Elucidated potential of immobilized Janibacter sp. for saline wastewater phenol removal.

Phenolic compounds are commonly found in industrial effluents and can be hazardous to organisms even at low concentrations. Over the years, researchers have demonstrated that bioremediation is a cost-effective and environmentally friendly alternative to physicochemical approaches used to remove phenol. The aim of this study was to investigate the removal of phenol from saline wastewaters by a halotolerant strain of the genus Janibacter. For this purpose, bacterial cells were immobilized on different supports, from which mica and zeolite were ultimately chosen due to their higher removal efficiency. The wet weight of immobilized cells per 1 g of mica and zeolite was 0.51 and 0.48 g, respectively. Free cells consumed 100 mg/L of phenol in 88 h, while immobilized cells used it in 40 h. Immobilized cells revealed a higher thermostability and could operate over a wider pH range and salinity. Unlike free cells, immobilized cells could remove 700 mg/L of phenol and could be reused for at least nine cycles. Interestingly the phenol removal efficiency of zeolite-immobilized cells remained unchanged after 4 months of storage at 4 and - 20 °C, which could be of great advantage for industrial applications. Complete destruction of phenol was observed through the meta pathway comprising phenol hydroxylase and catechol 2,3-dioxygenase enzymes. KEY POINTS: • Mica- and zeolite-immobilized cells were able to consume high concentrations of phenol. • Cells immobilized on mica and zeolite had considerable operational and storage stability. • Immobilized cells could be a good candidate for phenol removal in saline environments.

Engineering a Novel Metabolic Pathway for Improving Cellular Malonyl-CoA Levels in Escherichia coli.

Cellular pool of malonyl-CoA in Escherichia coli is small, which impedes its utility for overproduction of natural products such as phenylpropanoids, polyketides, and flavonoids. In this study, we report the use of a new metabolic pathway to increase the malonyl-CoA concentration as a limiting metabolite in E. coli. For this purpose, the malonate/sodium symporter from Malonomonas rubra, and malonyl-CoA synthetase (MCS) from Bradyrhizobium japonicum were co-expressed in E. coli. This new pathway allows the cell to actively import malonate from the culture medium and to convert malonate and CoA to malonyl-CoA via an ATP-dependent ligation reaction. HPLC analysis confirmed elevated levels of malonyl-CoA and (2S)-naringenin as a malonyl-CoA-dependent metabolite, in E. coli. A 6.8-fold and more than 3.5-fold increase in (2S)-naringenin production were achieved in the engineered host in comparison with non-engineered E. coli and previously reported passive transport MatBMatC pathway, respectively. This observation suggests that using active transporters of malonate not only improves malonyl-CoA-dependent production but also makes it possible to harness low concentrations of malonate in culture media.

Genome-resolved insight into the reservoir of antibiotic resistance genes in aquatic microbial community.

Aquatic microbial communities are an important reservoir of antibiotic resistance genes (ARGs). However, distribution and diversity of different ARG categories in environmental microbes with different ecological strategies is not yet well studied. Despite the potential exposure of the southern part of the Caspian Sea to the release of antibiotics, little is known about its natural resistome profile. We used a combination of Hidden Markov model (HMM), homology alignment and a deep learning approach for comprehensive screening of the diversity and distribution of ARGs in the Caspian Sea metagenomes at genome resolution. Detected ARGs were classified into five antibiotic resistance categories including prevention of access to target (44%), modification/protection of targets (30%), direct modification of antibiotics (22%), stress resistance (3%), and metal resistance (1%). The 102 detected ARG containing metagenome-assembled genomes of the Caspian Sea were dominated by representatives of Acidimicrobiia, Gammaproteobacteria, and Actinobacteria classes. Comparative analysis revealed that the highly abundant, oligotrophic, and genome streamlined representatives of taxa Acidimicrobiia and Actinobacteria modify the antibiotic target via mutation to develop antibiotic resistance rather than carrying extra resistance genes. Our results help with understanding how the encoded resistance categories of each genome are aligned with its ecological strategies.

Chitosan/PEO nanofibers containing Calendula officinalis extract: Preparation, characterization, in vitro and in vivo evaluation for wound healing applications.

Wound healing is a complex pathophysiological process, highlighting the importance of effective and thorough wound care along with the prevention of wound infection, a major barrier that can slow down or even disrupt the healing process. To date, there are plenty of herbal plants well known and historically supernatural, showing profound wound healing effects. Application of such herbal extracts/ingredients in electrospun nanofiber platforms has shown promising outcomes in improving wound healing process. Based on these facts, we loaded Calendula officinalis extract (CO) in chitosan/polyethylene oxide scaffolds (CS/PEO) by electrospinning. Using SEM, morphology of electrospun scaffolds showed a narrow range of fiber diameter, around 143--252 nm, with uniform and bead-free appearance. FT-IR spectroscopy confirmed the presence of CO extract in nanofibrous scaffolds. Of importance, incorporation of CO extract improved mechanical properties of CS/PEO nanofibers. A 1602 cP reduction in viscosity and a 0.892 ms/cm increase in the conductivity of the solution was observed after addition of the CO extract. CO extract showed strong antibacterial properties with 96% and 94% reduction in Gram positive and Gram negative bacteria, respectively. In vitro studies with fibroblast cells confirmed enhanced proliferation, growth and attachment of the cells. The in vivo and histological analysis of rat wounds, revealed excellent wound healing ability of CS/PEO/CO dressings (87.5 % wound closure after 14 days) via improving collagen synthesis, re-epithelization and remodeling of the tissue. In sum, our findings show that CS/PEO/CO scaffolds can be used as a promising dressing for the treatment of skin wounds.

Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2.

Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The Km and kcat values of 6.5 µM and 4.57 s-1, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.

Heterologous expression of recombinant urate oxidase using the intein-mediated protein purification in Pichia pastoris.

The potential of urate oxidase (uricase) for clinical use has been highlighted because of its role in lowering the blood uric acid levels for the treatment of tumor lysis syndrome. In the present study, the codon-optimized synthetic gene of Aspergillus flavus uricase was fused to the Mxe GyrA intein and chitin-binding domain. The construct was inserted into pPICZA and pPICZαA vectors and electroporated into Pichia pastoris GS115 for the cytosolic and secretory expression. Transformants were screened on gradients of Zeocin up to 2000 µg/ml to find multi-copy integrants. For both constructs, colonies with more resistance were screened for the highest uricase producers by enzyme assay. PCR analysis confirmed successful cassettes insertion into the genome and Mut + phenotype. The gene copy index was determined to be two and five for cytosolic and secretory strains, respectively. Productivity of the cytosolic and secretory strains was found to be 0.74 and 0.001 U/ml culture media in order while the cytosolic recombinant enzyme accounted for about 6% of total proteins. One-step purification of the expressed uricase was done with the aid of the chitin affinity column, followed by DTT induction for intein on-column cleavage. The yield of 40.8 mg/L and K m of 0.22 mM was obtained for intracellular expression. It seems that the intracellular production of uricase can indeed serve as an effective alternative to secretory expression. Moreover, this is the first report considering cytosolic production of uricase using the intein-mediated protein purification in the methylotrophic yeast, P. pastoris.

Metagenomic discovery and functional validation of L-asparaginases with anti-leukemic effect from the Caspian Sea.

By screening 27,000 publicly available prokaryotic genomes, we recovered ca. 6300 type I and ca. 5200 type II putative L-asparaginase highlighting the vast potential of prokaryotes. Caspian water with similar salt composition to the human serum was targeted for in silico L-asparaginase screening. We screened ca. three million predicted genes of its assembled metagenomes that resulted in annotation of 87 putative L-asparaginase genes. The L-asparagine hydrolysis was experimentally confirmed by synthesizing and cloning three selected genes in E. coli. Catalytic parameters of the purified enzymes were determined to be among the most desirable reported values. Two recombinant enzymes represented remarkable anti-proliferative activity (IC50 <1IU/ml) against leukemia cell line Jurkat while no cytotoxic effect on human erythrocytes or human umbilical vein endothelial cells was detected. Similar salinity and ionic concentration of the Caspian water to the human serum highlights the potential of secretory L-asparaginases recovered from these metagenomes as potential treatment agents.

Developing a Fluorescent Hybrid Nanobiosensor Based on Quantum Dots and Azoreductase Enzyme forMethyl Red Monitoring.

Background: Azo dyes are the most widely used synthetic colorants in the textile, food, pharmaceutical, cosmetic, and other industries, accounting for nearly 70% of all dyestuffs consumed. Recently, much research attention has been paid to efficient monitoring of these hazardous chemicals and their related metabolites because of their potentially harmful effect on environmental issues. In contrast to the complex and expensive instrumental procedures, the detection system based on the quantum dots (QDs) with the superior optochemical properties provides a new era in the pollution sensing and prevention. Methods: We have developed a QD-enzyme hybrid system to probe methyl red (MR) in aqueous solutions using a fluorescence quenching procedure. Results: The azoreductase enzyme catalyzed the reduction of azo group in MR, which can efficiently decrease the Förster resonance energy transfer between the QDs and MR molecules. The correlation between the QDs photoluminescence recovery and MR enzymatic decolorization at the neutral phosphate buffer permitted the creation of a fluorescence quenching-based sensor. The synthesized biosensor can be used for the accurate detection of MR in a linear calibration over MR concentrations of 5-84 µM, with the limit of detection of 0.5 µM in response time of three minutes. Conclusion: Our findings revealed that this fluorometric sensor has the potential to be successfully applied for monitoring a wide linear range of MR concentration with the relative standard deviation of 4% rather than the other method.

Heterologous production of porcine derived antimicrobial peptide PR-39 in Escherichia coli using SUMO and intein fusion systems.

About half a century after antibiotics discovery, multi-antibiotic-resistant bacteria posed a new challenge to medicine. Attempts to discover new antibiotics have drawn the attention to Antimicrobial Peptides (AMPs). The rapid growth, besides its known genetic and manipulation systems, makes E. coli the preferred host system for production of recombinant proteins on an industrial scale. To produce AMPs in E. coli, the application of fusion-tags with the aim of stability, solubility, and prevention of antimicrobial activity is one of the best practices in this regard. In this study, we presented two different expression systems for the production of PR-39 in E. coli; one in fusion with intein-Chitin binding domain (CBD) and another in fusion with SUMO accompanied by polyhistidine affinity tag. Both were cloned in the NdeI-XhoI sites of pET-17b and transformed to E. coli BL21 (DE3) pLysS. Recombinant bacteria were cultured and induced with 0.4 mM IPTG at 30 °C. Expression and purification of target proteins were confirmed by Tricine- SDS-PAGE and dot blot analysis. Recovery of 250 µg PR-39/L from SUMO fusion system and 280 µg PR-39/L from the intein fusion system was achieved. Both purified peptides showed antibacterial activity using MIC/MBC demonstrating their functionality after SUMO and intein mediated purification.

Improving the thermal stability of azoreductase from Halomonas elongata by introducing a disulfide bond via site-directed mutagenesis.

Azoreductases mainly reduce azo dyes, the largest class of colorants, to colorless aromatic amines. AzoH, a new azoreductase from the halophilic bacterium, Halomonas elongata, has been recently cloned and expressed in Escherichia coli. The aim of this study was to improve thermal stability of this enzyme by introducing new disulfide bonds. Since X-ray crystallography was not available, homology modeling and molecular dynamics was used to construct the enzyme three-dimensional structure. Potential disulfide bonds for increasing thermal stability were found using DIScover online software. Appropriate mutations (L49C/D108C) to form a disulfide bond were introduced by the Quik-Change method. Mutant protein expressed in E. coli showed increased thermal stability at 50 °C (increased half-life from 12.6 Min in AzoH to 26.66 Min in a mutated enzyme). The mutated enzyme could also tolerate 5% (w/v) NaCl and retained 30% of original activity after 24 H incubation, whereas the wild-type enzyme was completely inactivated. According to circular dichroism studies, the secondary structure was not altered by this mutation; however, a blue shift in intrinsic florescent graph revealed changes in the tertiary structure. This is the first study to improve thermal stability and salt tolerance of a halophilic azoreductase.

Exploring the potential of halophilic archaea for the decolorization of azo dyes.

Azo dyes are being extensively used in textile industries, so finding a proper solution to decolorize them is of high importance. In order to find azo dye decolorizing strains among haloarchaea, which are well known for their tolerance to harsh environmental conditions, fifteen haloarchaeal strains were screened. Halogeometricum sp. strain A and Haloferax sp. strain B with the highest decolorization ability (95% and 91% for Remazol black B; both about 60% for Acid blue 161, respectively) were selected for further studies. It was shown that both strains were able to grow and decolorize the dye in a medium containing up to 5 M NaCl, with optimum decolorization activity at 2.5-3.4 M, pH 7, and a wide temperature range between 30 to 45 °C. Moreover, both strains were able to tolerate and decolorize up to 1,000 mg l-1 Remazol black B. Also, they were able to survive in 5,000 mg l-1 of the dye after 20 days' incubation. Glucose and yeast extract were found to be the best carbon and nitrogen sources in the decolorization medium for both strains. This is the first report studying decolorization of azo dyes using halophilic archaea.

Cloning and characterization of Halomonas elongata L-asparaginase, a promising chemotherapeutic agent.

L-asparaginase has been used in the treatment of patients with acute lymphoblastic leukemia (ALL) for more than 30 years. Rapid clearance of the enzyme from blood stream and its L-glutaminase-dependent neurotoxicity has led to searching for new L-asparaginases with more desirable properties. In the present study, L-asparaginase coding gene of Halomonas elongata was isolated, expressed in Escherichia coli, purified, and characterized. The purified protein was found to have a molecular mass of 39.5 kDa and 1000-folds more activity towards L-asparagine than L-glutamine. Enzyme-specific activity towards L-asparagine was determined to be 1510 U/mg, which is among the highest reported values for microbial L-asparaginases. K m , Vmax, and k cat values were 5.6 mM, 2.2 µmol/min, and 1.96 × 103 1/S, respectively. Optimum temperature was found to be 37 °C while the enzyme showed maximum activity at a wide pH range (from 6 to 9). Enzyme half-life in the presence of human serum at 37 °C was 90 min which is three times higher when compared with reported values for E. coli L-asparaginase. Enzyme showed cytotoxic effects against Jurkat and U937 cell lines with an IC50 of 2 and 1 U/ml, respectively. Also, no toxic effects on human erythrocytes and Chinese hamster ovary cell lines were detected, and just minor inhibitory effects on human umbilical vein endothelial cells were observed. This is the first report describing the therapeutic potentials of a recombinant L-asparaginase isolated from a halophilic bacterium as an anticancer agent.

Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme.

Uric acid, a side product of nucleotide metabolism, should be cleared from blood stream since its accumulation can cause cardiovascular diseases and gout. Uricase (urate oxidase) converts uric acid to 5-hydroxyisourate, but it is absent in human and other higher apes. Yet, the recombinant form of uricase, Rasburicase, is now commercially available to cure tumor lysis syndrome by lowering serum uric acid level. Developing new methods to efficiently purify pharmaceutical proteins like uricase has attracted researchers' attention. Self-cleaving intein mediated single column purification is one of these novel approaches. Self-cleaving inteins are modified forms of natural inteins that can excise and join only at one junction site. In this study, the synthetic gene of Aspergillus flavus uricase, a homotetrameric protein, was cloned into pTXB1 vector as a fusion with the N-terminal of Mxe GyrA intein and chitin-binding domain (CBD) for simple purification. Expression was confirmed by western blot analysis. The fusion protein containing uricase-intein-CBD was purified on a chitin column. The cleavage was induced by adding DTT,1 as a reducing agent to release uricase. The purity of uricase and complete excision of the intein and CBD were confirmed by SDS-PAGE2 while its proper folding was proved by circular dichroism and fluorescent emission studies. Isoelectric focusing further confirmed its homogeneity when a single protein band was observed at the predicted pI value. This is the first report of successful purification of a multimeric therapeutic enzyme by intein-mediated protein cleaving using a well-established and facile system.

The potential of halophilic and halotolerant bacteria for the production of antineoplastic enzymes: L-asparaginase and L-glutaminase.

L-asparaginase and L-glutaminase can be effectively used for the treatment of patients who suffer from accute lymphoblastic leukemia and tumor cells. Microbial sources are the best source for the bulk production of these enzymes. However, their long-term administration may cause immunological responses, so screening for new enzymes with novel properties is required. Halophilic and halotolerant bacteria with novel enzymatic characteristics can be considered as a potential source for production of enzymes with different immunological properties. In this study, L-asparaginase and L-glutaminase production by halophilic bacteria isolated from Urmia salt lake was studied. Out of the 85 isolated halophilic and halotolerant bacterial strains, 16 (19 %) showed L-asparaginase activity and 3 strains (3.5 %) showed L-glutaminase activity. Strains with the highest activities were selected for further studies. Based on 16S rDNA sequence analysis, it was shown that the selected isolates for L-asparaginase and L-glutaminase production belong to the genus Bacillus and Salicola, respectively. Both enzymes were produced extracellularly. The strain with the most L-asparaginase production did not show L-glutaminase production which is medically important. The effects of key parameters including temperature, initial pH of the solution, and concentrations of glucose, asparagine or glutamine, and sodium chloride were evaluated by means of response surface methodology (RSM) to optimize enzymes production. Under the obtained optimal conditions, L-asparaginase and L-glutaminase production was increased up to 1.5 (61.7 unit/mL) and 2.6 fold (46.4 unit/mL), respectively.

Isolation, cloning and characterization of an azoreductase from the halophilic bacterium Halomonas elongata.

Azo dyes are a major class of colorants used in various industries including textile, paper and food. These dyes are regarded as pollutant since they are not readily reduced under aerobic conditions. Halomonas elongata, a halophilic bacterium, has the ability to decolorize different mono and di-azo dyes in anoxic conditions. In this study the putative azoreductase gene of H. elongata, formerly annotated as acp, was isolated, heterologously expressed in Escherichia coli, purified and characterized. The gene product, AzoH, was found to have a molecular mass of 22 kDa. The enzyme requires NADH, as an electron donor for its activity. The apparent Km was 63 µM for NADH and 12 µM for methyl red as a mono-azo dye substrate. The specific activity for methyl red was 0.27 µmol min(-1)mg(-1). The optimum enzyme activity was achieved in 50mM sodium phosphate buffer at pH 6. Although increased salinity resulted in reduced activity, AzoH could decolorize azo dye at NaCl concentrations up to 15% (w/v). The enzyme was also shown to be able to decolorize remazol black B as a representative of di-azo dyes. This is the first report describing the sequence and activity of an azo-reducing enzyme from a halophilic bacterium.

Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis.

Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses.

Genome-scale reconstruction of the metabolic network in Pseudomonas stutzeri A1501.

Pseudomonas stutzeri A1501 is an endophytic bacterium capable of nitrogen fixation. This strain has been isolated from the rice rhizosphere and provides the plant with fixed nitrogen and phytohormones. These interesting features encouraged us to study the metabolism of this microorganism at the systems-level. In this work, we present the first genome-scale metabolic model (iPB890) for P. stutzeri, involving 890 genes, 1135 reactions, and 813 metabolites. A combination of automatic and manual approaches was used in the reconstruction process. Briefly, using the metabolic networks of Pseudomonas aeruginosa and Pseudomonas putida as templates, a draft metabolic network of P. stutzeri was reconstructed. Then, the draft network was driven through an iterative and curative process of gap filling. In the next step, the model was evaluated using different experimental data such as specific growth rate, Biolog substrate utilization data and other experimental observations. In most of the evaluation cases, the model was successful in correctly predicting the cellular phenotypes. Thus, we posit that the iPB890 model serves as a suitable platform to explore the metabolism of P. stutzeri.

Phenol removal from refinery wastewater by mutant recombinant horseradish peroxidase.

Application of mutated recombinant horseradish peroxidase (HRP) for phenol removal from refinery effluents is reported. Recombinant HRP produced in Escherichia coli suffers from the disadvantage of lacking glycosylation, which affects its catalytic efficiency and stability toward inactivating parameters such as increased temperature and enhanced amounts of hydrogen peroxide. In the present study, the previously reported variant (in which Asn268 was substituted with Asp, N268D) with improved stability characteristics and catalytic efficiency was used to remove phenol from a petroleum refinery effluent. The presence and removal of phenol was studied by high-performance liquid chromatography; the precipitated oxidized phenol was also observed and removed from the sample by centrifugation. Results showed that the N268D variant can remove 61%, 67%, and 81% of phenol from effluent in 1, 2, and 16 H, respectively. By exploiting the N268D mutant, removal of 50% phenol could be achieved in 42 Min, which was more than 22 times less than the treatment time required by native recombinant enzyme.

Studies on the refolding process of recombinant horseradish peroxidase.

Horseradish peroxidase (HRP) is an important heme-containing glyco-enzyme that has been used in many biotechnological fields. Valuable proteins like HRP can be obtained in sufficient amounts using Escherichia coli as an expression system. However, frequently, the expression of recombinant enzyme results in inclusion bodies, and the refolding yield is generally low for proteins such as plant peroxidases. In this study, a recombinant HRP was cloned and expressed in the form of inclusion bodies. Initially, the influence of few additives on HRP refolding was assessed by the one factor at a time method. Subsequently, factors with significant effects including glycerol, GSSG/DTT, and the enzyme concentration were selected for further optimization by means of the central composite design of response surface methodology (RSM). Under the obtained optimal condition, refolding increased about twofold. The refolding process was then monitored by the intrinsic fluorescence intensity under optimal conditions (0.35 mM GSSG, 0.044 mM DTT, 7 % glycerol, 1.7 M urea, and 2 mM CaCl2 in 20 mM Tris, pH 8.5) and the reconstitution of heme to the refolded peroxidase was detected by the Soret absorbance. Additionally, samples under unfolding and refolding conditions were analyzed by Zetasizer to determine size distribution in different media.

Phosphate buffer effects on thermal stability and H2O2-resistance of horseradish peroxidase.

Horseradish peroxidase (HRP) has attracted intense research interest due to its potential applications in biotechnological fields. However, inadequate stability under prevalent conditions such as elevated temperatures and H(2)O(2) exposure, has limited its industrial application. In this study, stability of HRP was investigated in the presence of different buffer systems (potassium phosphate and Tris-HCl) and additives. It was shown that the concentration of phosphate buffer severely affects enzyme thermostability in a way that in diluted potassium phosphate buffer (10mM) half-life (from 13 to 35 min at 80 °C) and T(m) (from 73 to 77.5 °C) increased significantly. Among additives tested, trehalose had the most thermostabilizing effect. Exploring the role of glycosylation in stabilizing effect of phosphate buffer, non-glycosylated recombinant HRP was also examined for its thermal and H(2)O(2) stability in both diluted and concentrated phosphate buffers. The recombinant enzyme was more thermally stable in diluted buffer in accordance to glycosylated HRP; but interestingly recombinant HRP showed higher H(2)O(2) tolerance in concentrated buffer.