Synthesis and characterization of carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrid for amylase immobilization

Carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrids have been synthesized in a modified Stöber process. The hybrid synthesis was optimized to obtain an efficient immobilization matrix for diastase alpha amylase, a multimeric enzyme of high technological significance. The synthesized hybrids were characterized using FTIR, XRD, SEM, TGA and BET studies. The enzyme immobilization was done by adsorption and using the immobilized enzyme, the hydrolysis of soluble starch has been optimized in comparison to free enzyme. The optimum usable pH for the immobilized enzyme ranged from pH 4 to 5, while pH 5 was optimum pH for the free enzyme activity. The kinetic parameters for the immobilized, (K _M = 3.4610 mg ml^?1; V _max = 6.3540 mg ml^?1 min^?1) and free enzyme (K _M = 4.1664 mg ml^?1; V _max = 4.291 mg ml^?1 min^?1) hydrolysis indicated that the immobilization at the nanohybrid has significantly improved the catalytic property of the enzyme. In the immobilized state, the enzyme remained usable for many repeated cycles like our previous material, gum acacia-gelatin-AgNp-silica. Storage experiments indicated that the immobilization has increased the stability of the enzyme and also that AgNps play a role in stabilizing the immobilized enzyme.     

Immobilization and Kinetics of Catalase on Calcium Carbonate Nanoparticles Attached Epoxy Support

A novel hybrid epoxy/nano CaCO₃ composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO₃ nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO₃ support with a conjugation yield of 0.67?±?0.01 mg/cm² and 92.63?±?0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of K _m for H₂O₂ was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in V _max value from 1,500 to 421.10 μmol (min mg protein)^?1 was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

Amperometric Biosensor for Evaluation of Competitive Cholinesterase Inhibition by the Reactivator HI‐6

Abstract

Amperometric biosensors containing enzymes butyrylcholinesterase or acetylcholinesterase were prepared. The biosensors were employed for studying of cholinesterase reactivator: HI‐6. Competitions between HI‐6 and acetylthiocholine as enzyme substrate were used for determination of IC₅₀ value. Biosensors with butyrylcholinesterase from human serum determined IC₅₀ as (1.00±0.02)×10^?6 M; the biosensor with acetylcholinesterase from human erythrocytes performance provided IC₅₀ (3.31±0.13)×10^?6 M, the one with human recombinant acetylcholinesterase (2.00±0.06)×10^?6 M and finally biosensor with acetylcholinesterase from electric eel (6.17±0.17)×10^?6 M when 5 mM acetylthiocholine as substrate was used. We are encouraged to consider presented biosensors as a very useful for evaluation of newly prepared cholinesterase reactivators.     

Removal of iodide from aqueous solutions by polyethylenimine-epichlorohydrin resins

The iodide removal from aqueous solutions (initial I^?-concentration: 300–5,000 mg/L) by a low and a high molecular weight polyethylenimine-epichlorohydrin resin was investigated both in absence and presence of background electrolytes (NaCl and Na₂SO₄, ionic strength due to background electrolyte 0.1 M) using a batch technique, ¹³¹I as radioactive tracer and high-resolution γ-ray spectrometry. The experiments in absence of background electrolyte were performed using solutions of initial pH 3 and 7, whereas those in presence using solutions of initial pH 3. The results, which demonstrated the high iodide-removal efficiency of both resins, were modeled using the Langmuir and Freundlich isotherm equations. The experimental data were better reproduced using the Langmuir equation. Using this equation maximum sorption capacity values (Q _max) of 638.8 and 603.3 mg/g were obtained for the high molecular weight resin from solutions of initial pH 3 and 7 respectively, whereas the corresponding values for the low molecular weight one were slightly lower (552.4 and 507.5 mg/g respectively). The iodide uptake by the resins strongly depended on the presence of competing anions and especially of sulfates. The examination of sections of the I-loaded resins grains by scanning electron microscopy/energy dispersive spectroscopy (SEM/EDS) revealed that iodine was evenly distributed throughout the bulk of the resins and not only bound to their surface.     

Purification of Peroxidase from Red Cabbage (Brassica oleracea var. capitata f. rubra) by Affinity Chromatography

Peroxidase was purified in a single step using 4-amino benzohydrazide affinity chromatography from red cabbage (Brassica oleracea var. capitata f. rubra), and some important biochemical characteristics of the purified enzyme were determined. The enzyme, with a specific activity of 3,550 EU/mg protein, was purified 120.6-fold with a yield of 2.9 % from the synthesized affinity matrix. The molecular weight of the enzyme was found to be 69.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited maximum activity at pH 7.0 and 30 °C. For guaiacol substrate, the K _m and V _max values were found as 0.048 mM and 1.46 EU/mL/min, respectively. Additionally, the IC₅₀ and K _i values for 4-amino benzohydrazide were calculated to be 1.047 and 0.702?±?0.05 mM, respectively, and 4-amino benzohydrazide showed noncompetitive inhibition.     

Isolation,Purification and Characterisation of an Organic Solvent-Tolerant Ca2+-Dependent Protease from Bacillus megaterium AU02

A new organic solvent-tolerant strain Bacillus megaterium AU02 which secretes an organic solvent-tolerant protease was isolated from milk industry waste. Statistical methods were employed to achieve optimum protease production of 43.6 U/ml in shake flask cultures. The productivity of the protease was increased to 53 U/ml when cultivated under controlled conditions in a 7-L fermentor. The protease was purified to homogeneity by a three-step process with 24 % yield and specific activity of 5,375 U/mg. The molecular mass of the protease was found to be 59 kDa. The enzyme was active over a wide range of pH (6.0–9.0), with an optimum activity at pH 7.0 and temperature from 40 to 70 °C having an optimum activity at 50 °C. The thermal stability of the enzyme increased significantly in the presence of CaCl₂, and it retained 90 % activity at 50 °C for 3 h. The K _m and V _max values were determined as 0.722 mg/ml and 0.018 U/mg respectively. The metalloprotease exhibited significant stability in the presence of organic solvents with log P values more than 2.5, nonionic detergents and oxidising agent. An attempt was made to test the synthesis of aspartame precursor (Cbz-Asp-Phe-NH₂) which was catalysed by AU02 protease in the presence of 50 % DMSO. These properties of AU02 protease make it an ideal choice for enzymatic peptide synthesis in organic media.     

Partial Purification and Characterization of a Novel Extracellular Tyrosinase from Auricularia auricula

Extracellular tyrosinase from Auricularia auricula RF201 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 12.6 kDa on SDS-PAGE. The optimum pH for tyrosinase activity was 7, and the enzyme was stable between pH 6 and 9. Tyrosinase has optimal activity at 40 °C and retained most of its activity between 4 and 50 °C. A. auricula tyrosinase could oxidize l-tyrosine, l-DOPA, catechol, and caffeic acid and displayed dark brown or peach color. However, the enzyme was unable to catalyze l-phenylalanine and ferulic acid. In comparison with other substrates, l-tyrosine displayed the highest affinity (K _m of 0.11 mM) and the maximal reaction velocity (V _max of 102.58 μmol/min). Tyrosinase activity was reduced in the presence of numerous tested compounds. Particularly SDS, it significantly inhibited enzyme activity. CuSO₄ and NaCl showed an activation effect on enzyme activity, with the maximum activation found in the presence of CuSO₄.     

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties

A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50?°C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30?°C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg²⁺ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55?°C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K _m and V _max values of β-galactosidase using ONPG as substrate at 50?°C were 2.805 mM and 37.45?×?10^?3?mM/min/mg, respectively.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Zingipain, a Ginger Protease with Acetylcholinesterase Inhibitory Activity

In order to search for new acetylcholinesterase inhibitors (AChEIs), 15 Zingiberaceae plants were tested for AChEI activity in rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Zingiber officinale contained a significant AChEI activity. Eighty percent saturation ammonium sulfate precipitation and diethylaminoethyl cellulose ion exchange chromatography (unbound fraction) enriched the protein to a single band on nondenaturing and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (approximately 33.5 kDa). Gelatin-degrading zymography showed that the AChEI-containing band also contained cysteine protease activity. The AChEI activity was largely stable between ?20 and 60 °C (at least over 120 min) and over a broad pH range (2–12). The AChEI activity was stimulated strongly by Mn²⁺ and Cu²⁺ at 1–10 mM and weakly by Ca²⁺, Fe²⁺, Mg²⁺, and Zn²⁺ at 1 mM, but was inhibited at 10 mM. In contrast, Hg²⁺ and ethylenediaminetetraacetic acid were very and moderately strongly inhibitory, respectively. In-gel tryptic digestion with liquid chromatography–tandem mass spectroscopy resolution revealed two heterogeneous peptides, a 16-amino-acid-long fragment with 100 % similarity to zingipain-1, which is a cysteine protease from Z. officinale, and a 9-amino-acid-long fragment that was 100 % identical to actinidin Act 2a, suggesting that the preparation was heterogeneous. AChEI exhibited noncompetitive inhibition of AChE for the hydrolysis of acetylthiocholine iodide with a K _i value of 9.31 mg/ml.     

Colorimetric determination of the activity of acetylcholinesterase and its inhibitors by exploiting the iodide-catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine by hydrogen peroxide

We describe a sensitive and selective colorimetric method for the determination of the activity of the enzyme acetylcholinesterase (AChE) and its inhibitors. Detection is based on the fact that acetylthiocholine iodide (ATCI) catalyzes the oxidation of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by H₂O₂ to form a blue product (ox-TMB) with an absorption peak at 652 nm, but that oxidation is suppressed if ACTI previously is hydrolyzed by AChE to form thiocholine which decolorizes ox-TMB. In the presence of inhibitor, the activity of AChE is inhibited, thereby inducing the recovery of the blue coloration. Based on these findings, a highly sensitive method is developed for the determination of AChE and its inhibitors. The assay only requires mixing of buffer, solutions of ATCI, TMB, H₂O₂ and a sample containing AChE and photometric measurement. It works in the 0.05 to 5 mU?mL^?1 enzyme activity range and has a detection limit as low as 30 μU?mL^?1. The inhibitor neostigmine causes 50 % enzyme inhibition in 14.5 nM concentration. This analytical system has a wide scope in that it may be applied to the determination of the activity of various other hydrolases with proper substrates.

Open image in new window

Graphical abstract The blue product formed by the iodide-catalyzed oxidation of 3,3′5,5′-tetramethylbenzidine (TMB) by hydrogen peroxide is decolorized if acetylthiocholine iodide (ATCI) is hydrolyzed by acetylcholinesterase (AChE) to form thiocholine. If, however, AChE is inhibited, color formation will take place again.

     

Purification and Side Chain Selective Chemical Modifications of Glutamate Dehydrogenase from Bacillus subtilis Natto

Glutamate dehydrogenase (GDH) from Bacillus subtilis natto was purified to apparent homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, size exclusion chromatography, and hydroxyapatite (HA) affinity chromatography. The GDH was purified 34-fold, with a yield of 41 % of total activity and a specific activity of 34.29 U/mg proteins. The molecular weight (M_r) of was measured at 47 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and 264 kDa with high-performance liquid chromatography (HPLC). The optimum pH and temperature for the deammoniation reaction were measured to be 7.5 and 30 °C, respectively. The active-site amino acid residues of GDH were investigated by chemical modification. The compounds 2,4,6-trinitrobenzenesulfonic acid (TNBS), phenylglyoxal (PG), and phenylmethanesulfonyl fluoride (PMSF) were used to modify lysine, arginine, and serine active site residues, respectively. After treatment with modifying reagents at concentrations of 1 mM, GDH activity fell to 10.7 % with TNBS, 83.3 % with PG, and 12.8 % with PMSF. However, with substrate protection, there was almost no loss in GDH activity following treatment with any modifying reagent. The kinetic parameters K _m and V _max were determined in each case. K _m values for native GDH, 50 % TNBS-inactivated GDH, and 50 % PMSF-inactivated GDH were 0.037, 0.104, and 0.017 mM, respectively. V _max values were 0.048, 0.022, and 0.031 mM/s, respectively. These results suggest that the active site contains one or more lysine residues that play a role in substrate binding and one or more serine residues that may maintain the enzyme conformation. However, arginine residues played less of a role in the activity of GDH.     

Purification and properties of a SDS-resistant xylanase from halophilic Streptomonospora sp. YIM 90494

An extracellular xylanase from halophilic Streptomonospora sp. YIM 90494 was purified to homogeneity from a fermentation broth by ammonium sulphate precipitation, gel filtration chromatography and ion exchange chromatography. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of approximately 50 kDa. The xylanase had maximum activity at pH 7.5 and 55 °C. The enzyme was stable over a broad pH range (pH 4.0–10.0) and showed good thermal stability when being incubated at 60 °C for 2 h. Kinetic experiments indicated that the enzyme had K _m and V _max values of 19.24 mg/mL and 6.1 μmol/min/mg, respectively, using birch wood xylan as substrate. The inhibitory effects of various metal ions and chemical agents on the xylanase activity were investigated. It is greatly interesting to note that Ag⁺ ion and SDS, which strongly inhibited most xylanases reported previously increases the xylanase activity in this study. These characteristics suggest that the enzyme with new properties has considerable potential in industrial applications.     

Purification and Characterization of Hyaluronate Lyase from <Emphasis Type="Italic">Arthrobacter globiformis</Emphasis> A152

A hyaluronate lyase was obtained by cultivating Arthrobacter globiformis strain A152. The enzyme was purified to homogeneity from the supernatant by ammonium sulfate fractionation, Q Sepharose Fast Flow, and Sephadex G-100 chromatography. The purification resulted in a 32.78-fold increase in hyaluronate lyase activity with specific activity of 297.2 U/mg. The molecular weight of the enzyme determined by SDS-PAGE was approximately 73.7 kDa. Using hyaluronic acid (HA) as a substrate, the maximal reaction rate (V_max) and the Michaelis–Menten constant (K_m) of hyaluronate lyase were found to be 4.76 μmol/min/ml and 0.11 mg/ml, respectively. The optimum pH and temperature values for hyaluronate lyase activity were pH 6.0 and 42 °C, respectively. This enzyme was stable at pH 4–10, 5–7, and 5–7 at 4, 37, and 42 °C, respectively. Investigation about temperature effects on hyaluronate lyase displayed that it was stable at 30–37 °C and also showed high activity at 37 °C. The enzymatic activity was enhanced by Ca²⁺ and was strongly inhibited by Cu²⁺ and SDS. These properties suggested that the hyaluronate lyase in this study could bring promising prospects in medical and industry applications.     

Design and Properties of an Immobilization Enzyme System for Inulin Conversion

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0–5.5 and 45–50 °C for the immobilized enzyme. The kinetic parameters of V _max and K _m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.     

Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe₃O₄) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of K_m and V_max. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The V_max values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg^?1 min^?1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.     

Purification and Characterization of Agarase from Marine Bacteria <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. PS12B and Its Use for Preparing Bioactive Hydrolysate from Agarophyte Red Seaweed <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis>

Acinetobacter strain PS12B was isolated from marine sediment and was found to be a good candidate to degrade agar and produce agarase enzyme. The extracellular agarase enzyme from strain PS12B was purified by ammonium sulfate precipitation followed by DEAE-cellulose ion-exchange chromatography. The specific activity of the crude enzyme which was 1.52 U increased to 45.76 U, after two-stage purification, with an enzyme yield of 9.76%. Purified enzyme had a molecular mass of 24 kDa. The optimum pH and temperature for activity of purified agarase were found to be 8.0 and 40 °C, respectively. The K_m and V_max values for agarase were 4.69 mg/ml and 0.5 μmol/min, respectively. Treatment with EDTA reduced the agarase activity by 58% at 5 mM concentration. The enzyme activity was stimulated by the presence of Fe²⁺, Mn²⁺, and Ca2⁺ ions while reducing reagents (β-mercaptoethanol and dithiothreitol, DTT) enhanced its activity by 30–40%. The purified agarase exhibited tolerance to both detergents and organic solvents. Major hydrolysis products of agar were DP4 and also a mixture of longer oligosaccharides DP6 and DP7. The enzyme hydrolysed seaweed (Gracilaria verrucosa) exhibited strong antioxidant activity in vitro. Successful hydrolysis of seaweed indicates the potential use of the enzyme to produce seaweed hydrolysate having health benefits as well as the industrial application like the production of biofuels.     

Purification and Biochemical Characterization of a Novel Alkaline Protease Produced by Penicillium nalgiovense

Penicillium nalgiovense PNA9 produces an extracellular protease during fermentation with characteristics of growth-associated product. Enzyme purification involved ammonium sulfate precipitation, dialysis, and ultrafiltration, resulting in 12.1-fold increase of specific activity (19.5 U/mg). The protein was isolated through a series of BN-PAGE and native PAGE runs. ESI-MS analysis confirmed the molecular mass of 45.2 kDa. N-Terminal sequencing (MGFLKLLKGSLATLAVVNAGKLLTANDGDE) revealed 93 % similarity to a Penicillium chrysogenum protease, identified as major allergen. The protease exhibits simple Michaelis-Menten kinetics and K _m (1.152 mg/ml), V _max (0.827 mg/ml/min), and k _cat (3.2?×?10²) (1/s) values against azocasein show that it possesses high substrate affinity and catalytic efficiency. The protease is active within 10–45 °C, pH 4.0–10.0, and 0–3 M NaCl, while maximum activity was observed at 35 °C, pH 8.0, and 0.25 M NaCl. It is active against the muscle proteins actin and myosin and inactive against myoglobin. It is highly stable in the presence of non-ionic surfactants, hydrogen peroxide, BTNB, and EDTA. Activity was inhibited by SDS, Mn²⁺ and Zn²⁺, and by the serine protease inhibitor PMSF, indicating the serine protease nature of the enzyme. These properties make the novel protease a suitable candidate enzyme in meat ripening and other biotechnological applications.     

Recombinant Expression and Characterization of l-Asparaginase II from a Moderately Thermotolerant Bacterial Isolate

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5?°C, was isolated from cow dung; l-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37?°C and in a buffer system of pH?6 to 7. The enzyme exhibited thermostability at 50?°C with a 33% and 28% of activity retention after 45 and 60?min. The kinetic parameters for the enzyme were calculated from Lineweaver?CBurk plot, and K _m and V _max were 0.89?mM and 0.18?U/mg, respectively.