Vmax Values Research Articles

Enzyme Kinetic of Lipase Catalysed Acidolysis of Used Cooking Palm Oil

Enzymatic acidolysis of various fats and oils has successfully shown a promising ability to alter specific positions of lipids and incorporate desirable fatty acids at specific positions. However, used cooking oil utilization for enzymatic acidolysis is still lacking scientific investigation. Hence, this study aims to utilize used cooking palm oil (UCPO) by enhancing the oleic acid content via enzymatic acidolysis using immobilized C. rugosa lipase. The optimum substrate molar ratio (mol/mol) was identified based on the analysis of the peroxide, iodine, and acid values. Then, kinetic parameters, Km and Vmax for the enzymatic acidolysis of UCPO were calculated. Substrate molar ratio of 1:1, 1:2, 1:3, 1:4, and 1:5 mol/mol (oil: acid) was varied to evaluate the peroxide, acid, and iodine values. The reaction conditions such as reaction temperature (50 °C), reaction time (24 hours), enzyme concentration (0.05 g), agitation speed (250 rpm), and pH (7) were fixed throughout the experiment. The Michaelis-Menten kinetic model was selected to describe the kinetic of enzymatic acidolysis of UCPO. The result showed that incorporation of oleic acid in UCPO has been successfully achieved with the increased in IV value from 39.47 I2/g to 110.53 I2/g. The optimum substrate molar ratio obtained was 1:3 mol/mol with the highest iodine value of 110.53 I2/g. The best linear regression approach is Lineweaver-Burk plot with the values of Vmax and Km were 34.5658 μmol/ml.min and 0.06 mol/L, respectively. The enzyme activity for C. rugosa lipase was obtained at 0.5804 μmol/min.ml.

Affinity-based and in a single step purification of recombinant horseradish peroxidase A2A isoenzyme produced by Pichia pastoris.

Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZαC. So, obtained pPICZαC-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.

Characterization of Immobilized β-Amylase Enzyme Isolated from Sweet Potato and prepared by Entrapment Method

Aim: This study attempted to isolate β-amylase from sweet potato and enzyme immobilized by encapsulation method, and characterized with various parameters. 
 Methods: The enzyme β-amylase was isolated with phosphate-buffered saline and purified by centrifugation with ammonium sulfate. The purified enzyme was immobilized on chitosan (0.25 g) and sodium alginate (0.25 g) polymers by entrapment method in the presence of calcium chloride (0.5 M). The immobilized enzyme was characterized by a starch hydrolysis test, the optimal pH and temperature were studied and the stability of the immobilized enzyme was also determined. SEM analysis was performed and Vm and Km were also found. 
 Results: The starch hydrolysis test showed positive results on the starch agar plates for immobilized enzymes. The thermal inactivation showed a severe loss in the activity of the free enzymes (49.3 %) while the temperature profile of the immobilized enzymes was much broader (84.55 %) at higher temperatures (80° C). The optimal pH and stability indicated that the immobilized enzyme has higher stability in the pH range of 5-8. The Km and Vmax value of free and immobilized enzyme was 7.67 mmol, 21.15 µmol (R2 0.8880), and 4.72 mmol,16.79 µmol (R2 0.8446) respectively. The storage of free and immobilized enzymes for one month showed that 83.5 % and 40 % of free enzymes and 11.6 % and 8.6 % of immobilized enzymes lost activity at 25° C and 4° C, respectively. SEM analysis shows the smooth, porous surface. 
 Conclusion: Immobilized enzymes (natural polymers) exhibit higher thermal stability the optimal pH and stability indicate immobilized enzyme has higher stability in the pH range of 5-8, and achieves a relative activity of 69.7 %. After 6 uses, the reuse efficiency of the immobilized enzyme decreased from 99.8 % to 52.3 %. The storage of the immobilized enzyme showed much higher stability than the found-free enzyme.

β-Xylosidase SRBX1 Activity from Sporisorium reilianum and Its Synergism with Xylanase SRXL1 in Xylose Release from Corn Hemicellulose.

Sposisorium reilianum is the causal agent of corn ear smut disease. Eleven genes have been identified in its genome that code for enzymes that could constitute its hemicellulosic system, three of which have been associated with two Endo-β-1,4-xylanases and one with α-L-arabinofuranosidase activity. In this study, the native protein extracellular with β-xylosidase activity, called SRBX1, produced by this basidiomycete was analyzed by performing production kinetics and its subsequent purification by gel filtration. The enzyme was characterized biochemically and sequenced. Finally, its synergism with Xylanase SRXL1 was determined. Its activity was higher in a medium with corn hemicellulose and glucose as carbon sources. The purified protein was a monomer associated with the sr16700 gene, with a molecular weight of 117 kDa and optimal activity at 60 °C in a pH range of 4-7, which had the ability to hydrolyze the ρ-nitrophenyl β-D-xylanopyranoside and ρ-Nitrophenyl α-L-arabinofuranoside substrates. Its activity was strongly inhibited by silver ions and presented Km and Vmax values of 2.5 mM and 0.2 μmol/min/mg, respectively, using ρ-nitrophenyl β-D-xylanopyranoside as a substrate. The enzyme degrades corn hemicellulose and birch xylan in combination and in sequential synergism with the xylanase SRXL1.

Enantioselective resolution of (R,S)-DMPM to prepare (R)-DMPM by an adsorbed-covalent crosslinked esterase PAE07 from Pseudochrobactrum asaccharolyticum WZZ003.

(R)-N-(2,6-dimethylphenyl) aminopropionic acid methyl ester ((R)-DMPM) is an important chiral intermediate of the fungicide N-(2,6-Dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester ((R)-Metalaxyl). In this study, (1) D3520 (macroporous acrylic anion resin), selected from the ten resins, was used to immobilize the esterase from Pseudochrobactrum asaccharolyticum WZZ003 (PAE07) for resoluting the (R,S)-DMPM to obtain (R)-DMPM. (2) Up to 20g/L PAE07 could be immobilized onto D3520 with a high enzymatic activity of 32.4 U/g. Moreover, the Km and Vmax values of 19.1mM and 2.8mM/min for D3520-immobilized PAE07 indicated its high activity and stereoselectivity. (3) The optimal temperature and pH for the immobilized PAE07 were 40 ℃ and 8.0, and substrate concentration was up to 0.35M. After 15h reaction, the conversion rate from (R,S)-DMPM to (R)-DMPM was 48.0% and the e.e.p and E values were 99.5% and 1393.0, respectively. In scale-up resolution, 200g/L substrate and 12.5g immobilized esterase PAE07 condition, a conversion rate from substrate to product of 48.1% and a product e.e.p of 98% were obtained within 12h, with the activity of immobilized PAE07 retained 80.2% after 5 cycles of reactions. These results indicated that the D3520-immobilized esterase PAE07 had great potential for enzymatic resolution of (R,S)-DMPM to prepare (R)-Metalaxyl.

Vicagrel is hydrolyzed by Raf kinase inhibitor protein in human intestine.

As an analog of clopidogrel and prasugrel, vicagrel is completely hydrolyzed to intermediate thiolactone metabolite 2-oxo-clopidogrel (also the precursor of active thiol metabolite H4) in human intestine, predominantly by AADAC and CES2; however, other unknown vicagrel hydrolases remain to be identified. In this study, recombinant human Raf kinase inhibitor protein (rhRKIP) and pooled human intestinal S9 (HIS9) fractions and microsome (HIM) preparations were used as the different enzyme sources; prasugrel as a probe drug for RKIP (a positive control), vicagrel as a substrate drug of interest, and the rate of the formation of thiolactone metabolites 2-oxo-clopidogrel and R95913 as metrics of hydrolase activity examined, respectively. In addition, an IC50 value of inhibition of rhRKIP-catalyzed vicagrel hydrolysis by locostatin was measured, and five classical esterase inhibitors with distinct esterase selectivity were used to dissect the involvement of multiple hydrolases in vicagrel hydrolysis. The results showed that rhRKIP hydrolyzed vicagrel in vitro, with the values of Km , Vmax , and CLint measured as 20.04±1.99μM, 434.60±12.46nM/min/mg protein, and 21.69±0.28ml/min/mg protein, respectively, and that an IC50 value of locostatin was estimated as 1.24±0.04mM for rhRKIP. In addition to locostatin, eserine and vinblastine strongly suppressed vicagrel hydrolysis in HIM. It is concluded that RKIP can catalyze the hydrolysis of vicagrel in the human intestine, and that vicagrel can be hydrolyzed by multiple hydrolases, such as RKIP, AADAC, and CES2, concomitantly.

Covalent Immobilisation of an Aspergillus niger Derived Endo-1,4-β-Mannanase, Man26A, on Glutaraldehyde-Activated Chitosan Nanoparticles for the Effective Production of Prebiotic MOS from Soybean Meal

An Aspergillus niger endo-1,4-β-mannanase, Man26A, was confirmed by FTIR and XRD to be immobilised on glutaraldehyde-activated chitosan nanoparticles via covalent bonding. The immobilisation (%) and activity yields (%) were 82.25% and 20.75%, respectively. The biochemical properties (pH, temperature optima, and stability) were then comparatively evaluated for both the free and immobilised Man26A. The optimal activity of Man26A shifted to a lower pH after immobilisation (pH 2.0–3.0, from pH 5 for the free enzyme), with the optimum temperature remaining unchanged (60 °C). The two enzymes exhibited identical thermal stability, maintaining 100% activity for the first 6 h at 55 °C. Substrate-specific kinetic analysis showed that the two enzymes had similar affinities towards locust bean gum (LBG) with varied Vmax values. In contrast, they showed various affinities towards soybean meal (SBM) and similar Vmax values. The immobilised enzyme was then employed in the enhancement of the functional feed/prebiotic properties of SBM from poultry feed, increasing mannooligosaccharides (MOS) quantities. The SBM main hydrolysis products were mannobiose (M2) and mannose (M1). The SBM-produced sugars could be utilised as a carbon source by probiotic bacteria; Streptococcus thermophilus, Bacillus subtilis, and Lactobacillus bulgaricus. The results indicate that the immobilised enzyme has the potential for use in the sustainable and cost-effective production of prebiotic MOS from agricultural biomass.

Characterization of thermo/halo stable cellulase produced from halophilic Virgibacillus salarius BM-02 using non-pretreated biomass

The production of extremozymes from halophilic bacteria has increased significantly due to their stability and efficiency in catalyzing a reaction, as well as their capacity to display optimum activity at various salt concentrations. In the current study, the halophilic bacterium Virgibacillus salarius strain BM-02 could utilize many non-pretreated substrates including cellulose, corn stover, sugarcane bagasse and wheat bran as a sole carbon source. However, wheat bran was the best substrate for achieving optimum saccharification yield (90.1%). The partially purified cellulase was active and stable at a wide range of pH (5–8) with residual activities > 58%. Moreover, it was stable at 5–12% of NaCl. Metal ions have a variable impact on the activity of partially purified cellulase however, Fe+3 exhibited the highest increase in the cellulase activity. The enzyme exhibited a thermal stability at 40, 50 and 60 °C with half-lives of 1049.50, 168.14 and 163.5 min, respectively. The value of Vmax was 22.27 U/mL while Km was 2.1 mM. The activation energy of denaturation Ed 69.81 kJ/mol, the enthalpy values (ΔHd) were positive, and the entropy values (ΔS) were negative. Therefore, V. Salarius is recommended as a novel promising halophilic extremozyme producer and agricultural waste remover in the bio-industrial applications.Graphical abstract

Methane production from ethanolic and acid fermentations of the organic fraction of municipal solid waste under different pH and reaction times

Thermodynamically, lactic acid and ethanol allow the best energy recovery among all common metabolites for methane production. When faster methane production is required, previously fermented organic fraction of municipal solid waste (OFMSW) can be essential for methane production. This research deals with OFMSW fermentation in batch reactors at pH values of 4, 5, and 6 at 35 °C. Samples were taken at reaction times of 1, 3, and 6 days for every pH, characterized, and exposed to methane production at 35 °C. The results show that lower pH and shorter fermentation times allow better ethanol and lactic acid production. Without exception, the fermented liquid fractions produce more methane than the solid ones. Fermented OFMSW produced 19% more methane than unfermented ones. The first methanization stage of the liquid fractions required 1–2 days and the solid fractions from 4 to 8 days. According to Michaelis & Menten, the reaction rate Vmax values are significantly higher for the liquid fractions than for the solid ones. Vmax shows that the lowest tested pH (4) during fermentation delivers the best substrate for methane production. The liquid digestates show significantly lower Km values than the solid ones, indicating a good affinity of the methanogens for the substrate. These results show the possibility of producing methane fast and efficiently using liquid fermented digestates. The solid fraction produces methane, but only slower than the liquid fraction.

Differential expression and activities of cytochrome P450 3A in the rat brain microsomes and mitochondria.

Midazolam (MDZ), a benzodiazepine derivative, is metabolized to 1'- and 4-hydroxylated metabolites (1'-OH-MDZ and 4-OH-MDZ, respectively) by cytochrome P450 3A (CYP3A). The purpose of this study was to investigate the CYP3A-mediated hydroxylation of MDZ in the rat brain mitochondria (MT). Brain microsomes (MC) and MT fractions were prepared from rats (n = 8) using differential and density gradient centrifugations, and the purity of the fractions was evaluated using VDAC1 and calreticulin as markers of MT and MC, respectively. The formation rates of 1'-OH-MDZ and 4-OH-MDZ in the rat brain MC and MT samples were determined using an LC-MS/MS method after validation. Subsequently, Michaelis-Menten kinetics of 1'- and 4-hydroxylation of MDZ were estimated. Western blot (WB) analysis was used to determine the protein expression of CYP3A in the rat brain MC and MT. The MC fractions had 5.93% ± 3.01% mitochondrial impurity, and the MT fractions had 19.3% ± 7.8% microsomal impurity (mean ± SD). The maximum velocity (Vmax ) values of the formation of the hydroxylated metabolites in the brain MT were 2.4-9-fold higher than those in MC. Further, the Vmax values of 4-OH-MDZ in both MC and MT fractions were substantially higher than those of 1'-OH-MDZ. The WB analysis showed that the intensity of the CYP3A immunoreactive band in MT was more than twofold higher than that in MC. It is concluded that compared with MC, rat brain MT contains substantial CYP3A, which may affect the pharmacology or toxicology of centrally acting xenobiotic and endogenous substrates of this enzyme.

Immobilisation of β-galactosidase onto double layered hydrophilic polymer coated magnetic nanoparticles: Preparation, characterisation and lactose hydrolysis

The surface of polydopamine (PDA) coated magnetic particles was grafted spontaneously with diaminopolyethylene glycol (DAPEG) as a second polymer layer by conjugation of PDA with DAPEG without using any activating agent. The amount of immobilised β-galactosidase was found to be 49.7 mg g−1 support. The optimum pH for the free and immobilised enzyme was observed at 5.5 and 6.0, respectively. The optimal temperature for the immobilised β-galactosidase was 15 °C higher compared with the free counterpart. The apparent Km values for the free and immobilised enzymes were found to be 3.69 and 4.35 mmol L−1, with Vmax values of 7.72 and 5.54 U mg−1, respectively. Thermal stability of β-galactosidase was increased upon immobilisation. Finally, the operational stability of the immobilised β-galactosidase was studied for lactose hydrolysis in a continuous reactor. After 96 h continuous operation, the remaining activity of the immobilised enzyme was 94%.

Development of Carbon-Based Support Using Biochar from Guava Seeds for Lipase Immobilization

Enzymes are promising tools for achieving an environmentally benign process. However, enzymes are required to be immobilized on economically competent supports to be reusable and maintain their activity. In this work, the aim was to evaluate the application of the biochar for immobilization of Burkholderia cepacia lipase (BCL) by physical adsorption (PA) and covalent binding (CB). Additionally, it was observed that regarding the biochemical properties, the optimal pH was 4.0 for the BCL immobilized by PA and pH 7.0 for the BCL free and immobilized by CB. Among the kinetic parameters, the maximal velocity (Vmax) for the free enzyme was 2500 µmol g−1·min−1, and for the PA- and CB-immobilized biocatalyst the values of Vmax were 2000 and 3333 µmol g−1·min−1, respectively. The Michaelis-Menten constant (Km) value for the free lipase was 665 mM and for the biocatalysts immobilized by PA and CB the Km values were 219 and 369 mM, respectively. Immobilized LBC exhibited superior thermal stability. The reusability tests showed that the LBC immobilized by PA preserved 50% of the initial activity after 16 cycles. Thus, biochar is a by-product of a renewable source; therefore, it is a promising alternative for lipase immobilization demonstrating its potential for use in a wide range of greener industrial processes.

Specific and efficient hydrolysis of all outer glucosyls in protopanaxadiol type and protopanaxatriol type ginsenosides by a β-glucosidase from Thermoclostridium stercorarium

To develop a new method for enzymatic preparation of minor ginsenosides, T. stercorarium β-glucosidase (Tsbgl) was characterized and its activities of deglycosylation towards natural ginsenosides were examined. The substrates of 1 mmol l−1 were incubated with the enzyme of 38.3 U ml−1 at 65 ℃ and pH 5.0. The Km values of Tsbgl for ginsenosides Rb1, Rg1 and pNPG were 0.37 ± 0.03, 3.26 ± 0.19, and 1.24 ± 0.03 mmol l−1, and the Vmax values were 183.63 ± 7.15, 85.03 ± 4.90, and 117.66 ± 1.96 μmol mg−1 min−1, respectively. The molar conversion of ginsenosides Rb1, Rb2, Rb3, Rc, Re, Rg1, and Rf by Tsbgl within 6 h was 100%, 50.1%, 42.7%, 92.0%, 57.3%, 67.9%, and 76.8%, respectively. The yield of aglycone protopanaxadiol was 35.5 μmol l−1 h−1 for Rb1, while the yields of aglycone protopanaxatriol were 64.2 and 70.4 μmol l−1 h−1 for Rg1 and Rf. Tsbgl with good organic solvent tolerance, mild reaction conditions and broad substrate specificity, could completely remove all outer glucosyls at the C-3 and C-20 hydroxyls of protopanaxadiol-type ginsenosides, and the C-6 and C-20 hydroxyls of protopanaxatriol-type ginsenosides through various pathways, providing a specific and efficient way to produce minor ginsenosides.

Citrus limon peroxidase-assisted biocatalytic approach for biodegradation of reactive 1847 colfax blue P3R and 621 colfax blue R dyes

One of the big environmental problems in today’s world is dye-contaminated toxic waste. Peroxidase is known as highly efficient for the degradation of various pollutants, including dyes. Environmental contamination caused by the discharge of dyes into water bodies is an onerous challenge that poses both human and ecological hazards. In the current studies, biocatalysts used for enzyme decolorization (1847 Colafx Blue P3R and 621 Colafx Blue) are regarded as an eco-friendly method utilizing commonly available low-cost material lemon peels (Citruslimon peroxidase). Peroxidase was extracted in a phosphate buffer of pH 7.0 and partially purified by 20–80% ammonium sulfate precipitation technique from Citruslimon peels. The soluble enzyme was characterized in terms of kinetic and thermodynamic parameters. The values of Km and Vmax (23.16 and 204.08 μmol/ml/min) were determined, respectively. The enzyme showed maximum activity at pH 5.0 and a temperature of 55 °C. Citruslimon efficiently degraded 1847 Colafx Blue P3R and 621 Colafx Blue R dyes with maximum degradation of 83 and 99%, respectively, with an initial dye concentration of 200 ppm at pH 4 and 35 °C temperature within 5–10 min of incubation time. The effect of the redox mediator on the degradation process was examined. Results showed that the peroxidase HOBT system efficiently enhanced the degradation of dyes from water. Hence, Citruslimon peroxidase is an efficient biocatalyst for the treatment of effluents.

A Novel Endo-Polygalacturonase from Penicillium rolfsii with Prebiotics Production Potential: Cloning, Characterization and Application.

In this study, a potential producer of prebiotics, a novel endo-polygalacturonase pePGA from Penicillium rolfsii BM-6, was successfully expressed in Komagataella phaffii, characterized and applied to produce pectic oligosaccharides. The optimum temperature and pH of pePGA were 60 °C and 6.0. The purified recombinant enzyme showed a good pH stability and was stable from pH 3.5 to 8.0. The Km, Vmax and kcat values of pePGA were 0.1569 g/L, 12,273 μmol/min/mg and 7478.4 s-1, respectively. More importantly, pePGA-POS, the pePGA hydrolysis products from commercial pectin, had good prebiotic and antibacterial activities in vitro. The pePGA-POS was able to significantly promote the growth of probiotics; meanwhile, the growth of Escherichia coli JM109, Staphylococcus aureus and Bacillus subtilis 168 was effectively inhibited by pePGA-POS. In addition, pePGA-POS also had the DPPH radical scavenging capacity. These properties of pePGA-POS make pePGA attractive for the production of prebiotics.

Single-walled carbon nanotube conjugated cytochrome c as exogenous nano catalytic medicine to combat intracellular oxidative stress.

Mitochondrial dysfunction has been reported to be one of the main causes of many diseases including cancer, type2 diabetes, neurodegenerative disorders, cardiac ischemia, sepsis, muscular dystrophy, etc. Under in vitro conditions, Cytochrome C (Cyt C) maintains mitochondrial homeostasis and stimulates apoptosis, along with being a key participant in the life-supporting function of ATP synthesis. Hence, the medicinal importance of Cyt C as catalytic defense is immensely important in various mitochondrial disorders. Here, we have developed a nanomaterial via electrostatically conjugating oxidized single-wall carbon nanotube with Cyt C (Cyt C@cSWCNT) for the exogenous delivery of Cyt C. The chemical and morphological characterization of the developed Cyt C@cSWCNT was done using UV-vis, FTIR, XPS, powder XRD, TGA/DSC, TEM, etc. The developed Cyt C@cSWCNT exhibited bifunctional catalase and peroxidase activity with Km (∼ 642.7 μM and 351.6 μM) and Vmax (∼0.33 μM/s and 2.62 μM/s) values, respectively. Also, through this conjugation Cyt C was found to retain its catalytic activity even at 60°C, excellent catalytic recyclability (at least up to 3 times), and wider pH activity (pH=3 to 9). Cyt C@cSWCNT was found to promote intracellular ROS quenching and maintain mitochondrial membrane potential and cellular membrane integrity via Na+/K+ ion homeostasis during the H2O2 stress. Overall the present strategy provides an alternative approach for the exogenous delivery of Cyt C which can be used as nano catalytic medicine.

Agarose-Degrading Characteristics of a Deep-Sea Bacterium Vibrio Natriegens WPAGA4 and Its Cold-Adapted GH50 Agarase Aga3420.

Up until now, the characterizations of GH50 agarases from Vibrio species have rarely been reported compared to GH16 agarases. In this study, a deep-sea strain, WPAGA4, was isolated and identified as Vibrio natriegens due to the maximum similarity of its 16S rRNA gene sequence, the values of its average nucleotide identity, and through digital DNA-DNA hybridization. Two circular chromosomes in V. natriegens WPAGA4 were assembled. A total of 4561 coding genes, 37 rRNA, 131 tRNA, and 59 other non-coding RNA genes were predicted in the genome of V. natriegens WPAGA4. An agarase gene belonging to the GH50 family was annotated in the genome sequence and expressed in E. coli cells. The optimum temperature and pH of the recombinant Aga3420 (rAga3420) were 40 °C and 7.0, respectively. Neoagarobiose (NA2) was the only product during the degradation process of agarose by rAga3420. rAga3420 had a favorable stability following incubation at 10-30 °C for 50 min. The Km, Vmax, and kcat values of rAga3420 were 2.8 mg/mL, 78.1 U/mg, and 376.9 s-1, respectively. rAga3420 displayed cold-adapted properties as 59.7% and 41.2% of the relative activity remained at 10 3 °C and 0 °C, respectively. This property ensured V. natriegens WPAGA4 could degrade and metabolize the agarose in cold deep-sea environments and enables rAga3420 to be an appropriate industrial enzyme for NA2 production, with industrial potential in medical and cosmetic fields.

Genome-Wide Identification and Expression Analysis of AMT and NRT Gene Family in Pecan (Carya illinoinensis) Seedlings Revealed a Preference for NH4+-N.

Nitrogen (N) is a major limiting factor for plant growth and crop production. The use of N fertilizer in forestry production is increasing each year, but the loss is substantial. Mastering the regulatory mechanisms of N uptake and transport is a key way to improve plant nitrogen use efficiency (NUE). However, this has rarely been studied in pecans. In this study, 10 AMT and 69 NRT gene family members were identified and systematically analyzed from the whole pecan genome using a bioinformatics approach, and the expression patterns of AMT and NRT genes and the uptake characteristics of NH4+ and NO3- in pecan were analyzed by aeroponic cultivation at varying NH4+/NO3- ratios (0/0, 0/100,25/75, 50/50, 75/25,100/0 as CK, T1, T2, T3, T4, and T5). The results showed that gene duplication was the main reason for the amplification of the AMT and NRT gene families in pecan, both of which experienced purifying selection. Based on qRT-PCR results, CiAMTs were primarily expressed in roots, and CiNRTs were majorly expressed in leaves, which were consistent with the distribution of pecan NH4+ and NO3- concentrations in the organs. The expression levels of CiAMTs and CiNRTs were mainly significantly upregulated under N deficiency and T4 treatment. Meanwhile, T4 treatment significantly increased the NH4+, NO3-, and NO2- concentrations as well as the Vmax and Km values of NH4+ and NO3- in pecans, and Vmax/Km indicated that pecan seedlings preferred to absorb NH4+. In summary, considering the single N source of T5, we suggested that the NH4+/NO3- ratio of 75:25 was more beneficial to improve the NUE of pecan, thus increasing pecan yield, which provides a theoretical basis for promoting the scale development of pecan and provides a basis for further identification of the functions of AMT and NRT genes in the N uptake and transport process of pecan.

Purification and characterization of a robust thermostable protease isolated from Bacillus subtilis strain HR02 as an extremozyme.

Since the hot water of Genow, a village in Isin rural district in the central district of Bandar Abbas, Hormozgan Province, Iran, has a rich source of thermophilic bacteria, the current study aimed to find a new thermophilic protease enzyme with suitable properties to be used in different industries. Water and sediment samples were collected from the hot water of Genow, and finally, 20 colonies were isolated. Among these isolated colonies, two bacterial strains grew on the skim milk agar medium, and a clear halo was formed around the colony, which was accurately identified by 16S rRNA gene sequence analysis. The comparison of 16S rRNA gene sequence analyses of isolated strains HR01 and HR02 with registered sequences of 16S rRNA genes in NCBI showed that the two isolates had the most similarity to Bacillus sonorensis and Bacillus subtilis, respectively. Among the two bacterial strains, the highest enzymatic activity was observed in B. subtilis strain HR02, from which the protease purification process was performed. A putative native B. subtilis strain HR02 protease (BSHR02PR) was purified by the UNO Q-6 ionic exchange chromatography method. Biochemical analyses revealed a monomeric enzyme, BsHR02Pro, with a molecular weight of 25 kDa, showing the maximum activity at 70°C and pH8.0. Moreover, the purified enzyme was stable up to 80 °C and in a pH range of 6.0-12.0. The steady-state kinetic analysis for colloidal casein showed that the Km , Vmax and kcat values of the purified enzyme were 25.7μM, 93.2μM min-1 and 2.18 s-1 , respectively. The hot water of Genow is a rich source of protease-producing bacteria. Sediments are a better source for the isolation of these types of bacteria than spring water. Overall, our results demonstrated a potential bacterial enzyme BsHR02Pro as a suitable catalyst to be used in the various industries.

Polyester fabric modification by chemical treatment to enhancing the β-glucosidase immobilization

This work reports the first approach to immobilizing the β-glucosidase enzyme on a modified polyester fabric support matrix. Herein, polyester fabric was successfully fabricated with hydrazide groups incorporated with graphene oxide, followed by glyoxal as the crosslinker. Various techniques, including Fourier transform infrared spectroscopy, scanning electron microscopy, thermogravimetric analysis, differential scanning calorimeter, and zeta potential analysis, were used to investigate their microstructural, dispersive, thermal, and physicochemical properties. β-glucosidase immobilization exhibited maximum activity at pH 6.0 with an immobilization yield (89.5%), immobilization efficiency (92%), and enzyme activity yields (82.3%). After fifteen reaction cycles, the remaining enzyme activity was 59%. Stored at 4 °C, immobilized β-glucosidase retained 74% of its activity, compared to a retain of 43% for soluble β-glucosidase, during the 6-weeks period. Soluble and immobilized enzyme exhibited similar optimal catalytic temperature at 60 °C, while the optimal catalytic pH was 5 and 6, respectively. Both soluble and immobilized β-glucosidase presented Michaelis–Menten kinetics with Vmax values of 1.82 and 2.94 U/mg, and Km values of 2.94 and 5.15 mM, respectively. This research provided a potential directed immobilization method for β-glucosidase, and robust biocatalyst for industrial applications.