Optimum pH For Activity Research Articles

Cloning Cellulase Genes from Victoria Falls Rainforest Decaying Logs Metagenome.

The Victoria Falls rainforest is a protected site whose forest floors harbor a host of cellulolytic microorganisms involved in biomass degradation. This study collected decaying logs and soil from the rainforest for bioprospecting cellulases from their metagenomes. Metagenomic DNA was isolated from the compound sample. Degenerate cellulase primers were used to amplify cellulase genes in the metagenome. The resulting amplicons cloned into Z-competent Escherichia coli DH5α were analyzed by functional screening for the production of cellulase extracellularly. Functional screening of the clones resulted in one clone (Clone-i) testing positive for extracellular cellulase production. Submerged fermentation of Clone-i was carried out for cellulase production. The cellulases were characterized to determine their activity's optimum pH and temperature. The diversity of the cellulases produced by Clone-i was determined. Clone-i's optimum enzyme activity was observed after 72 hours of incubation at 50°C and pH 5. Clone-i produced 80% more exoglucanases as compared to endoglucanases. The cellulolytic Clone-i' isolate shows Victoria Falls rainforest's potential as an enzyme bioprospecting site, reflecting that metagenomics is a valuable tool in microbial ecology.

Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

The Role of Gastric Lipase and Pepsin in Lipid Digestion of a Powder Infant Formula Using a Simulated Neonatal Gastric System

This study has sought to determine the impact of interfacial dynamics on the in vitro lipid digestion of a commercial infant formula; in particular, the specific role of interfacial proteolysis on the subsequent rates of reaction of droplet lipolysis. A powder infant formula was used as the as a protein-stabilised emulsion substrate during simulated infant gastric digestion at different pH level 3.5, 4.5 and 5.5. The digestate was treated with a fungal lipase and porcine pepsin (used to analogue human gastric lipase and pepsin) respectively and in a combined action. The study found that for fungal lipase treated digestate, the rate and extent of lipolysis were observed to be maxim at pH 5.5, in accordance with the optimal pH activity of the lipase. Findings also indicated that the proteinaceous interface did not appear to act as a barrier to lipolysis, since treatment with lipase and pepsin did not result in any significant increase in extent of lipolysis. However, it was observed that surface proteolysis did lead to alteration of the structural fate of the enzyme during digestion when compared to when the emulsion was digested solely by lipase. Findings suggest that lipolysis under these conditions may be independent of the structural dynamics of the emulsion during digestion, as observed within the context of this study design.

Isolation and Purification of Chickpea (Cicer arietinum) Seeds Protein, Testing Their Antibacterial Activity, and Using to Extend The Shelf Life of Beef Patties

Antimicrobial proteins (AMP) from chickpea (Cicer arietinum L.) seeds were isolated and purified using the saturation of (NH4)2So4 by 80% and gel filtration through Sephacryl S-200, the inhibition zone of the separated peak was 24, and 22 mm for growth of Escherichia coli and Salmonella typhimurium respectively. The molecular mass was 28385 Da estimated through Sephacryl S-200. The optimum pH for activity was 5.5. It was stable at 4.5-7.5, while it lost 21.07 and 55.65% from its activity at pH 3 and 8 respectively, the optimum temperature for activity was 35°C and it was stable at 35°C for 60 min, while it lost all inhibitory activity at 65°C for the same time. The use of AMP at 100 mg resulted in an inhibition zone of 37± 2.92, 35±1.68, 32±2.33, and 33±2.09 mm, with a significant difference at (P≤0.05) against E. coli and S. Typhimurium, Staphylococcus aureus, and Bacillus cereus, respectively. The use of AMP to extend the shelf life of beef patties at 100 mg.kg-1 resulted in a decrease in the total count of bacteria, as it reached 7.4×102±0.18, 4.6×102±0.22 and 2.8×102±0.19 CFU.g-1, while it was 7.4×102±0.23, 8.2×102±0.31, and 9.5×102±0.27 CFU.g-1 in the control sample during a storage period of 0, 3, and 6 d at 4°C. It was observed that there was no significant difference in the control treatment and AMP added on 0 d, while a significant difference was observed at (P≤0.05) for treatments at a storage period of 3 and 6 d at 4°C.

EXTRACTION, PARTIAL PURIFICATION AND CHARACTERIZATION OF BETA-GLUCOSIDASE FROM APRICOT DEFATTED SEEDS POWDER

The objective of this study, is characterization of partially purified beta-glucosidase (BGS) extracted from apricot defatted seeds powder. The enzyme was extracted using sodium phosphate solution (0.2M, pH 7) containing 2% sodium chloride with mixing ratio of (1:5 solid: liquid) for extraction period of 3 hours at 4C. The crude extract was precipitated by ammonium sulfate at 20-70% saturation, dialyzed then concentrated. The specific activity of the enzyme, folds of purification and the enzymatic yield were 5.45 units/mg, 7.8 fold and 84.5%, respectively. The results showed that the optimum conditions for the enzyme activity were at pH 5.5 and 6.5, the optimum temperature for the enzyme’s activity was 40°C and 55°C, while the thermal stability of the enzyme was between 25-45°C when the enzyme retained more than 90% of its activity over 30 minutes incubation at the optimum pH for activity, and retained 88% of its activity at 50° C. The results also revealed the inhibitory and activator effects of minerals and chemical compounds on the BGS activity.

Improvement of optimum pH and specific activity of pectate lyase from Bacillus RN.1 using loop replacement.

Background: Alkaline pectate lyase plays an important role in papermaking, biological refining and wastewater treatment, but its industrial applications are largely limited owing to its low activity and poor alkali resistance. Methods: The alkaline pectate lyase BspPel from Bacillus RN.1 was heterologously expressed in Escherichia coli BL21 (DE3) and its activity and alkali resistance were improved by loop replacement. Simultaneously, the effect of R260 on enzyme alkaline tolerance was also explored. Results: Recombinant pectate lyase (BspPel-th) showed the highest activity at 60°C and pH 11.0, and showed significant stability over a wide pH range (3.0-11.0). The specific enzyme activity after purification was 139.4U/mg, which was 4.4 times higher than that of the wild-type enzyme. BspPel-th has good affinity for apple pectin, since the V max and K m were 29μmol/min. mL and 0.46mol/L, respectively. Molecular dynamics simulation results showed that the flexibility of the loop region of BspPel-th was improved. Conclusion: The modified BspPel-th has considerable potential for industrial applications with high pH processes.

Formulation and Evaluation of Polyherbal Shampoo Containing Different Herbal extract

The aim of the present study is to formulate and evaluate polyherbal shampoo containing natural ingredients with an emphasis on safety and efficacy. It clears dirt and dandruff, promotes hair growth, luster, strengthens and darkens the hair. The shampoo is prepared by taking the polyherbal extract from Hibiscus Flowers, Pomegranate Peels, Neem Leaves, Curry Leaves, Shikakai and Amla Fruit in different proportions. Several physicochemical tests were performed for visual assessment, wetting time, pH, viscosity, assurance of solid contents, surface tension, dirt dispersion, conditioning performance and foam stability. The formulated polyherbal shampoo is brown in color with demonstrable good froth stability, good cleansing ability, low surface tension, optimum pH and conditioning activity. Dirt dispersion of polyherbal shampoo is light in colour along with 25 ml foam height. All these are the ideal characteristics of good quality polyherbal shampoo to be used in daily life. However, further scientific investigation is required for validation of its overall quality.

Heterologous Expression of Extracellular Proteinase pAsPs of Aspergillus pseudotamarii in Komagataella phaffii

Neutral protease pAsPs gene was obtained by sequence optimization of NpI protease from Aspergillus pseudotamarii. pAsPs was for the first time integrated in the genome of yeast strain Komagataella phaffii T07, and then produced in a 5 L bioreactor with an enzyme yield of 150,800 U/mL of culture liquid towards casein. The specific activity of the pAsPs was 7,657,000 U/mg toward casein, 2320 U/mg toward hemoglobin, and 25,344 U/mg toward azocasein per 1 mg of the protein. The enzyme was found to be inhibited by Cu2+. Optimal activity pH was shown in the range of pH 6.5-8.0, and optimal temperature-50-60 °C. The molecular mass of the recombinant protease pAsPs was shown to be 67.5 kDa. Mass-spectrometric analysis confirmed the identity of the amino acid sequence of the obtained pAsPs preparation with the predicted sequence, with 17% coverage and protein score 288. Thus, the novel neutral protease pAsPs is a promising candidate for large-scale use in manufacturing, including the food industry.

Serine-Carboxyl Peptidases, Sedolisins: From Discovery to Evolution.

Sedolisin is a proteolytic enzyme, listed in the peptidase database MEROPS as a founding member of clan SB, family S53. This enzyme, although active at low pH, was originally shown not to be inhibited by an aspartic peptidase specific inhibitor, S-PI (pepstatin Ac). In this Perspective, the S53 family is described from the moment of original identification to evolution. The representative enzymes of the family are sedolisin, kumamolisin, and TPP-1. They exhibit the following unique features. (1) The fold of the molecule is similar to that of subtilisin, but the catalytic residues consist of a triad, Ser/Glu/Asp, that is unlike the Ser/His/Asp triad of subtilisin. (2) The molecule is expressed as a pro-form composed of the amino-terminal prosegment and the active domain. Additionally, some members of this family have an additional, carboxy-terminal prosegment. (3) Their optimum pH for activity is in the acidic region, not in the neutral to alkaline region where subtilisin is active. (4) Their distribution in nature is very broad across the three kingdoms of life. (5) Some of these enzymes from fungi and bacteria are pathogens to plants. (6) Some of them have significant potential applications for industry. (7) The lack of a TPP-1 gene in human brain is the cause of incurable juvenile neuronal ceroid lipofuscinosis (Batten's disease).

Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation.

Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naïve and in silico metagenomic approaches, we retrieved 16 esterases from the α/β-hydrolase fold superfamily with the closest match from uncultured acidophilic Acidobacteria, Actinobacteria (Acidithrix, Acidimicrobium, and Ferrimicrobium), Acidiphilium, and other Proteobacteria inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of ∼2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in Escherichia coli. The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33–68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30–65°C, retaining 20–61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 ± 0.6°C). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 ± 75 U/g at pH 8.0 and 30°C) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system.

Biophysical characterization of the recombinant chitinase chi18-5 with potential biotechnological interest.

Chitinase chi18-5 is an enzyme able to hydrolyze chitin and chitosan producing chitooligosaccharides (COS) of potential technological interest. chi18-5 is produced naturally by the fungus Trichoderma atroviride. It belongs to the glycosyl hydrolase (GH) family 18 of the Carbohydrate Active Enzyme (CAZy) database and it has 83% identity compared to the well-characterized chi42 of Trichoderma harzianum. Several efforts have been made to characterize the biochemical activity of the enzyme and its structure. Here, we studied the biophysical properties of recombinant chi18-5. In order to gain insight into its structure and stability, we studied thermal denaturation by Circular Dichroism (CD), Intrinsic Fluorescence (FL), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) at several pH between 3 and 8. We observed that the conformation of chi18-5 changes near its pI, and the transitions as a function of the temperature involved an increment in β-sheet secondary structure at the expenses of ⍺-helix. We also performed amide hydrogen exchange dynamics in selected conditions. At pH ≤ 6, the proportion of fast exchanging residues are larger than at pH ≥ 6. Our results suggest that at pH below pI, chi18-5 is in a less compact structure which may have influence in the interaction with substrate and enzyme activity. KEY POINTS: • Characterization of enzyme behavior is critical for their wide applications • We produced and characterized biophysically a chitinase as a function of pH • The pH of optimum activity correlates with a less compact structure of chi18-5.

The Zoonotic Helminth Parasite Fasciola hepatica: Virulence-Associated Cathepsin B and Cathepsin L Cysteine Peptidases Secreted by Infective Newly Excysted Juveniles (NEJ).

Simple SummaryFasciolosis, caused by the worm parasite Fasciola hepatica (liver fluke), is a global disease of farm animals and a neglected disease of humans. Infection arises from the ingestion of resistant metacercariae that contaminate vegetation. Within the intestine, the parasite excysts as an active larvae, the newly excysted juvenile (NEJ), that borrows through the intestinal wall to infect the host and migrates to the liver. NEJ release, tissue penetration and migration are facilitated by enzymes secreted by the parasite, namely, cathepsin B1 (FhCB1), cathepsin B2 (FhCB2), cathepsin B3 (FhCB3) and cathepsin L3 (FhCL3). While our knowledge of these enzymes is growing, we have yet to understand why the parasites require all four of them to invade the host. In this study, we produced functional recombinant forms of these enzymes and demonstrated that they vary greatly in terms of activity, optimal pH and substrate specificity, suggesting that, combined, these enzymes provide the parasite with an efficient digestion system for different host tissues and molecules. We also identified several compounds that inhibited the activity of these enzymes, but did not affect the ability of the larvae to excyst or survive. However, this does not exclude these enzymes as targets for development of drugs or vaccines.Fasciolosis caused by Fasciola hepatica is a major global disease of livestock and an important neglected helminthiasis of humans. Infection arises when encysted metacercariae are ingested by the mammalian host. Within the intestine, the parasite excysts as a newly excysted juvenile (NEJ) that penetrates the intestinal wall and migrates to the liver. NEJ excystment and tissue penetration are facilitated by the secretion of cysteine peptidases, namely, cathepsin B1 (FhCB1), cathepsin B2 (FhCB2), cathepsin B3 (FhCB3) and cathepsin L3 (FhCL3). While our knowledge of these peptidases is growing, we have yet to understand why multiple enzymes are required for parasite invasion. Here, we produced functional recombinant forms of these four peptidases and compared their physio-biochemical characteristics. Our studies show great variation of their pH optima for activity, substrate specificity and inhibitory profile. Carboxy-dipeptidase activity was exhibited exclusively by FhCB1. Our studies suggest that, combined, these peptidases create a powerful hydrolytic cocktail capable of digesting the various host tissues, cells and macromolecules. Although we found several inhibitors of these enzymes, they did not show potent inhibition of metacercarial excystment or NEJ viability in vitro. However, this does not exclude these peptidases as targets for future drug or vaccine development.

Structural and functional analysis of broad pH and thermal stable protease from Penicillium aurantiogriseum URM 4622

This study aimed to better characterize a recently purified stable extracellular alkaline peptidase produced by Penicillium aurantiogriseum (URM 4622) through fluorescence spectroscopy, far-UV circular dichroism, kinetic and thermodynamic models to understand its’ structure-activity and denaturation. Fluorescence data showed that changing pH leads to tryptophan residues exposure to more hydrophilic environments at optimum activity pH 9.0 and 10.0. When thermally treated, it displayed less unfolding at these pH values, along with 4-fold less photoproducts formation than at neutral pH. Different pH CD spectra showed more β-sheet (21.5–43.0%) than α-helix (1–6.2%). At pH9.0, more than 2-fold higher α-helix content than any other pH. The melting temperature (T m) was observed between 50 and 60 °C at all pH studied, with lower T m at pH 9.0–11.0 (54.9–50.3 °C). The protease displayed two phase transition, with two energies of denaturation, and a 4-fold higher thermal stability (ΔH°m) than reports for other microorganism’s proteases. An irreversible folding transition occurs between 50 and 60 °C. It displayed energies of denaturation suggesting higher thermal stability than reported for other microorganism’s proteases. These results help elucidating the applicability of this new stable protease.

Production of xylanase by Aspergillus sp. ART500.1 on agroindustrial residues and its biochemical properties

The aim of this work was to evaluate the use of different agro-industrial residues for the production of xylanase by Aspergillus sp. ART 100.1, as well as analyzing the biochemical properties of the enzyme. Agroindustrial residues malt bagasse, pineapple crown, açaí bagasse and soybean husk present in large quantities in the Tocantins region were used to evaluate the production of xylanase. Cultivation conditions for xylanase production were evaluated in submerged and solid-state cultivation. The highest production of xylanase in submerged cultivation was obtained using soybean husk residue (23.60 U/mL), while, for solid-state cultivation conditions, the highest production of xylanase was obtained with malt bagasse (110.00 U/g). The effect of additives to the culture medium was also evaluated, with the best result for the use of the xylose additive in the pineapple crown in solid-state cultivation. The enzyme produced in solid-state cultivation was characterized in terms of pH and temperature. The optimum activity pH was observed at 5.0 and for temperature at 55 °C. The xylanase was stable in a pH range between 4 and 5 and retained 50% of its activity at 45 °C after 110 minutes. The Aspergillus sp. ART 500.1 presents potential for the production of xylanase using agro-industrial residues, enabling the development of bioprocesses for the scaling of production.

Poly aromatic hydrocarbons degradation by lignin peroxidase produced from local isolate Aspergillus terreus SG777

Lignin peroxidase produced from A. terreus SG777 precipitated with 80% ammonium sulfatesaturation then desalting by dialysis against Tris buffer solution and partially purified by ionexchange chromatography using DEAE- cellulose.The specific activity reached 56.67U/mg withpurification fold was 5.4 times and 13.7% recovery. The optimum pH for activity and stability was(3). The maximum activity was observed at 40°C and the enzyme maintained the activity when itwas incubated at (25-35)˚C.Degradation of poly aromatic hydrocarbons when incubated with 65.51 U/ml lignin peroxidaseat (40°C) for 4 hr compared with the Refinery treatment. Results showed (97.54%) removalefficiency of Di benzo (a, h) anthrancene by lignin peroxidase, while only (54.37%) was treated byRefinery treatment. Flourene and anthrancene degradation (95.98%) and (83.51%) respectively,while the flourene and anthrancene degradation treated by Refinery treatment only 84.51% and(46.83%) respectively.The aim of this study was degradation of poly aromatic hydrocarbons by using partialpurification lignin peroxidase.

Disulfide bond engineering of AppA phytase for increased thermostability requires co-expression of protein disulfide isomerase in Pichia pastoris

BackgroundPhytases are widely used commercially as dietary supplements for swine and poultry to increase the digestibility of phytic acid. Enzyme development has focused on increasing thermostability to withstand the high temperatures during industrial steam pelleting. Increasing thermostability often reduces activity at gut temperatures and there remains a demand for improved phyases for a growing market.ResultsIn this work, we present a thermostable variant of the E. coli AppA phytase, ApV1, that contains an extra non-consecutive disulfide bond. Detailed biochemical characterisation of ApV1 showed similar activity to the wild type, with no statistical differences in kcat and KM for phytic acid or in the pH and temperature activity optima. Yet, it retained approximately 50% activity after incubations for 20 min at 65, 75 and 85 °C compared to almost full inactivation of the wild-type enzyme. Production of ApV1 in Pichia pastoris (Komagataella phaffi) was much lower than the wild-type enzyme due to the presence of the extra non-consecutive disulfide bond. Production bottlenecks were explored using bidirectional promoters for co-expression of folding chaperones. Co-expression of protein disulfide bond isomerase (Pdi) increased production of ApV1 by ~ 12-fold compared to expression without this folding catalyst and restored yields to similar levels seen with the wild-type enzyme.ConclusionsOverall, the results show that protein engineering for enhanced enzymatic properties like thermostability may result in folding complexity and decreased production in microbial systems. Hence parallel development of improved production strains is imperative to achieve the desirable levels of recombinant protein for industrial processes.

Characterization of β-Galactosidase from Lactose Utilizing Yeast Isolated from the Dairy Sample

Objectives: The objective of the study was to isolate lactose positive yeasts from dairy samples collected from local markets of Kathmandu, to extract crude β-galactosidase from the lactose positive yeast and to characterize the enzyme for optimum time duration, pH, temperature, Michaelis-Menten constant (Km) and maximum activity (Vmax).
 Methods: Four lactose positive yeast strains were isolated from dairy samples collected from local market of Kathmandu by pour plate method. Single strain having maximum lactose positive activity was selected for the study. The mass culture of the lactose positive yeast strain was lysed by 2% Chloroform and the yeast cell lysate containing β-galactosidase (i.e. crude enzyme extract) was characterized by using ONPG (Ortho-Nitrophenyl-β-D-galactopyranoside) as substrate. ONPG is a colorless substrate for the enzyme assay which is hydrolyzed by the enzyme into yellow colored product ONP (Ortho-Nitrophenol). The concentration of product formed was monitored spectrophotometrically at 420 nm to determine the enzyme activity and to characterize the enzyme.
 Results: The enzyme had wide range of working temperature from 0-50ºC, with optimal temperature of 37ºC. However, greater than 50% hydrolyzing ability was maintained in the range of 14-40ºC. Optimum time of reaction was 70 min. The enzyme had maximum activity in the near neutral pH of 6.8. Michaelis-Menten constant of the enzyme was found to be 2.23 mM of ONPG and Vmax was 58.82 nmol/min/ml. Enzyme activity was 27.88 nmol/min/ml, Specific enzyme activity was 59.97 nmol/ min/mg and total enzyme activity was 3346.33 nmol/min.
 Conclusion: The activity over a wide range of temperature 0-50ºC with low Km value shows that the enzyme has a commercial application in clearance of lactose pollution in waste water in different environmental conditions.

Thermo stable tyrosinase purified from Pleurotus djamor grown in biomimetic calcium carbonate: A biological strategy to industrial waste remediation

A novel, neutral and heat-tolerant tyrosinase produced from Pleurotus djamor (P. djamor) using biomimetic calcium carbonate which was further used for the biodegradation of the phenolic waste of industrial effluents. Carbon-dioxide was sequestered/captured into calcium carbonate (CaCO 3 ) using carbonic anhydrase (2.1 U/mg protein) of Oceanobacillus with sequestration capacity as 24.7 CaCO 3 /mg protein in 25 s. This CaCO 3 was used as a substrate for cultivation of P. djamor (a mushroom) which was the potential source of tyrosinase. Mushroom tyrosinase was purified up to 51.7 folds purity which shown a molecular weight ∼ 90 kDa on SDS-PAGE. The optimal thermal and pH activity of the tyrosinase was observed at 50 °C and 7.0, respectively. The tyrosinase was stable within a pH range of 6.0–8.0, temperature 35 °C–55 °C and showed high substrate specificity for L-DOPA. Tyrosinase activity was drastically enhanced by Cu 2 + , Ca 2 + and Mn 2 + metal ions. K + ions were also found to inhibit the tyrosinase activity up to the moderate level. Mushroom tyrosinase was used to degrade phenol from industry effluents with 90% removal in 6 h. Extracellular nature as well as tyrosinase stability in the extreme conditions (temperature, pH, and presence of heavy metals) makes it preferable candidate for phenol bioremediation from industrial effluents. • Sequestration of carbon di oxide into calcium carbonate is clearly shown. • Biomimetically precipitated calcium carbonate is first time used for mushroom production. • Tyrosinase is purified from pre-mature phase of Pleurotus djamor . • Feasibility of degradation of phenol from industrial effluents by mushroom tyrosinase is presented. • A comprehensive mechanism for complete carbon-di-oxide utilization have been hypothesized.

One-pot synthesis and biochemical characterization of protease metal organic framework (protease@MOF) and its application on the hydrolysis of fish protein-waste

A protease from Bacillus sp. CHA410 was purified and immobilized by a one-step MOF-embedded approach. The immobilized protease characterized using transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), and scanning electron microscopy (SEM). The optimal pH activity of protease CHA410 and protease@MOF was obtained at pH 8.0 and 9.0, respectively. Thermostability results after 160 min incubation showed that a 25 and 41 % enhancement in the relative activity of protease@MOF observed at 60 and 70 °C, respectively, compared to free protease. Km of the free and immobilized protease@MOF in the presence of casein was 0.685 and 0.033 mg/mL, respectively. Also, Km of the free and immobilized protease@MOF in the presence of fibrin was 0.292 and 0.145 mg/mL, respectively. The Fibrinolytic activity/Caseinolytic activity ratio (F/C ratio) of the free and immobilized proteases was 0.36 and 0.43, respectively. Protease activity of both forms of the enzyme was increased in the presence of some divalent cations, including Ca2+, Mn2+, Mg2+, and Zn2+ ions, while it intensely diminished by phenylmethylsulfonyl fluoride (PMSF), proposed as serine-protease. A 10 and 82 % enhancement in protease activity of free and immobilized proteases was achieved in the presence of butanol, respectively. Storage stability results showed that the immobilized enzyme retained about 70 % of its original activity at the end of this period, while the free enzyme only showed 22 % of its initial activity. The hydrolysis degree of immobilized protease CHA410 in the hydrolysis of fish protein waste was obtained about 46 % after 2 h of incubation at 50 °C. In comparison, it was gained about 20 % for protease CHA410 at a similar situation. Finally, results indicated that the free and immobilized protease could be used in the food industry for the hydrolysis of fish protein waste.

Molecular characterization of Plasmodium falciparum DNA-3-methyladenine glycosylase

BackgroundThe emergence of artemisinin-resistant malaria parasites highlights the need for novel drugs and their targets. Alkylation of purine bases can hinder DNA replication and if unresolved would eventually result in cell death. DNA-3-methyladenine glycosylase (MAG) is responsible for the repair of those alkylated bases. Plasmodium falciparum (Pf) MAG was characterized for its potential for development as an anti-malarial candidate.MethodsNative PfMAG from crude extract of chloroquine- and pyrimethamine-resistant P. falciparum K1 strain was partially purified using three chromatographic procedures. From bio-informatics analysis, primers were designed for amplification, insertion into pBAD202/D-TOPO and heterologous expression in Escherichia coli of recombinant PfMAG. Functional and biochemical properties of the recombinant enzyme were characterized.ResultsPfMAG activity was most prominent in parasite schizont stages, with a specific activity of 147 U/mg (partially purified) protein. K1 PfMAG contained an insertion of AAT (coding for asparagine) compared to 3D7 strain and 16% similarity to the human enzyme. Recombinant PfMAG (74 kDa) was twice as large as the human enzyme, preferred double-stranded DNA substrate, and demonstrated glycosylase activity over a pH range of 4–9, optimal salt concentration of 100–200 mM NaCl but reduced activity at 250 mM NaCl, no requirement for divalent cations, which were inhibitory in a dose-dependent manner.ConclusionPfMAG activity increased with parasite development being highest in the schizont stages. K1 PfMAG contained an indel AAT (asparagine) not present in 3D7 strain and the recombinant enzyme was twice as large as the human enzyme. Recombinant PfMAG had a wide range of optimal pH activity, and was inhibited at high (250 mM) NaCl concentration as well as by divalent cations. The properties of PfMAG provide basic data that should be of assistance in developing anti-malarials against this potential parasite target.