Publications by authors named "Saeed Aminzadeh"

32 Publications

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

Cohnella 1759 cysteine protease shows significant long term half-life and impressive increased activity in presence of some chemical reagents.

Sci Rep 2021 Feb 25;11(1):4573. Epub 2021 Feb 25.

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

Thermostability and substrate specificity of proteases are major factors in their industrial applications. rEla is a novel recombinant cysteine protease obtained from a thermophilic bacterium, Cohnella sp.A01 (PTCC No: 1921). Herein, we were interested in recombinant production and characterization of the enzyme and finding the novel features in comparison with other well-studied cysteine proteases. The bioinformatics analysis showed that rEla is allosteric cysteine protease from DJ-1/ThiJ/PfpI superfamily. The enzyme was heterologously expressed and characterized and the recombinant enzyme molecular mass was 19.38 kD which seems to be smaller than most of the cysteine proteases. rEla exhibited acceptable activity in broad pH and temperature ranges. The optimum activity was observed at 50℃ and pH 8 and the enzyme showed remarkable stability by keeping 50% of residual activity after 100 days storage at room temperature. The enzyme K and V values were 21.93 mM, 8 U/ml, respectively. To the best of our knowledge, in comparison with the other characterized cysteine proteases, rEla is the only reported cysteine protease with collagen specificity. The enzymes activity increases up to 1.4 times in the presence of calcium ion (2 mM) suggesting it as the enzyme's co-factor. When exposed to surfactants including Tween20, Tween80, Triton X-100 and SDS (1% and 4% v/v) the enzyme activity surprisingly increased up to 5 times.
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http://dx.doi.org/10.1038/s41598-021-84267-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7907070PMC
February 2021

Structural and biochemical characterization of a novel thermophilic Coh01147 protease.

PLoS One 2020 23;15(6):e0234958. Epub 2020 Jun 23.

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

Proteases play an essential role in living organisms and represent one of the largest groups of industrial enzymes. The aim of this work was recombinant production and characterization of a newly identified thermostable protease 1147 from thermophilum indigenous Cohnella sp. A01. Phylogenetic tree analysis showed that protease 1147 is closely related to the cysteine proteases from DJ-1/ThiJ/PfpI superfamily, with the conserved catalytic tetrad. Structural prediction using MODELLER 9v7 indicated that protease 1147 has an overall α/β sandwich tertiary structure. The gene of protease 1147 was cloned and expressed in Escherichia coli (E. coli) BL21. The recombinant protease 1147 appeared as a homogenous band of 18 kDa in SDS-PAGE, which was verified by western blot and zymography. The recombinant protein was purified with a yield of approximately 88% in a single step using Ni-NTA affinity chromatography. Furthermore, a rapid one-step thermal shock procedure was successfully implemented to purify the protein with a yield of 73%. Using casein as the substrate, Km, and kcat, kcat/Km values of 13.72 mM, 3.143 × 10-3 (s-1), and 0.381 (M-1 S-1) were obtained, respectively. The maximum protease activity was detected at pH = 7 and 60°C with the inactivation rate constant (kin) of 2.10 × 10-3 (m-1), and half-life (t1/2) of 330.07 min. Protease 1147 exhibited excellent stability to organic solvent, metal ions, and 1% SDS. The protease activity was significantly enhanced by Tween 20 and Tween 80 and suppressed by cysteine protease specific inhibitors. Docking results and molecular dynamics (MD) simulation revealed that Tween 20 interacted with protease 1147 via hydrogen bonds and made the structure more stable. CD and fluorescence spectra indicated structural changes taking place at 100°C, very basic and acidic pH, and in the presence of Tween 20. These properties make this newly characterized protease a potential candidate for various biotechnological applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234958PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310833PMC
September 2020

Novel halo- and thermo-tolerant Cohnella sp. A01 L-glutaminase: heterologous expression and biochemical characterization.

Sci Rep 2019 12 13;9(1):19062. Epub 2019 Dec 13.

Faculty of Science, Payame Noor University, Tehran, Iran.

L-glutaminase importance to use in the food industry and medicine has attracted much attention. Enzymes stability has always been a challenge while working with them. We heterologously expressed and characterized a novel stable L-glutaminase from an extremophile bacterium (Cohnella sp. A01, PTCC No: 1921). K, V, catalytic efficiency and specific activity of rSAM were respectively 1.8 mM, 49 µmol/min, 1851 1/(S.mM) and 9.2 IU/mg. Activation energy for substrate to product conversion and irreversible thermo-inactivation were respectively 4 kJ/mol and 105 kJ/mol from the linear Arrhenius plot. rSAM had the highest activity at temperature 50 °C, pH 8 and was resistant to a wide range of temperature and pH. In compare to the other characterized glutaminases, rSAM was the most resistant to NaCl. Mg, glycerol, DTT, and BME enhanced the enzyme activity and iodoacetate and iodoacetamide inhibited it. rSAM had only been partially digested by some proteases. According to the Fluorimetry and Circular dichroism analysis, rSAM in pH range from 4 to 11 and temperatures up to 60 °C had structural stability. A cysteine residue in the enzyme active site and a thiol bond were predicted upon the modeled tertiary structure of rSAM. Present structural studies also confirmed the presence of a thiol bond in its structure.
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http://dx.doi.org/10.1038/s41598-019-55587-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910923PMC
December 2019

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

Comparative effects of quercetin and hydroalcoholic extract of boiss with atorvastatin on atherosclerosis complication in male wistar rats.

Food Sci Nutr 2019 Sep 16;7(9):2875-2887. Epub 2019 Aug 16.

Bioprocess Engineering Research Group National Institute of Genetic Engineering and Biotechnology Tehran Iran.

The use of herbal remedies is significantly considered in the atherosclerosis treatment, reduction of fatty elements, and prevention of activity of oxidative stress factors. The present study was conducted on 48 rats in 6 groups. The experimental and sham groups were fed with 2% cholesterol for 40 days; and experimental groups were separately treated by atorvastatin, quercetin, and hydroalcoholic extract for 4 weeks. After treatment procedure, some serum factors such as low-density lipoprotein (LDL), total cholesterol (TC), malondialdehyde (MDA), and reactive oxygen species (ROS) were evaluated. Serum levels of LDL, TC, MDA, and ROS were significantly lower in experimental groups than sham group ( < .01). There was a significant decrease in serum MDA levels of these two groups in comparison with the atorvastatin-treated group ( < .05). Blood pressure parameters were decreased in treated with quercetin and hydroalcoholic extract in comparison with the sham group ( < .05). Quercetin and hydroalcoholic extract similar to atorvastatin could decrease serum lipids [except high-density lipoprotein (HDL)], oxidative stress factors, aorta contraction, weight gain, and blood pressure. These reagents improved the vascular structure and prevented the plaque formation.
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http://dx.doi.org/10.1002/fsn3.1136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6766565PMC
September 2019

Hyaluronic acid production enhancement via genetically modification and culture medium optimization in Lactobacillus acidophilus.

Int J Biol Macromol 2019 Jan 17;121:870-881. Epub 2018 Oct 17.

National Institute for Genetic Engineering and Biotechnology (NIGEB), Institute of Industrial and Environmental Biotechnology, Bioprocess Engineering Research Group, Shahrak-e Pajoohesh km 15, Tehran-Karaj Highway, P. O. Box: 14965/161, Tehran, Iran.

Hyaluronic acid (HA) is a natural polymer with various molecular weights that specify multiple biological roles. Traditionally, HA is obtained from animal waste and conventional pathogenic streptococci. However, there are challenges in these processes such as the presence of exotoxins, hyaluronidase, and viral contamination. In order to reduce these problems, this study was conducted to produce HA using recombinant bacterium that is generally recognized as safe (GRAS), and thereafter increase production through experimental design. At first, some lactic acid bacteria were screened and evaluated for HA production. Accordingly, among the selected bacteria, Lactobacillus acidophilus PTCC1643 produced about 0.25 g HA/L in the 48th hour of cultivation, and was thus selected as an alternative host for heterologous HA production. An expression vector containing HA synthase genes was transformed into L. acidophilus by electroporation. Consequently, HA production increased to 0.4 g/L. Eventually, response surface method (RSM) was used, which increased HA production to 1.7 g/L. This is approximately 7-fold higher than that produced at first. The resulting HA was characterized by FTIR spectroscopy and its molecular weight was estimated using agarose gel electrophoresis. In conclusion, L. acidophilus could be a safe, effective, and novel HA producer with industrial potential and commercial prospects.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.10.112DOI Listing
January 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

Biochemical Characterization of Recombinant Thermostable Cohnella sp. A01 β-Glucanase

Iran Biomed J 2018 09 13;22(5):345-54. Epub 2018 Jan 13.

National Institute for Genetic Engineering and Biotechnology (NIGEB), Institute of Industrial and Environmental Biotechnology, Bioprocess Engineering Research Group, Shahrak-E-Pajoohesh km 15, Tehran-Karaj Highway, P. O. Box: 14965/161, Tehran, Iran.

Background: Typically, non-cellulytic glucanase, including fungi and yeast cell wall hydrolyzing enzymes, are released by some symbiotic fungi and plants during the mycoparasitic fungi attack on plants. These enzymes are known as the defense mechanisms of plants. This study intends to investigate the biochemical properties of β-1,6-glucanase (bg16M) from native thermophilic bacteria, Cohnella A01.

Methods: bg16M gene was cloned and expressed in E. coli BL21 (DE3). The enzyme was purified utilizing Ni-NTA nikcle sepharose column. Pustulan and laminarin were selected as substrates in enzyme assay. The purified bg16M enzyme was treated with different pH, temperature, metal ions, and detergents.

Results: The expressed protein, including 639 amino acids, showed a high similarity with the hydrolytic glycosylated family 30. The molecular weight of enzyme was 64 kDa, and purification yield was 46%. The bg16M demonstrated activity as 4.83 U/ml on laminarin and 2.88 U/ml on pustulan. The optimum pH and temperature of the enzyme were 8 and 50 °C, respectively. The enzyme had an appropriate stability at high temperatures and in the pH range of 7 to 9, showing acceptable stability, while it did not lose enzymatic activity completely at acidic or basic pH. None of the studied metal ions and chemical compounds was the activator of bg16M, and urea, SDS, and copper acted as enzyme inhibitors.

Conclusion: Biochemical characterization of this enzyme revealed that bg16M can be applied in beverage industries and medical sectors because of its high activity, as well as thermal and alkaline stability.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058188PMC
http://dx.doi.org/10.29252/ibj.22.5.345DOI Listing
September 2018

Significant increase in cyanide degradation by Bacillus sp. M01 PTCC 1908 with response surface methodology optimization.

AMB Express 2017 Nov 10;7(1):200. Epub 2017 Nov 10.

National Institute for Genetic Engineering and Biotechnology (NIGEB), Shahrak-e Pajoohesh km 15, Tehran-Karaj Highway, Tehran, P. O. Box: 14965/161, Iran.

Cyanide is used in many industries despite its toxicity. Cyanide biodegradation is affordable and eco-friendly. Sampling from cyanide-contaminated areas from the Muteh gold mine and isolation of 24 bacteria were performed successfully. The selected bacteria-'Bacillus sp. M01'-showed maximum tolerance (15 mM) to cyanide and deposited in Persian Type Culture Collection by PTCC No.: 1908. In the primary experiments, effective factors were identified through the Plackett-Burman design. In order to attain the maximum degradation by Bacillus sp. M01 PTCC 1908, culture conditions were optimized by using response surface methodology. By optimizing the effective factor values and considering the interaction between them, the culture conditions were optimized. The degradation percentage was calculated using one-way ANOVA vs t test, and was found to have increased 2.35 times compared to pre-optimization. In all of the experiments, R was as high as 91%. The results of this study are strongly significant for cyanide biodegradation. This method enables the bacteria to degrade 86% of 10 mM cyanide in 48 h. This process has been patented in Iranian Intellectual Property Centre under Licence No: 90533.
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http://dx.doi.org/10.1186/s13568-017-0502-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681455PMC
November 2017

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

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

Improving population health management with payer data analytics.

Authors:
Saeed Aminzadeh

Health Manag Technol 2016 Jun;37(4):21

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June 2016

Study the effect of F17S mutation on the chimeric lipase.

J Genet Eng Biotechnol 2016 Jun 3;14(1):83-89. Epub 2016 Sep 3.

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

Lipases (, EC 3.1.1.3) are one of the highest value commercial enzymes as they have potential applications in biotechnology for detergents, food, pharmaceuticals, leather, textiles, cosmetics, and paper industries; and are currently receiving considerable attention because of their potential applications in biotechnology. Lipase 2 (BTL2) is one of the most important research targets, because of its potential industrial applications. In this study, the effect of substitution Phe17 with Ser in mutated BTL2 lipase, which conserved pentapeptide (Ala-His-Ser-Gln-Gly) was replaced with similar sequences (Gly-Glu-Ser-Ala-Gly) of lipase (CLR) at the nucleophilic elbow region. Docking results confirmed the mutated lipase to be better than the chimeric lipase. So, cloning was conducted, and the mutated and chimeric btl2 genes were expressed in , and then the enzymes were purified by anion exchange chromatography. The mutation increased lipase lipolytic activity against most of the applied substrates, with the exception of tributyrin when compared with chimeric lipase. Further, the mutated lipase exhibited higher activity than the chimeric lipase at all temperatures. Optimum pH of the mutated lipase was obtained at pH 9.5, which was more than the chimeric one. Enzyme activity of the mutated lipase in the presence of organic solvents, detergents, and metal ions was also improved than the chimeric lipase.
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http://dx.doi.org/10.1016/j.jgeb.2016.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6299889PMC
June 2016

Toward the development of a single-round infection assay based on EGFP reporting for anti-HIV-1 drug discovery.

Rep Biochem Mol Biol 2015 Oct;4(1):1-9

Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.

Background: The rapid increase of HIV-1 strains resistant to current antiretroviral drugs is a challenge for successful AIDS therapy. This necessitates the development of novel drugs, and to this end, availability of screening systems for in vitro drug discovery is a priority. Herein, we report the modification of a previously developed system for increased sensitivity, ease of use, and cost-efficiency, based on the application of the EGFP marker.

Methods: A PCR-amplified gfp gene (gfp) was cloned into pmzNL4-3, the plasmid already designed to produce single-cycle replicable virions, in frame with the reverse-transcriptase gene to construct the pmzNL4-3/GFP plasmid. GFP-mzNL4-3 pseudo-typed virions, as the first progeny viruses, were recovered from the culture supernatant of HEK293T cells co-transfected with pmzNL4-3/GFP and the helper plasmids pSPAX2 and pMD2G, which respectively encode HIV-1 Gag-Pol and vesicular stomatitis virus glycoprotein. Single-cycle replication and virion production were assessed by syncytia formation, p24 antigen assays, and electron and fluorescence microscopy.

Results: The incorporation of EGFP into the viral particles allowed their quantification by fluorometry, flow-cytometry, and fluorescence microscopy; however, this modification did not affect the single-round infectivity or production rate of the GFP fluorescence-emitting virions.

Conclusions: Our results certify the development of a rapid, inexpensive, and safe GFP-reporting single-cycle replicable system for anti-HIV drug discovery. Further experiments are needed to measure the validity and robustness of the assay.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4757091PMC
October 2015

Bacterial Secretome Analysis in Hunt for Novel Bacteriocins with Ability to Control subsp. .

Iran J Biotechnol 2015 Sep;13(3):10-19

Department of Biotechnology, Faculty of New Technologies and Energy Engineering, Shahid Beheshti University, Tehran, Iran.

Background: subsp. the causative agent of bacterial citrus canker, has affected citriculture worldwide. Varieties of means have been used to minimize its devastating effects, but no attention has been given to bacteriocins.

Objectives: Here and for the first time, we report the isolation and characterization of two novel bacteriocins.

Materials And Methods: Secretome containing bacteriocins of isolated bacteria was separated via SDS-PAGE. Each isolated protein band was characterized and checked for its efficacy in controlling two pathogenic isolates of Xcc via disk diffusion assay. The effects of varieties of carbon, nitrogen and phosphate sources were evaluated on both bacterial growth and bacteriocin production via Taguchi orthogonal method.

Results: The two bacteriocins showed an activity up to 55ºC that were sensitive to proteases suggesting being protein in nature. Analysis of SDS-PAGE purified protein bands of bacterial secretomes with demonstrated potency against revealed the presence of peptides with relative molecular masses of 16.9 and 17 kDa for and , respectively. Sequence analysis of peptides revealed an HCP1 family VI secretion system homologue for (YP_001439956) and pilin FimA homologue for (CBK85798.1). A Taguchi orthogonal array was also implemented to determine the effect of temperature and eight other chemical factors on bacteriocin production for each bacterium.

Conclusions: Two peptides with novel antibacterial activities effective against were isolated, characterized and conditions were optimized for their higher production.
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http://dx.doi.org/10.15171/ijb.1123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435018PMC
September 2015

The Effect of pH on Globular State of Lipase-3646; an Appropriate Model for Molten Globule Investigations.

Protein J 2015 Aug;34(4):267-74

Bioprocess Engineering Group, Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Sharek-e Pajoohesh Km 15, Tehran-Karaj Highway, P.O. Box: 14965/161, Tehran, Iran.

Secondary structure content of proteins in molten globule state is relatively constant while the quantity of tertiary structures clearly declines due to alterations in side-chain packing. In the present study, we analyze the MG state of lipase-3646 for the first time. We introduce lipase-3646 as an appropriate model for investigating the properties and behavior of a protein in MG state as well as folding pathway. Applying fluorescence spectroscopy we measured both intrinsic and extrinsic fluorescence of lipase-3646 in a pH range from 1.0 to 12.0. It was found that at pH 3.0 the protein acquires a MG state. Applying far-UV circular dichroism (CD), our analysis on the secondary structure of lipase-3646 revealed a slight change in the MG state intermediate (pH 3.0) compared to the native state (pH 8.5), which this amount of change is common for MG. Measurements in near-UV CD also showed a significant change in the enzyme conformation at pH 3.0 in comparison with the pH 8.5 wherein the protein acquires its native structure. Quenching the fluorescence by applying acrylamide, the amount 23 and 35 M(-1) were measured at pHs 8.5 and 3.0 respectively for stern-volmer constant (KSV). An increase in the enzyme molecular volume in the MG state was confirmed by gel filtration chromatography.
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http://dx.doi.org/10.1007/s10930-015-9622-1DOI Listing
August 2015

A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method.

Iran J Biotechnol 2015 Jun;13(2):56-59

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

Background: Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function.

Objectives: We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR.

Materials And Methods: Genomic DNA from was extracted. Nested PCR was used to amplify lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first and second PCR products are slightly longer than PCR products in a conventional SOE. PCR products obtained from the first round of PCR are used for the second PCR by applying the nested and mutated primers. Following to the purification of the amplified fragments, they will be subject of the further purification and will be used as template to perform the third round of PCR using gene specific primers. In the end, the products will be cloned into a suitable vector for subsequent application.

Results: In comparison to the conventional SOE-PCR, the improved method (i.e. N-SOE-PCR) increases the yield and specificity of the products. In addition, the proposed method shows a large reduction in the non-specific products.

Conclusions: By applying two more primers in the conventional SOE, the specificity of the method will be improved. This would be in part due to annealing of the primers further inside the amplicon that increases both the efficiency and a better attachment of the primers. Positioning of the primer far from both ends of an amplicon leads to an enhanced binding as well as increased affinity in the third round of amplification in SOE.
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http://dx.doi.org/10.15171/ijb.1090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5435007PMC
June 2015

Cloning, expression, purification, and characterization of lipase 3646 from thermophilic indigenous Cohnella sp. A01.

Protein Expr Purif 2015 May 12;109:120-6. Epub 2014 Oct 12.

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

Lipases form one of the most important groups of biocatalysts used in biotechnology. We studied the lipase from the bacterium Cohnella sp. A01 due to the versatility of thermophilic lipases in industry. In this study lipase 3646 gene from the thermophilic bacterium Cohnella sp. A01 was expressed in Escherichia coli and the enzyme was purified by a two-steps anion exchange chromatography. The purified lipase appeared to have a molecular weight of approximately 29.5kDa on SDS-PAGE. The values of Km and Vmax, calculated by the Michaelis-Menten equation, were 1077μM and 61.94U/mg, respectively. The kinetic characterization of the purified enzyme exhibited maximum activity at 70°C and pH 8.5. Activities at 50, 55 and 60°C for 120min were measured 58%, 47% and 41%, respectively. The enzyme was also highly stable at the pH range of 8.5-10.0 for 180min. The effect of EDTA indicated that the enzyme is not a metalloenzyme. The stability of lipase 3646 in the presence of organic solvents, detergents, metal ions and inhibitors suggested that this lipase could be exploited in certain industries such as detergent and leather. Lipase 3646 was determined structurally to be 37.5% α-helix, 12.8% β-sheet, 22.7% β-turn and 27% random coil.
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http://dx.doi.org/10.1016/j.pep.2014.10.002DOI Listing
May 2015

Protein engineering of Bacillus thermocatenulatus lipase via deletion of the α5 helix.

Appl Biochem Biotechnol 2014 Sep 27;174(1):339-51. Epub 2014 Jul 27.

Department of Biotechnology, Isfahan University of Technology, Isfahan, 84156-8311, Iran.

Lipases from Bacillus thermocatenulatus are a member of superfamily of α/β hydrolase, but there are structural differences between them. In this work, we focused on the α5 helix of B. thermocatenulatus lipase (BTL2) which is absent in canonical α/β hydrolase fold. In silico study showed that the α5 helix is a region that causes disorder in BTL2 protein. So, the α5 helix (residues 131 to 150) has been deleted from the B. thermocatenulatus lipase gene (BTL2) and the remain (Δα5-BTL2) has been expressed in Pichia pastoris yeast. The α5 deletion results in increase of enzyme-specific activity in the presence of tributyrin, tricaproin, tricaprylin, tricaprin, trilaurin, and olive oil (C18) substrates by 1.4-, 1.7-, 2.0-, 1.2-, 1.75-, and 1.95-fold, respectively. Also, deletion leads to increase in enzyme activity in different temperatures and pHs, whereas it did not significantly affect on enzyme activity in the presence of organic solvents, metal ions, and detergents.
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http://dx.doi.org/10.1007/s12010-014-1063-3DOI Listing
September 2014

Evaluation of gene expression changes of serotonin receptors, 5-HT3AR and 5-HT2AR as main stress factors in breast cancer patients.

Asian Pac J Cancer Prev 2014 ;15(11):4455-8

Department of Medical Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran E-mail :

Breast cancer is a serious and potentially lethal multi-factor disease among 40-50 aged women in both developed and developing countries. Also, various studies have pointed to roles of neurotransmitters like serotonin in development of cancers, through action on various types of receptors. This study was conducted to evaluate serotonin receptor (5HT2AR and 5HT3AR) genes expression in peripheral blood mononuclear cells (PBMCs) of breast cancer patients in comparison with the healthy people and in the MCF7 cell line. Peripheral blood samples were obtained from 30 patients and 30 healthy individuals. Total RNA was extracted from PBMCs and MCF-7 cells. and 5HT2AR and 5HT3AR were detected by RT-PCR techniques. Finally, serotonin receptor gene expression variation in breast cancer patients and MCF-7 cells were determined by real time-PCR. This latter indicated significant promotion in expression of 5HT3AR and 5HT2AR in PBMCs in breast cancer patients but expression of 5HT2AR in the MCF-7 cell line was significantly decreased. In conclusion, after performing complimentary tests, determine of gene expression changes in serotonin receptors (5HT2AR and 5HT3AR) may be useful as a new approach in treatment of breast cancer based on use of antagonists.
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http://dx.doi.org/10.7314/apjcp.2014.15.11.4455DOI Listing
March 2015

Purification and characterization of pepsin-solubilized collagen from skin of sea cucumber Holothuria parva.

Appl Biochem Biotechnol 2014 May 28;173(1):143-54. Epub 2014 Mar 28.

Department of Animal and Marine Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), P.O. Box: 14965/161, Sharek-e Pajoohesh Km 15, Tehran-Karaj Highway, Tehran, Iran.

Pepsin-solubilized collagen (PSC) was extracted from the skin of sea cucumber Holothuria parva and was fractionally characterized. The PSC from H. parva skin consisted of three α1 chains (α1)3, in contrast to calf skin collagen type I with two α1 and one α2 chains (α1)2α2 with approximately 130 kDa each. The maximum transition (Tm) and denaturation temperature (Td) of PSC were determined to be 46.94 and 32.5 °C, respectively. The amino acid composition analysis revealed that glycine, proline, alanine, and hydroxyproline were the abundant amino acids available in extracted PSC. The results showed that the isolated collagen from H. parva has some similar characteristics to previously reported collagens used in food and pharmaceutical industries.
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http://dx.doi.org/10.1007/s12010-014-0823-4DOI Listing
May 2014

Study of the effect of F17A mutation on characteristics of Bacillus thermocatenulatus lipase expressed in Pichia pastoris using in silico and experimental methods.

Biotechnol Appl Biochem 2014 May-Jun;61(3):264-73. Epub 2014 Mar 18.

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

Bacillus thermocatenulatus lipase 2 (BTL2), a thermoalkalophilic lipase, is the best studied enzyme for its particular properties, which make it useful in different industries. Displacement of conserved phenylalanine 17 (Phe-17) residue in the active site of BTL2 has a critical role in oxyanion hole formation, which is important for enzyme activity. In this study, to facilitate oxyanion hole formation, Phe-17 was substituted with Alanine residue (F17A). The best structures of the opened form of the native and mutated lipases were garnered based on the crystal structures of 2W22. To evaluate catalytic activity, both lipases were docked to a set of ligands using Hex 6.3 software. Following in silico study, both native and mutant btl2 genes were cloned and expressed in Pichia pastoris. Based on the results obtained, the mutation increased lipase lipolytic activity against most of the applied substrates, especially for tributyrin and tricaprylin, by 1.9 and 2.15 fold, respectively. However, optimum temperature and pH were the same for both lipases (60 °C and pH 8.0). As previously reported, it is believed that F17A mutation simplifies oxyanion hole formation and declines steric hindrance in the enzyme active site, which might ultimately lead to more efficient accessibility of substrates.
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http://dx.doi.org/10.1002/bab.1164DOI Listing
April 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

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

Characterization of the newly isolated Geobacillus sp. T1, the efficient cellulase-producer on untreated barley and wheat straws.

Bioresour Technol 2012 Sep 19;120:99-105. Epub 2012 Jun 19.

Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14155-6343, Iran.

A thermophile cellulase-producing bacterium was isolated and identified as closely related to Geobacillus subterraneus. The strain, named Geobacillus sp. T1, was able to grow and produce cellulase on cellobiose, microcrystalline cellulose, carboxymethylcellulose (CMC), barley straw, wheat straw and Whatman No. 1 filter paper. However, barley and wheat straws were significantly better substrates for cellulase production. When Geobacillus sp. T1 was cultivated in the presence of 0.5% barley straw, 0.1% Tween 80 and pH 6.5 at 50°C, the maximum level of free cellulase up to 143.50 U/mL was produced after 24h. This cellulase (≈ 54 kDa) was most active at pH 6.5 and 70°C. The enzyme in citrate phosphate buffer (10mM) was stable at 60°C for at least 1h. Geobacillus sp. T1 with efficient growth and cellulase production on straws seems a potential candidate for conversion of agricultural biomass to fuels.
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http://dx.doi.org/10.1016/j.biortech.2012.06.027DOI Listing
September 2012

Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A.

Braz J Microbiol 2011 Jul 1;42(3):1017-29. Epub 2011 Sep 1.

Department of Animal and Marine Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB) , Shahrak-e Pajoohesh Km 15, Tehran-Karaj Highway, Tehran , Iran ; Faculty of Marine Sciences, Khorramshahr Marine Sciences and Technology University , Khorramshahr , Iran.

Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. In the following investigation, a novel chitinase with antifungal activity was characterized from a native Serratia marcescens B4A. Partially purified enzyme had an apparent molecular mass of 54 kDa. It indicated an optimum activity in pH 5 at 45°C. Enzyme was stable in 55°C for 20 min and at a pH range of 3-9 for 90 min at 25°C. When the temperature was raised to 60°C, it might affect the structure of enzymes lead to reduction of chitinase activity. Moreover, the Km and Vmax values for chitin were 8.3 mg/ml and 2.4 mmol/min, respectively. Additionally, the effect of some cations and chemical compounds were found to stimulate the chitinase activity. In addition, Iodoacetamide and Idoacetic acid did not inhibit enzyme activity, indicating that cysteine residues are not part of the catalytic site of chitinase. Finally, chitinase activity was further monitored by scanning electronic microscopy data in which progressive changes in chitin porosity appeared upon treatment with chitinase. This enzyme exhibited antifungal activity against Rhizoctonia solani, Bipolaris sp, Alternaria raphani, Alternaria brassicicola, revealing a potential application for the industry with potentially exploitable significance. Fungal chitin shows some special features, in particular with respect to chemical structure. Difference in chitinolytic ability must result from the subsite structure in the enzyme binding cleft. This implies that why the enzyme didn't have significant antifungal activity against other Fungi.
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http://dx.doi.org/10.1590/S1517-838220110003000022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768790PMC
July 2011
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