Publications by authors named "Kamahldin Haghbeen"

29 Publications

  • Page 1 of 1

Biodegradation of cyanide to ammonia and carbon dioxide by an industrially valuable enzyme from the newly isolated zs.

J Environ Sci Health A Tox Hazard Subst Environ Eng 2021 Sep 14:1-7. Epub 2021 Sep 14.

Bioprocess Engineering Group, Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

The biodetoxification of cyanide-rich wastewater has been suggested as an appropriate technique due to its environmental friendliness and cost effectiveness. In this research, zs that was newly isolated from cyanide-polluted wastewater was selected to catalyze cyanide via an enzymatic mechanism. Enzyme was purified and its activity was also determined by ammonia assay. Subsequently, the operational procedure was optimized to enhance cyanide biodegradation at variable pH values, temperatures and cyanide concentrations using response surface methodology (RSM). The results revealed that the interactions between pH and temperature, as well as those between pH and cyanide concentration, were significant, and the concentration of cyanide in a 650 mg.L solution was decreased by 73%. According to this study, it can be proposed that due to its higher activity level compared with those of similar enzymes, this enzyme can prove useful in enzymatic biodegradation of cyanide which is a promising approach in the treatment of industrial effluent.
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http://dx.doi.org/10.1080/10934529.2021.1967653DOI Listing
September 2021

Thermophilic iron containing type superoxide dismutase from Cohnella sp. A01.

Int J Biol Macromol 2021 Sep 27;187:373-385. Epub 2021 Jul 27.

Bioprocess Engineering Group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran. Electronic address:

Superoxide dismutases (SODs) (EC 1.15.1.1) are well known antioxidant enzymes that play critical roles in cellular defenses of living organisms against harmful superoxide radicals during oxidative stress. This study details on cloning, biochemical and functional characterization of an iron containing type superoxide dismutase (SOD) from a novel thermophilic bacteria Cohnella sp. A01 (CaSOD). The secondary and three dimensional structure of the protein were predicted. CaSOD gene was subsequently cloned into pET-26b(+) expression vector and expression of the recombinant protein (rCaSOD) was optimized in E. coli BL21 (DE3) and the purified recombinant SOD showed a single band with an apparent molecular weight of 26 kDa by SDS-PAGE. The half-life and thermodynamic parameters including ΔH, ΔS, and ΔG were 187 min at 60 °C, 7.3 kJ.mol, -76.8 kJ.mol.°K, and 84.1 kJ.mol, respectively. The rCaSOD exhibited catalytic activity in a very broad range of pH (6.0-10.0) and temperatures (35-75 °C), as well as stability in a broad pH range, from 3.0 to 11.0, and wide range of temperature, different concentrations of detergent agents, metal ions, organic solvents and other chemicals. The results suggest that this novel enzyme could be used for various industrial applications in cosmetic, food, and pharmaceutical industries.
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http://dx.doi.org/10.1016/j.ijbiomac.2021.07.150DOI Listing
September 2021

New Insight into the Interactions of Arbutin with Mushroom Tyrosinase.

Protein J 2021 May 28. Epub 2021 May 28.

National Institute for Genetic Engineering and Biotechnology, P.O. Box: 14965/161, Tehran, Iran.

As a safe substitute for hydroquinone, β-arbutin, a natural plant substance, and its synthetic counterpart, α-arbutin, are used in depigmentation formulations. However, there are debatable points regarding the impact of arbutin on tyrosinase and the pigmentation process. To shed light on this issue, the effects of Pyrus biossieriana leaves extract (PbLE) and β-arbutin, extracted from PbLE, on mushroom tyrosinase (MT) were comprehensively examined. The study was focused on cresolase activity as the characteristic reaction of a tyrosinase. Kinetics studies disclosed that β-arbutin can modulate MT monophenolase activity from inhibition to activation or vice versa. β-Arbutin inhibited L-tyrosine (LTy) oxidation at concentrations < 0.3 mM but it increased (more than 400%) the enzymatic oxidation of L-tyrosine at the concentrations > 0.3 mM. An opposite pattern (activation then inhibition) was observed when a synthetic substrate was used instead of LTy. Computational studies, focused on the heavy chain of MT, indicated that β-arbutin effect could be overruled by the enzyme's ability to provide the ligand with a non-specific binding site (MTPc). A plausible mechanism was presented to show the influence of MTPc on the substrate pose in the active site. The possible determinant correlation between the findings of this research and the current studies on human tyrosinase role in the pigmentation process has been presented.
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http://dx.doi.org/10.1007/s10930-021-10004-xDOI Listing
May 2021

New Provisional Function of OmpA from sp. Strain SA01 Based on Environmental Challenges.

mSystems 2021 Jan 12;6(1). Epub 2021 Jan 12.

Department of Energy & Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

An outer membrane protein A (OmpA) from sp. strain SA01 was identified and characterized in-depth based on the structural and functional characteristics already known of its homologues. structural studies showed that this protein can be a slow porin, binds to peptidoglycan, and exhibits emulsifying properties. Characterization of the recombinant SA01-OmpA, based on its emulsifying properties, represented its promising potentials in biotechnology. Also, the presence of SA01-OmpA in outer membrane vesicles (OMV) and biofilm showed that this protein, like its homologues in , can be secreted into the extracellular environment through OMVs and play a role in the formation of biofilm. After ensuring the correct selection of the protein of interest, the role of oxidative stress induced by cell nutritional parameters (utilization of specific carbon sources) on the expression level of OmpA was carefully studied. For this purpose, the oxidative stress level of SA01 cell cultures in the presence of three nonrelevant carbon sources (sodium acetate, ethanol, and phenol) was examined under each condition. High expression of SA01-OmpA in ethanol- and phenol-fed cells with higher levels of oxidative stress than acetate suggested that oxidative stress could be a substantial factor in the regulation of SA01-OmpA expression. The significant association of SA01-OmpA expression with the levels of oxidative stress induced by cadmium and HO, with oxidative stress-inducing properties and lack of nutritional value, confirmed that the cells tend to harness their capacities with a possible increase in OmpA production. Collectively, this study suggests a homeostasis role for OmpA in sp. SA01 under oxidative stress besides assuming many other roles hitherto attributed to this protein. OmpA is known as a multifaceted protein with multiple functions, including emulsifying properties. Bioemulsifiers are surface-active compounds that can disperse hydrophobic compounds in water and help increase the bioavailability of hydrophobic hydrocarbons to be used by degrading microorganisms. In this study, an OmpA from sp. SA01 was identified and introduced as an emulsifier with a higher emulsifying capacity than rhamnolipid. We also showed that the expression of this protein is not dependent on the nutritional requirements but is more influenced by the oxidative stress caused by stressors. This finding, along with the structural role of this protein as a slow porin or its role in OMV biogenesis and biofilm formation, suggests that this protein can play an important role in maintaining cellular homeostasis under oxidative stress conditions. Altogether, the present study provides a new perspective on the functional performance of OmpA, which can be used both to optimize its production as an emulsifier and a target in the treatment of multidrug-resistant strains.
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http://dx.doi.org/10.1128/mSystems.01175-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901484PMC
January 2021

Neuroprotective effect of Lithospermum officinale callus extract on inflamed primary microglial cells.

Curr Pharm Biotechnol 2020 Nov 4. Epub 2020 Nov 4.

Department of Plant Bioproducts, National Institute for Genetic Engineering and Biotechnology, P.O.Box:14965/161, Tehran. Iran.

Background And Objective: Lithospermum officinale is a famous medicinal herb in the traditional medicine of India. However, the medicinal use of its root extract is limited due to the presence of pyrrolizidine alkaloids (PzAl). It was recently shown that PzAl are not accumulated in the cell culture of L. officinale while the biosynthetic pathway of phenolic acids remains active so that rosmarinic acid (RsA) is the main product in the proliferated callus. Considering the existing literature on the anti-inflammatory effects of caffeic acid (CfA) and its derivatives, this research was devoted to the evaluation of the anti-inflammatory capacity of methanolic extracts of L. officinale callus (LoE) on the rat microglial cells as the immune cells of the Central Nervous System, which play an essential role in the responses to neuroinflammation.

Methods: primary microglia were obtained from Wistar rat, then they were subjected to various amounts of CfA and methanolic extracts of 17 and 31-day L. officinale callus prior to stimulation by LPS. In addition to HPLC analysis of the extracts, viability, nitric oxide production, evaluation of the pro-inflammatory genes and cytokines in the inflamed microglia were investigated.

Results: Methanolic extract of the 17-day old callus of L. officinale exhibited anti-inflammatory effects on the LPS- stimulated microglial cells much higher than that was observed for CfA. The data was further supported by the decreased expression of NOS2, TNF-α, and Cox-2 mRNA and the suppression of TNF-α and IL-1β release in the activated microglial cells pretreated with the effective dose of LoE (0.8 mg mL-1).

Conclusion: It was assumed that better anti-neuroinflammatory performance of LoE than CfA in LPS-activated primary microglia could be a result of synergism of the components of the extract and the lipophilic nature of RsA as the main phenolic acid of LoE. Considering the fact that LoE shows high antioxidant capacity and lacks PzAl, it is anticipated that LoE is considered as a reliable substitute to the extract of the natural root of L. officinale and plays a key role in the preparation of neuroprotective pharmaceutical formula.
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http://dx.doi.org/10.2174/1389201021666201104145439DOI Listing
November 2020

Production of phenolic acids in hairy root cultures of medicinal plant L. in response to elicitors.

Mol Biol Res Commun 2020 Apr;9(1):23-34

Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

In this study, hairy root induction in leaf and stem explants of using various strains was established for the first time. Although inoculation of explants by immersion method resulted in tissue necrosis, direct injection of explants by all examined strains (A13,R318,A4,GMI 9534 and ATCC15834) was effective. All different parts of the stem were susceptible to infection. However, the middle and lower internodes showed a higher rate of transformation. Among the different strains, the strain A13 exhibited the highest infection efficiency (almost 75% of the explants). A13 and R318-infected hairy roots showed the highest biomass production (close to 60 mg/flask), while infection with GMI 9534 produced the highest content of phenolic acids. Finally, the effect of phytohormone elicitation on hairy root growth and phenolic acid biosynthesis was investigated. A substantial increase in root growth and phenolic acids accumulation was obtained followed by 0.3 mg L IBA and 100 µM MeJA treatment, respectively.
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http://dx.doi.org/10.22099/mbrc.2020.36031.1475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275824PMC
April 2020

A new sensitive spectrophotometric method for determination of saliva and blood glucose.

Spectrochim Acta A Mol Biomol Spectrosc 2020 Mar 4;229:117897. Epub 2019 Dec 4.

National Institute for Genetic Engineering and Biotechnology, P.O. Box: 14965, /161, Tehran, Iran. Electronic address:

There is an increasing need for accurate and inexpensive glucometers as the world moves toward personalized medicine. Among the existing technologies, photometric based devices are more desired due to the cost-effectiveness, ease-of-use and the potential to be adopted in the smart-phone technology for remote sensing and self-monitoring purposes. However, the accuracy, precision, and reproducibility of the results of these devices are heavily dependent on the details of the chosen glucose measuring method. Considering the delicate problems with the current spectrophotometric methods, a new method was developed for more precise, accurate, and fast measurement of blood glucose via the coupled reactions of glucose oxidase and peroxidase using 4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid (GASA) as the substrate. Stability of GASA and its oxidized products along with its direct and fast consumption by peroxidase, made it possible to determine blood glucose concentration in <20 s with high reproducibility. The low detection limit of GASA method (0.36 mg dL) with a linear range from 0.36 to 399.6 mg.dL also allowed determination of salivary glucose concentration (SGC). As compared with the blood samples, the SGC results were more dispersed, especially for the diabetic participants, assumingly due to the diverse nature of salivary samples. However, a good correlation coefficient of 0.81 for non-diabetic individuals showed that it is accurate enough to recognize non-diabetic from diabetic condition. Results of this study disclose the potential application of GASA method as a reliable alternative for the current spectrophotometric methods with the ability to be adopted in miniaturized glucometers.
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http://dx.doi.org/10.1016/j.saa.2019.117897DOI Listing
March 2020

sp. A01 laccase: thermostable, detergent resistant, anti-environmental and industrial pollutants enzyme.

Heliyon 2019 Sep 30;5(9):e02543. Epub 2019 Sep 30.

Bioprocess Engineering Group, Institute of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Iran.

Laccase (EC 1.10.3.2; benzenediol; oxygen oxidoreductases) is a multi-copper oxidase that catalyzes the oxidation of phenols, polyphenols, aromatic amines, and different non-phenolic substrates with concomitant reduction of O to HO. Enzymatic oxidation techniques have the potential of implementation in different areas of industrial fields. In this study, the sp. A01 laccase gene was cloned into pET-26 (b+) vector and was transformed to BL21. Then it was purified using His tag affinity (Ni sepharose resin) chromatography. The estimated molecular weight was approximately 60 kDa using SDS-PAGE. The highest enzyme activity and best pH for 2,6-dimethoxyphenol (DMP) oxidation were recorded as 8 at 90 °C respectively. The calculated half-life and kinetic values including K, V, turn over number (k), and catalytic efficiency (k/K) of the enzyme were 106 min at 90 °C and 686 μM, 10.69 U/ml, 20.3 S, and 0.029 s μM, respectively. The DMP was available as the substrate in all the calculations. Enzyme activity enhanced in the presence of Cu, NaCl, SDS, n-hexane, Triton X-100, tween 20, and tween 80, significantly. The binding residues were predicted and mapped upon the modeled tertiary structure of identified laccase. The remaining activity and structural properties of sp. A01 laccase in extreme conditions such as high temperatures and presence of metals, detergents, and organic solvents suggest the potential of this enzyme in biotechnological and industrial applications. This process has been patented in Iranian Intellectual Property Centre under License No: 91325.
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http://dx.doi.org/10.1016/j.heliyon.2019.e02543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819783PMC
September 2019

High phenol degradation capacity of a newly characterized Acinetobacter sp. SA01: Bacterial cell viability and membrane impairment in respect to the phenol toxicity.

Ecotoxicol Environ Saf 2018 Nov 22;164:455-466. Epub 2018 Aug 22.

Division of Industrial & Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P. O. Box 14155-6343, Tehran, Iran. Electronic address:

An efficient phenol-degrading bacterial strain, belonging to Acinetobacter genus, was isolated and selected to study the impact of different environmentally relevant phenol concentrations on the degradation process. The bacterial isolate, labeled as Acinetobacter sp. SA01 was able to degrade the maximum phenol concentration of 1 g/l during 60 h at optimum condition of pH 7, 30 °C and 180 rpm. Aeration and initial cell density, the two important factors, were carefully examined in the optimal growth conditions. The results showed that these two variables related proportionally with phenol degradation rate. Further investigations showed no effect of inoculum size on the enhancement of degradation of phenol at over 1 g/l. Flow cytometry (FCM) study was performed to find out the relationship between phenol-induced damages and phenol degradation process. Single staining using propidium iodide (PI) showed increased cell membrane permeability with an increase of phenol concentration, while single staining with carboxyfluorescein diacetate (cFDA) demonstrated a considerable reduction in esterase activity of the cells treated with phenol at more than 1 g/l. A detailed investigation of cellular viability using concurrent double staining of cFDA/PI revealed that the cell death increases in cells exposed to phenol at more than 1 g/l. The rate of cell death was low but noticeable in the presence of phenol concentration of 2 g/l, over time. Phenol at concentrations of 3 and 4 g/l caused strong toxicity in living cells of Acinetobacter sp. SA01. The plate count method and microscopy analysis of the cells treated with phenol at 1.5 and 2 g/l confirmed an apparent reduction in cell number over time. It was assumed that the phenol concentrations higher than 1 g/l have destructive effects on membrane integrity of Acinetobacter sp. SA01. Our results also revealed that the toxicity did not reduce by increasing initial cell density. Scanning electron microscopy (SEM) examination of bacterial cells revealed the surface morphological changes following exposure to phenol. The bacterial cells, with wizened appearance and wrinkled surface, were observed by exposing to phenol (1 g/l) at lag phase. A morphological change occurred in the mid-logarithmic phase as the bacterial cells demonstrated coccobacilli form as well as elongated filamentous shape. The wrinkled cell surface were totally disappeared in mid-stationary phase, suggesting that the complete degradation of phenol relieve the stress and direct bacterial cells toward possessing smoother cell membrane.
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http://dx.doi.org/10.1016/j.ecoenv.2018.08.051DOI Listing
November 2018

Pyrrolizidine Alkaloids-Free Extract from the Cell Culture of Lithospermum officinale with High Antioxidant Capacity.

Appl Biochem Biotechnol 2019 Mar 27;187(3):744-752. Epub 2018 Jul 27.

National Institute for Genetic Engineering and Biotechnology, P.O. Box: 14965/161, Tehran, Iran.

The benefits of Lithospermum officinale has encouraged people to continue using its extract (CAS 90063-58-4) in both medicinal and cosmetic industries despite the fact that chemical analysis confirms the presence of pyrrolizidine alkaloids (PAs) in the extract. While the cultivation of L. officinale takes, at least, 2 years to produce usable crops, its callus culture proliferated 8.3 times with 4.9-fold biomass in less than 30 days under the applied conditions in this study. Under the applied conditions, the cell extract contained no toxic PAs while phenylpropanoid pathway was active toward phenolic acids formation not toward naphthoquinone derivatives. Rosmarinic acid was produced as the main constituent. Total phenolic content and antioxidant capacity of the proliferated cell extracts were similar to those of the extracts of the natural plant tissues, in particular from the root. These results support the idea that the extract of L. officinale cells can be a reliable substitute for the extract of the natural plant tissues.
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http://dx.doi.org/10.1007/s12010-018-2830-3DOI Listing
March 2019

Improving the thermostability of Serratia marcescens B4A chitinase via G191V site-directed mutagenesis.

Int J Biol Macromol 2018 Sep 4;116:64-70. Epub 2018 May 4.

Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology, (NIGEB), Tehran, Iran.

Chitinases with high thermostability are important for many industrial and biotechnological applications. This study was conducted to enhance the stability of Serratia marcescens B4A chitinase by site directed mutagenesis of G191 V. Further characterization showed that the thermal stability of the mutant showed marked increase of about 5 and 15 fold at 50 and 60 °C respectively, while the optimum temperature and pH was retained. Kinetic analysis showed decreased K and V of the mutant in comparison with the wild type chitinase of about 1.3 and 3 fold, respectively. Based on structural prediction, it was speculated that this replacement shortened an important loop concomitant with the extension of adjacent β sheets. Accordingly, a higher thermostability of G191 V up to 90 °C supporting the decreased flexibility of unfolded state was also indicated. Finally, a practical proof of kinetic and thermal stabilization of chitinase was provided through decreased flexibility and entropic stabilization of its surface loops.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.05.014DOI Listing
September 2018

New insight into the allosteric effect of L-tyrosine on mushroom tyrosinase during L-dopa production.

Int J Biol Macromol 2018 Jul 3;114:821-829. Epub 2018 Apr 3.

National Institute for Genetic Engineering and Biotechnology, P.O. Box:14965-161, Tehran, Iran. Electronic address:

Kinetics studies of L-tyrosine (LTy) ortho-hydroxylation by mushroom tyrosinase (MT) confirmed that MT was severely, but not completely, inhibited at higher concentrations of LTy. Despite the availability of the crystal structure reports, no allosteric site has been identified on MT. To examine the assumption that a non-specific binding site works as a regulatory site, docking simulations were run for the second molecule of L-tyrosine (LTy) on the complexes of the first L-tyrosine molecule (LTy) with the heavy chain (H) of MT (LTy/HMT) and its dimer with the light chain (Ty/LHMT). In both, LTy occupied a non-specific binding site (MTPc). MD simulations revealed LTy/HMT/LTy and LTy/LHMT/LTy were stable. Binding free-energy analysis supported the formation of LTy/HMT/LTy and LTy/LHMT/LTy at higher concentrations of LTy and disclosed the importance of ΔE and ΔG during binding of LTy to MTPc. Upon LTy binding to MTPc, the Cu-Cu distance remained unchanged while the spatial position of LTy in the active site (MTPa) changed so that it would not be able to participate in ortho-hydroxylation. This study suggests a tuning role for L chain during binding of the ligands to MTPa and MTPc. Given these results, a plausible mechanism was proposed for the MT substrate inhibition.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.03.185DOI Listing
July 2018

A cold-adapted endoglucanase from camel rumen with high catalytic activity at moderate and low temperatures: an anomaly of truly cold-adapted evolution in a mesophilic environment.

Extremophiles 2018 Mar 12;22(2):315-326. Epub 2018 Jan 12.

Department of Energy and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

Endoglucanases are important enzymes in plant biomass degradation. They have current and potential applications in various industrial sectors including human and animal food processing, textile, paper, and renewable biofuel production. It is assumed that the cold-active endoglucanases, with high catalytic rates in moderate and cold temperatures, can improve the cost-effectiveness of industrial processes by lowering the need for heating and, thus, energy consumption. In this study, the endoglucanase CelCM3 was procured from a camel rumen metagenome via gene cloning and expression in Escherichia coli BL21 (DE3). The maximum activity of the enzyme on carboxymethyl cellulose (CMC) was obtained at pH 5 and 30 °C with a V and K of 339 U/mg and 2.57 mg/ml, respectively. The enzyme with an estimated low melting temperature of 45 °C and about 50% activity at 4 °C was identified to be cold-adapted. A thermodynamic analysis corroborated that CelCM3 with an activation energy (E), enthalpy of activation (ΔH), and Gibb's free energy (ΔG) of, respectively, 18.47 kJ mol, 16.12 kJ mol, and 56.09 kJ mol is a cold-active endoglucanase. In addition, CelCM3 was tolerant of metal ions, non-ionic detergents, urea, and organic solvents. Given these interesting characteristics, CelCM3 shows promise to meet the requirements of industrial applications.
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http://dx.doi.org/10.1007/s00792-018-0999-6DOI Listing
March 2018

Cohnella amylopullulanases: Biochemical characterization of two recombinant thermophilic enzymes.

PLoS One 2017 10;12(4):e0175013. Epub 2017 Apr 10.

Department of Industrial and Environmental Biotechnology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases (EC 3.2.1.41, GH57) from an endogenous Cohnella sp., [Coh00831 (KP335161; 1998 bp) and Coh01133 (KP335160: 3678 bp)] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3). The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2), maltotriose (G3) and maltotetraose (G4). The enzymes hydrolyzed pullulan to maltotriose (G3) only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175013PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386253PMC
August 2017

Catalytic efficiency and thermostability improvement of Suc2 invertase through rational site-directed mutagenesis.

Enzyme Microb Technol 2017 Jan 11;96:14-22. Epub 2016 Sep 11.

Department of Plant Bioproducts, National Institute of Genetic Engineering and Biotechnology. P.O. Box: 149651/161, Tehran, Iran.

Engineering of invertases has come to attention because of increasing demand for possible applications of invertases in various industrial processes. Due to the known physicochemical properties, invertases from micro-organisms such as Saccharomyces cerevisiae carrying SUC2 gene are considered as primary models. To improve thermostability and catalytic efficiency of SUC2 invertase (SInv), six influential residues with Relative Solvent Accessibility<5% were selected through multiple-sequence alignments, molecular modelling, structural and computational analyses. Consequently, SInv and 5 mutants including three mutants with single point substitution [Mut1=P152V, Mut2=S85V and Mut3=K153F)], one mutant with two points [Mut4=S305V-N463V] and one mutant with three points [Mut5=S85V-K153F-T271V] were developed via site-directed mutagenesis and produced using Pichia pastoris as the host. Physicochemical studies on these enzymes indicated that the selected amino acids which were located in the active site region mainly influenced catalytic efficiency. The best improvement belonged to Mut1 (54% increase in K/K) and Mut3 exhibited the worst effect (90% increase in K). These results suggest that Pro152 and Lys153 play key role in preparation of the right substrate lodging in the active site of SInv. The best thermostability improvement (16%) was observed for Mut4 in which two hydrophilic residues located on the loops, far from the active site, were replaced by Valines. These results suggest that tactful simultaneous substitution of influential hydrophilic residues in both active site region and peripheral loops with hydrophobic amino acids could result in more thermostable invertases with enhanced catalytic efficiency.
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http://dx.doi.org/10.1016/j.enzmictec.2016.09.004DOI Listing
January 2017

Thermostable chitinase from Cohnella sp. A01: isolation and product optimization.

Braz J Microbiol 2016 Oct - Dec;47(4):931-940. Epub 2016 Jul 26.

National Institute of Genetic Engineering and Biotechnology, Department of Industrial and Environmental Biotechnology, Bioprocess Engineering Group, Tehran, Iran.

Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and KHPO represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70°C, with K and V values of chitinase to be 5.6mg/mL and 0.87μmol/min, respectively. Ag, Co, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn, Cu, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052389PMC
http://dx.doi.org/10.1016/j.bjm.2016.07.009DOI Listing
January 2017

Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

Eur J Med Chem 2016 Oct 15;122:138-148. Epub 2016 Jun 15.

Department of Chemistry, Faculty of Science, Semnan University, Semnan, Iran.

Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible.
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http://dx.doi.org/10.1016/j.ejmech.2016.06.013DOI Listing
October 2016

Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties.

3 Biotech 2016 Dec 8;6(2):129. Epub 2016 Jun 8.

Department of Biology, Central Tehran Branch, Islamic Azad University, Tehran, Islamic Republic of Iran.

Invertase (EC.3.2.1.26) catalyzes the hydrolysis of sucrose to an equimolar mixture of D-glucose and D-fructose which is of interest for various industrial applications. In this research, Saccharomyces cerevisiae invertase gene (SUC2) was optimized based on Pichia pastoris codon preference. The synthetic gene was introduced into the methylotrophic yeast Pichia pastoris under the control of the inducible AOX1 promoter. High level of the extracellular recombinant invertase (R-inv) production was achieved via methanol induction for 4 days and purified by His-Tag affinity chromatography which appeared to be a mixture of glycosylated proteins with various sizes of 85-95 kDa on SDS-PAGE. Deglycosylation of the proteins by Endo-H resulted in the proteins with average molecular weight of 60 kDa. The purified recombinant invertase biochemical properties and kinetic parameters determined a pH and temperature optimum at 4.8 and 60 °C, respectively, which in comparison with native S. cerevisiae invertase, thermal stability of recombinant invertase is highly increased in different heating treatment experiments. The purification of recombinant invertase resulted in an enzyme with specific activity of 178.56 U/mg with 3.83-fold of purification and the kinetic constants for enzyme were Km value of 19 mM and Vmax value of 300 μmol min mg With kinetic efficiency (Kcat/Km) of 13.15 s mmol it can be concluded that recombinant P. pastoris invertase can be more effective for industrial quality criteria. We conclude that recombinant P. pastoris enzyme with broad pH stability, substrate specificity and proper thermal stability can fulfil a series of predefined industrial quality criteria to be used in food, pharmaceutical and bio ethanol production industries.
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http://dx.doi.org/10.1007/s13205-016-0441-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909026PMC
December 2016

Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates.

J Enzyme Inhib Med Chem 2016 Dec 2;31(6):1162-9. Epub 2015 Nov 2.

f Department of Chemical Engineering , University of Waterloo , Waterloo , Ontario , Canada.

Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λmax = 390 nm, ɛ = 32 880 M(-1) cm(-1) at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05 nmol mL(-1) was achieved for peroxidase.
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http://dx.doi.org/10.3109/14756366.2015.1103234DOI Listing
December 2016

Characterization of inhibitory effects of the potential therapeutic inhibitors, benzoic acid and pyridine derivatives, on the monophenolase and diphenolase activities of tyrosinase.

Iran J Basic Med Sci 2015 Feb;18(2):122-9

Department of Clinical Biochemistry and Genetics, Qazvin University of Medical Science, Qazvin, Iran.

Objectives: Involvement of tyrosinase in the synthesis of melanin and cell signaling pathway has made it an attractive target in the search for therapeutic inhibitors for treatment of different skin hyperpigmentation disorders and melanoma cancers.

Materials And Methods: In the present study, we conducted a comprehensive kinetic analysis to understand the mechanisms of inhibition imposed by 2-amino benzoic acid, 4-amino benzoic acid, nicotinic acid, and picolinic acid on the monophenolase and diphenolase activities of the mushroom tyrosinase, and then MTT assay was exploited to evaluate their toxicity on the melanoma cells.

Results: Kinetic analysis revealed that nicotinic acid and picolinic acid competitively restricted the monophenolase activity with inhibition constants (Ki) of 1.21 mM and 1.97 mM and the diphenolase activity with Kis of 2.4 mM and 2.93 mM, respectively. 2-aminobenzoic acid and 4-aminobenzoic acid inhibited the monophenolase activity in a non-competitive fashion with Kis of 5.15 µM and 3.8 µM and the diphenolase activity with Kis of 4.72 µM and 20 µM, respectively.

Conclusion: Our cell-based data revealed that only the pyridine derivatives imposed cytotoxicity in melanoma cells. Importantly, the concentrations of the inhibitors leading to 50% decrease in the cell density (IC50) were comparable to those causing 50% drop in the enzyme activity, implying that the observed cytotoxicity is highly likely due to the tyrosinase inhibition. Moreover, our cell-based data exhibited that the pyridine derivatives acted as anti-proliferative agents, perhaps inducing cytotoxicity in the melanoma cells through inhibition of the tyrosinase activities.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366722PMC
February 2015

Physicochemical study of a novel chimeric chitinase with enhanced binding ability.

Acta Biochim Biophys Sin (Shanghai) 2013 Oct 26;45(10):845-56. Epub 2013 Aug 26.

National Institute of Genetic Engineering and Biotechnology, PO Box: 149651/161, Tehran, Iran.

Chitinases are slow-reacting but important enzymes as they are anticipated to have diverse applications. The role of a chitin-binding domain (ChBD) in enhancing the quality of binding is essential information for purposeful engineering of chitinases. The idea of making hybrid chitinases by fusing a known ChBD to a chitinase, which naturally lacks ChBD is of interest especially for bio-controlling purposes. Therefore, in the present study, the ChBD of Serratia marcescens chitinase B was selected and fused to the fungal chitinase, Trichoderma atroviride Chit42. Both Chit42 and chemric Chit42 (ChC) showed similar activity towards colloidal chitin with specificity constants of 0.83 and 1.07 min(-1), respectively, same optimum temperatures (40°C), and similar optimum pH (4 and 4.5, respectively). In the presence of insoluble chitin, ChC showed higher activity (70%) and obtained a remarkably higher binding constant (700 times). Spectroscopic studies indicated that chimerization of Chit42 caused some structural changes, which resulted in a reduction of α-helix in ChC structure. Chemical and thermal stability studies suggested that ChC had a more stable structure than Chit42. Hill analysis of the binding data revealed mixed-cooperativity with positive cooperativity governing at ChC concentrations below 0.5 and above 2 µM in the presence of insoluble chitin. It is suggested that the addition of the ChBD to Chit42 affords structural changes which enhance the binding ability of ChC to insoluble chitin, improving its catalytic efficiency and increasing its thermal and chemical stability.
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http://dx.doi.org/10.1093/abbs/gmt089DOI Listing
October 2013

A novel ∼34-kDa α-amylase from psychrotroph Exiguobacterium sp. SH3: production, purification, and characterization.

Biotechnol Appl Biochem 2014 Mar-Apr;61(2):118-25. Epub 2013 Nov 14.

Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran.

An amylase-producing psychrotroph bacterium was isolated from soil and identified as belonging to the genus Exiguobacterium. A novel cold-adapted α-amylase, Amy SH3, was purified from culture medium of this bacterium using acetone precipitation and DEAE-Sepharose anion-exchange chromatography. The molecular mass of the enzyme was estimated about 34 kDa using SDS-PAGE. Biochemical characterization of Amy SH3 revealed that the optimum temperature for maximum activity of Amy SH3 was 37°C. However, Amy SH3 was also active at cold temperatures, showing 13% and 39% activity at 0 and 10°C, respectively. The optimum pH for maximum activity of Amy SH3 was pH 7, whereas the amylase was active over a pH range of 5 to 10. The activity of Amy SH3 was enhanced by Co²⁺ but decreased by Mg²⁺, Mn²⁺, Zn²⁺, Fe²⁺, and Ca²⁺. Amy SH3 was able to retain 76% of its activity in the presence of 0.5% SDS. The K(m) and V(max) of the enzyme were calculated to be 0.06 mg/mL and 4,010 U/mL, respectively. The cold-adapted Amy SH3 seems very promising for applications at ambient temperature.
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http://dx.doi.org/10.1002/bab.1140DOI Listing
December 2014

In silico and experimental characterization of chimeric Bacillus thermocatenulatus lipase with the complete conserved pentapeptide of Candida rugosa lipase.

Appl Biochem Biotechnol 2013 Feb 29;169(3):773-85. Epub 2012 Dec 29.

Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology-NIGEB, P.O. Box 14965/161, Tehran, Iran.

Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (¹¹²Ala-His-Ser-Gln-Gly¹¹⁶) was replaced with similar sequences (²⁰⁷Gly-Glu-Ser-Ala-Gly²¹¹) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca²⁺ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (¹¹²Gly-Glu-Ser-Ala-Gly¹¹⁶) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.
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http://dx.doi.org/10.1007/s12010-012-0014-0DOI Listing
February 2013

Anti-inflammatory effect of seeds and callus of Nigella sativa L. extracts on mix glial cells with regard to their thymoquinone content.

AAPS PharmSciTech 2013 Mar 19;14(1):160-7. Epub 2012 Dec 19.

National Institute of Genetic Engineering and Biotechnology, P.O.Box: 149651/161, Tehran, Iran.

Anti-inflammatory effect of the alcoholic extracts of N. sativa seeds and its callus on mix glial cells of rat with regard to their thymoquinone (TQ) content was investigated. Callus induction was achieved for explants of young leaf, stem, petiole, and root of N. sativa on solid Murashige and Skoog (MS) medium containing 2,4-D (1 mg/l) and kinetin (2.15 mg/l). TQ content of the alcoholic extracts was measured by HPLC. Total phenols were determined using Folin-Ciocalteu method and antioxidant power was estimated using FRAP tests. The mix glial cells, inflamed by lipopolysaccharide, were subjected to anti-inflammatory studies in the presence of various amounts of TQ and the alcoholic extracts. Viability of the cells and nitric oxide production were measured by MTT and Griess reagent, respectively. The leaf callus obtained the highest growth rate (115.4 mg/day) on MS medium containing 2,4-D (0.22 mg/l) and kinetin (2.15 mg/l). Analyses confirmed that TQ content of the callus of leaf was 12 times higher than that measured in the seeds extract. However, it decreased as the calli aged. Decrease in the TQ content of the callus was accompanied with an increase in its phenolic content and antioxidant ability. Studies on the inflamed rat mix glial cells revealed significant reduction in the nitric oxide production in the presence of 0.2 to 1.6 mg/ml of callus extract and 1.25 to 20 μl/ml of the seed extracts. However, the extent of the effects is modified assumingly due to the presence of the other existing substances in the extracts.
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http://dx.doi.org/10.1208/s12249-012-9899-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581679PMC
March 2013

Anionic peroxidase production by Arnebia euchroma callus.

Biotechnol Appl Biochem 2011 Nov-Dec;58(6):456-63. Epub 2011 Oct 31.

Department of Basic Sciences, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran.

Arnebia euchroma callus, obtained from the root cell culture of an Iranian native specimen, has gained a doubling time of 63 H after regular subculturing on Linsmaier-Skoog (LS) medium containing sugar (50 g/L), 2,4-dichlorophenoxyacetic acid (10(-6) M), and kinetin (10(-5) M) under darkness at 25°C. Despite the observed somaclonal variations, peroxidase production by the A. euchroma calli has been stable over 4 years under the aforementioned conditions. Isoelectric focusing experiments revealed that the partially purified A. euchroma peroxidases (AePoxs) are mainly anionic with pI values of about 5.5 and 6.6. AePox reaches its optimal activity at 55°C and pH 7.5. Results of the various kinetic studies suggest that AePox belongs to the type III plant peroxidases with no activity for the oxidation of 3-indoleacetic acid, but seems to play a role in the lignin biosynthesis and H(2) O(2) regulation during the proliferation of the A. euchroma cells on LS medium. Comparing the biochemical properties of AePox with horseradish peroxidase and in view of the ease of solid cell culture, the A. euchroma callus could be considered as a source of plant peroxidase for some biotechnological applications.
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http://dx.doi.org/10.1002/bab.42DOI Listing
August 2012

Direct spectrophotometric assay of laccase using diazo derivatives of guaiacol.

Anal Chem 2011 Jun 11;83(11):4200-5. Epub 2011 May 11.

Faculty of Basic Sciences, National Institute for Genetic Engineering and Biotechnology, P.O. Box 149651/161, Tehran, Iran.

Laccase (EC 1.10.3.2) is a widespread cuproenzyme able to oxidize various types of phenols and similar aromatic compounds through a one-electron transfer mechanism. The enzyme has already found its way into the market as a biocatalyst. Because of its ability to be paired by electron mediators, the expectation for employing laccases in versatile processes is very high. There are a few spectrophotometric methods for assaying the laccase activity; however, all of them are based on the formation of product(s) resulting from the enzymatic and inevitable succeeding chemical reactions. Use of diazo derivatives of guaiacol (DdG) was developed as a new spectrophotometric method based on substrate depletion allowing direct assessment of enzyme activity has been introduced. This method allows accurate comprehensive kinetic studies of laccases and provides reliable information about the quality of docking of different substrates or one substrate to the active sites of different laccases. Using this method, the kinetic parameters of various DdG carrying different electron donating and withdrawing substituents were used to assay laccase from Neurospora crassa. 2-Methoxy-4-[(4-phenyl)azo]-phenol (K(m) = 93.5 μM and V = 1.98 μM/min) was identified as an appropriate substrate for the accurate and routine spectrophotometric based assay of laccases.
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http://dx.doi.org/10.1021/ac200501wDOI Listing
June 2011

Direct spectrophotometric assay of laccase using diazo derivatives of guaiacol.

Anal Chem 2011 Jun 11;83(11):4200-5. Epub 2011 May 11.

Faculty of Basic Sciences, National Institute for Genetic Engineering and Biotechnology, P.O. Box 149651/161, Tehran, Iran.

Laccase (EC 1.10.3.2) is a widespread cuproenzyme able to oxidize various types of phenols and similar aromatic compounds through a one-electron transfer mechanism. The enzyme has already found its way into the market as a biocatalyst. Because of its ability to be paired by electron mediators, the expectation for employing laccases in versatile processes is very high. There are a few spectrophotometric methods for assaying the laccase activity; however, all of them are based on the formation of product(s) resulting from the enzymatic and inevitable succeeding chemical reactions. Use of diazo derivatives of guaiacol (DdG) was developed as a new spectrophotometric method based on substrate depletion allowing direct assessment of enzyme activity has been introduced. This method allows accurate comprehensive kinetic studies of laccases and provides reliable information about the quality of docking of different substrates or one substrate to the active sites of different laccases. Using this method, the kinetic parameters of various DdG carrying different electron donating and withdrawing substituents were used to assay laccase from Neurospora crassa. 2-Methoxy-4-[(4-phenyl)azo]-phenol (K(m) = 93.5 μM and V = 1.98 μM/min) was identified as an appropriate substrate for the accurate and routine spectrophotometric based assay of laccases.
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http://dx.doi.org/10.1021/ac200501wDOI Listing
June 2011

Successful resonance Raman study of cresolase activity of mushroom tyrosinase.

Biochem Biophys Res Commun 2004 Feb;314(4):925-30

Biochemistry Department, National Research Center for Genetic Engineering and Biotechnology, Tehran, Iran.

Mono-oxygenase (cresolase) activity of mushroom tyrosinase (MT) in the presence of 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide (HPABS) was successfully studied by resonance Raman (rR) spectroscopy. HPABS is a synthetic competitive inhibitor (K(i)=7.17 x 10(-6)M) for the cresolase activity with a large extinction coefficient at 365 nm. Upon reacting with MT, HPABS produced an enzyme-inhibitor (EI) complex with sufficiently long life span. Analyzing the ensuing spectrum indicates that the azo tautomer of HPABS binds to the enzyme and retains its geometrical isomeric form in the EI complex. The observed changes in the rR spectrum of HPABS after binding to MT support the idea that an electrophilic attack on the inhibitor has happened. Similar experiments were designed for studying the oxidase activity of MT. However, the enzymatic reaction, even in the presence of 4-[(2,4-dinitrophenyl)azo]-1,2-benzenediols was still fast enough to tan the reaction solution quickly and render its rR spectrum impregnable background.
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http://dx.doi.org/10.1016/j.bbrc.2003.12.197DOI Listing
February 2004

Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates.

Anal Biochem 2003 Jan;312(1):23-32

National Research Center for Genetic Engineering and Biotechnology, PO Box 14155-6343, Tehran, Iran.

Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their lambda(max)(s) with the least interference of the intermediates' or products' absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver-Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented.
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http://dx.doi.org/10.1016/s0003-2697(02)00408-6DOI Listing
January 2003
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