lysozyme (LL) is a particular protein contained in C. procera plant having a promising antibacterial property. The process applied for extraction implicated ammonium sulfate precipitation, water extraction, ion exchange and gel filtration column chromatography evidencing 14.4 kDa for LL as a molecular mass. LL after purification showed 397.18 U mg−1 as specific activity (10.30 purification fold), and with 37.9% as a yield. In addition, the chemical and physical analyses showed that LL had its best realization when the pH was 5.5 at 50 °C. Moreover when metal ions such as Ca2+ and Mg2+ were incorporated the relative activity of the enzyme resulted ampliflied. When a purification was conducted with CM-Cellulose and Sephadex G-100 chromatography, LL revealed an antibacterial property versus both Escherichia coli (MICs 14 µg ml−1) and Bacillus cereus (MICs 13 µg ml−1). HR-TEM analysis displayed an antimicrobial potential of LL after its purification; that analysis showed the deterioration of the cell wall (external membrane of the cell) of both E. coli and B. cereus. Therefore, the LL obtained from C. procera maintains ability as an antibacterial enzyme applicable in various domains such as biotechnological and pharmaceutical industries.Graphical abstract

Isolation and Characterization of Paenibacillus polymyxa B7 and Inhibition of Aspergillus tubingensis A1 by Its Antifungal Substances.

Screening of Bacillus with antagonistic effects on paddy mold pathogens to provide strain resources for biological control of mold in Oryza sativa L. screening of Bacillus isolates antagonistic towards Aspergillus tubingensis from rhizosphere soil of healthy paddy; classification and identification of antagonistic strains by biological characteristics and 16S rDNA sequence analysis; transcriptome sequencing after RNA extraction from Bacillus-treated Aspergillus tubingensis; and extraction of inhibitory crude proteins of Bacillus by ammonium sulfate precipitation; inhibitory crude protein and Bacillus spp. were treated separately for A. tubingensis and observed by scanning electron microscopy (SEM). An antagonistic strain of Bacillus, named B7, was identified as Paenibacillus polymyxa by 16S rDNA identification and phylogenetic evolutionary tree comparison analysis. Analysis of the transcriptome results showed that genes related to secondary metabolite biosynthesis such as antifungal protein were significantly downregulated. SEM results showed that the mycelium of A. tubingensis underwent severe rupture after treatment with P. polymyxa and antifungal proteins, respectively. In addition, the sporocarp changed less after treatment with P. polymyxa, and the sporangium stalks had obvious folds. P. polymyxa B7 has a good antagonistic effect against A. tubingensis and has potential for biocontrol applications of paddy mold pathogens.

Recent Advances Regarding Polyphenol Oxidase in Camellia sinensis: Extraction, Purification, Characterization, and Application.

Polyphenol oxidase (PPO) is an important metalloenzyme in the tea plant (Camellia sinensis). However, there has recently been a lack of comprehensive reviews on Camellia sinensis PPO. In this study, the methods for extracting PPO from Camellia sinensis, including acetone extraction, buffer extraction, and surfactant extraction, are compared in detail. The main purification methods for Camellia sinensis PPO, such as ammonium sulfate precipitation, three-phase partitioning, dialysis, ultrafiltration, ion exchange chromatography, gel filtration chromatography, and affinity chromatography, are summarized. PPOs from different sources of tea plants are characterized and systematically compared in terms of optimal pH, optimal temperature, molecular weight, substrate specificity, and activators and inhibitors. In addition, the applications of PPO in tea processing and the in vitro synthesis of theaflavins are outlined. In this review, detailed research regarding the extraction, purification, properties, and application of Camellia sinensis PPO is summarized to provide a reference for further research on PPO.