Molecular cloning, expression and characterization of poxa1b gene from Pleurotus ostreatus.

In recent decades, fungus laccases (p-diphenol-dioxygen oxidoreductases; EC 1.10.3.2) have attracted the attention of researches due to their wide range of biotechnological and industrial applications. In the present study, we have cloned a gene encoding laccase (poxa1b) from Pleurotus ostreatus and then heterologously expressed in Escherichia coli BL21. The biochemical properties of POXA1b were characterized using ABTS as a typical substrate of laccases. Moreover, the in vitro oxidation of the benzo[a]pyrene was investigated in the presence or absence of ABTS. The codon-optimized poxa1b showed higher expression yields and efficiency in comparison with the wild-type (p < 0.01). The maximum activity of POXA1b (2075 UL-1) was observed after incubation at 50 °C for 0.5 h and the enzyme retained more than 85% of its initial activity after 2 h incubation at 25-45 °C. The optimum pH of the enzyme was pH4 and the enzyme was stable when being incubated at pH range from 2.5 to 4.5 for 2 h in the absence of ABTS, the enzyme oxidized a little amount of benzo[a]pyrene, whereas its oxidation enhanced following the ABTS addition. These findings indicate POXA1b of P. ostreatus as a promising candidate for further biotechnological approaches.

Investigation of a robust pretreatment technique based on ultrasound-assisted, cost-effective ionic liquid for enhancing saccharification and bioethanol production from wheat straw.

Application of cost-effective pretreatment of wheat straw is an important stage for massive bioethanol production. A new approach is aimed to enhance the pretreatment of wheat straw by using low-cost ionic liquid [TEA][HSO4] coupled with ultrasound irradiation. The pretreatment was conducted both at room temperature and at 130 °C with a high biomass loading rate of 20% and 20% wt water assisted by ultrasound at 100 W-24 kHz for 15 and 30 min. Wheat straw pretreated at 130 °C for 15 and 30 min had high delignification rates of 67.8% and 74.9%, respectively, and hemicellulose removal rates of 47.0% and 52.2%. Moreover, this pretreatment resulted in producing total reducing sugars of 24.5 and 32.1 mg/mL in enzymatic saccharification, respectively, which corresponds to saccharification yields of 67.7% and 79.8% with commercial cellulase enzyme CelluMax for 72 h. The ethanol generation rates of 38.9 and 42.0 g/L were attained for pretreated samples for 15 and 30 min, equivalent to the yields of 76.1% and 82.2% of the maximum theoretical yield following 48 h of fermentation. This demonstration provided a cheap and promising pretreatment technology in terms of efficiency and shortening the pretreatment time based on applying low-cost ionic liquid and efficient ultrasound pretreatment techniques, which facilitated the feasibility of this approach and could further develop the future of biorefinery.

Lipid Production Capacity of a Newly Characterized Cyanobacterial Strain Synechocystissp. MH01: A Comparative Performance Evaluation of Cyanobacterial Lipid-Based Biodiesel.

BACKGROUND: Cyanobacteria have been the focus of extensive researches because of their high potential for the development of new generations of useful natural compounds with vast applications. For the entire last ten years, a lot of attention has been dedicated to the cyanobacterial lipids as a main source of valuable materials for clean energy production. OBJECTIVES: As there is a direct relationship between biofuel properties and compositional characteristics of fatty acids, a selected lipid-producing cyanobacterial strain was examined and analyzed in terms of fatty acid composition. The biodiesel quality parameters were carefully examined as well. MATERIALS AND METHODS: A cyanobacterial strain was isolated from waterfalls in the northern part of Iran and identified as Synechocystis sp. MH01. The fatty acids profile of the selected strain, as tested in various culture conditions, was analyzed by gas chromatography (GC) and compared with control subjects to further validating the biodiesel quality parameters. RESULTS: The autotrophic cultivation of Synechocystissp. MH01 resulted in biomass and lipid productivity of 109 mg.L-1 day-1 and 22.89 mg.L-1 day-1, respectively. The mixotrophic cultivation of MH01 strain in sucrose-containing medium led to an approximately 1.8 and 1.22 fold increase in biomass and lipid productivity compared with the autotrophic condition. The addition of glycine to BG11 medium caused up to ~1.3 and ~1.18 fold increase in biomass and lipid productivity compared with control subjects. The analysis of qualitative parameters of the biodiesel, as derived from the lipids, indicated that Synechocystis sp. MH01 has a high ability for lipid production under optimal culture conditions. CONCLUSIONS: It seems feasible to evolve the Synechocystissp. MH01 further particularly for more lipid production as a promising primary raw material for biofuel production through fine-tuning of medium composition.

Growth optimization and identification of an ω-transaminase by a novel native PAGE activity staining method in a Bacillus sp. strain BaH isolated from Iranian soil.

ω-Transaminases' (ω-TAs) importance for synthesizing chiral amines led to the development of different methods to quickly identify and characterize new sources of these enzymes. Here we describe the optimization of growth and induction of such an enzyme in a wild type strain of Bacillus sp. strain BaH (IBRC-M 11337) isolated from Iranian soil in shaking flasks by the response surface methodology (RSM). Optimum conditions were set in a multiplexed bench-top bioreactor system (Sixfors). ω-TA activity of obtained biomass was checked by an innovative efficient colorimetric assay for localizing ω-TAs in crude extracts on acrylamide gel by using ortho-xylylenediamine (OXD) as amino donor. The application of the established OXD assay is thereby expanded from high-throughput activity screenings and colony-based screenings of heterologously expressed mutants to a direct identification of ω-TAs in wild-type strains: This assay can be used to detect the protein band of the respective enzyme in crude extracts of novel isolates by visual inspection of native PAGEs without any upstream protein purification, thus enabling subsequent further investigations of a newly discovered enzyme directly from the crude extract.

A novel thermostable alkaline histamine oxidase from Glutamicibacter sp. N1A3101, induced by histamine and its analogue betahistine.

Biogenic amines (BAs) are low molecular weight organic bases formed by natural amino acids decarboxylation and trigger an array of toxicological effects in humans and animals. Bacterial amine oxidases enzymes are determined as practical tools to implement the rapid quantification of BAs in foods. Our study set out to obtain a new efficient, amine oxidase enzyme for developing new enzyme-based quantification of histamine. The soils from different sources were screened using histamine as sole carbon and nitrogen sources, and histamine oxidase producing bacteria were selected and identified using specific primers for histamine oxidase (HOD) gene. The HOD gene of six strains, out of 26 isolated histamine-utilizing bacteria, were amplified using our designed primers. The HOD enzyme from Glutamicibacter sp. N1A3101, isolated from nettle soil, was found to be thermostable and showed the highest substrate specificity toward the histamine and with no detected activity in the presence of putrescine, cadaverine, spermine, and spermidine. Its oxidation activity toward tyramine was lower than other HOD reported so far. The isolated enzyme was stable at 60 °C for 30 min and showed pH stability ranging from 6 to 9. Furthermore, we indicated the induction of identified HOD activity in the presence of betahistine as well, with nearly equal efficiency and without the consumption of the substrate.

Introducing a thermotolerant Gluconobacter japonicus strain, potentially useful for coenzyme Q10 production.

In this report, Gluconobacter strains were screened for coenzyme Q10 (CoQ10) production. A thermotolerant strain, Gluconobacter japonicus FM10, was eventually employed for CoQ10 production optimization. To do so, a two-step optimization strategy was used. The first step focused on biomass increase and the second step focused on increase in CoQ10 production. Factors including temperature, pH, carbon, and nitrogen sources were optimized at the first step, and temperature, pH, and aeration were optimized at the second step. The batch culture fermentation was used with the optimized factors of the first phase (30 °C, pH 6.5, D-sorbitol, and yeast extract-peptone as the carbon and nitrogen sources). After 18 h, the temperature, pH, and aeration were shifted to the optimized values of the second step (36 °C, pH 7, and no aeration). By this strategy, the dry cell mass (17.1 g/L) and CoQ10 (23.2 mg/L) were obtained after 20 h, which the latter was 2.3 times higher than that of the first step of optimization. Among the conditions tested, carbon source was the most important factor on the cell growth at the first step while no aeration was the key factor for CoQ10 production in the second step of optimization.

Molecular mechanism of enzyme tolerance against organic solvents: Insights from molecular dynamics simulation.

Biocatalysis in presence of organic solvents has numerous industrially attractive advantages in comparison to traditional aqueous solvents. In some cases, the presence of organic molecules such as methanol in the processes such as enzymatic production of biodiesel is inevitable. However, enzyme inactivation and/or instability in organic solvents limits such biotechnological processes. Although it was found that some enzymes are more and others are less tolerant against organic solvents, the structural basis of such differences is relatively unknown. In this work, using molecular dynamics simulations, we have investigated the structural behavior of enzymes with completely different structural architecture including lipase, laccase and lysozyme in the presence of methanol as polar and hexane as non-polar organic solvents. In agreement with the previous experimental observations, simulations showed that lipase is more tolerant against both polar and non-polar organic solvents. It is found that lipase has high stability in pure hexane even higher than that obtained in the aqueous solvent. In contrast, laccase shows better stability in the aqueous conditions. To obtain general mechanism of enzyme inactivation in the presence of methanol and hexane, we have treated lysozyme as model enzyme in the different percentages of these solvents in long MD simulations. It is found that lysozyme is completely denatured at high concentration- of methanol, but it remains native at low concentration of this solvent. Interestingly, the concentration-dependence structural behavior of enzyme was completely different in the presence of hexane. It was obtained that low concentrations of hexane may impose more instability on the enzyme conformation than higher percentages. Results also showed that presence of water is determining factor in the enzyme stability at high concentrations of hexane. Pure hexane may also lead to the surface denaturation of the enzymes. Both methanol and hexane denaturation mechanisms were initiated by diffusion of organic solvent in hydrophobic core. However, enzyme denaturation in hexane was continued by a collapse of hydrophobic core and entering hexane molecules to the core, but in methanol it was completed by decomposition of the secondary structures. In both cases it was found that beta structures are more prone to destabilize than helix structures. This may be a reason for obtained results about lower stability of laccase with ß-barrel architecture than lipase with multiple helixes at it surface. In total, by our extensive structural data, it was found that the forces which stabilize tertiary structure have pivotal role in enzyme tolerance against both polar and non-polar organic solvents.

Preliminary Study on Cost-Effective L-Tryptophan Production from Indole and L-Serine by E. coli Cells.

BACKGROUND: L-tryptophan is used widespread in the pharmaceutical industry. The majority of L-Trp production depends on microbial processes that produce L-tryptophan from indole and L-serine. These processes are very costly due to the costs of precursors, especially L-serine. Use of inexpensive substitutions as the L-serine source of L-tryptophan production enables us to reach a cost-effective process. In this paper, effect of Triton X-100 on L-Trp production and the ability to use Iranian cane molasses as inexpensive L-serine source was investigated. METHODS: Escherichia coli (E. coli) ATCC 11303 cells were grown in 10-L fermenter containing minimal medium supplemented with beet molasses as an inexpensive carbon source and indole as tryptophan synthase inducer. Whole cells of stationary phase were used as biocatalyst for L-Trp production. Triton X-100 addition to the production medium as indole reservoir was investigated. Then, cane molasses was used as L-Ser source in L-Trp production medium. Amount of L-Tryptophan and theoretical yield of L-Trp production was determined by HPLC and by a colorimetrically method on the basis of remaining indole assay, respectively. RESULTS: As a result, triton X-100 increased L-Trp production three times. Also, the result showed that 0.68 mM L-Tryptophan was produced in the presence of cane molasses at 37°C for 8 hr. CONCLUSION: This result showed that cane molasses of Qazvin sugar factory includes significant amounts of L-Ser that makes it a suitable substitution for L-Ser in L-Trp production. Therefore, it has the potential to be used for cost-effective L-Trp production in industrial scale.

Optimization of L-Tryptophan Biosynthesis From L-Serine of Processed Iranian Beet and Cane Molasses and Indole by Induced Escherichia coli ATCC 11303 Cells.

BACKGROUND: L-tryptophan is an important ingredient in medicines, especially in neuromedicines such as antidepressants. Many commercial processes employ various microorganisms with high tryptophan synthase activity to produce L-tryptophan from indole and L-serine, but these processes are very costly due to the costs of precursors, especially L-serine. OBJECTIVES: For this reason, we studied the ability to use processed Iranian cane and beet molasses as L-serine sources for L-tryptophan production, which enables us to reach a cost-effective process. MATERIALS AND METHODS: Whole cells of Escherichia coli ATCC 11303 were induced for L-tryptophan synthase by addition of indole to the growth medium and bacterial cells harvested from the growth medium were used as biocatalysts in the production medium. Conditions of the production medium were optimized and Iranian cane and beet molasses were processed by solvent extraction with ethanol and n-butanol and used as L-serine sources of the production medium. Amount of L-tryptophan and theoretical yield of L-tryptophan production were determined by High Performance Liquid Chromatography and by a colorimetrical method on the basis of the remaining indole assay, respectively. RESULTS: L-tryptophan production increased by 15 folds, when indole was used as an inducer. L-tryptophan was produced from processed Iranian beet molasses in satisfactory amounts (0.53 mM) and no exogenous pyridoxal phosphate was required as a cofactor under our experimental conditions. CONCLUSIONS: The obtained results proved that Iranian beet molasses include significant amounts of L-serine that makes them a suitable substitution for L-serine. Findings of the present study give impetus to use of Iranian beet molasses for cost-effective L-Trp production in the amino acid industry.

First report of a lipopeptide biosurfactant from thermophilic bacterium Aneurinibacillus thermoaerophilus MK01 newly isolated from municipal landfill site.

A biosurfactant-producing thermophile was isolated from the Kahrizak landfill of Tehran and identified as a bacterium belonging to the genus Aneurinibacillus. A thermostable lipopeptide-type biosurfactant was purified from the culture medium of this bacterium and showed stability in the temperature range of 20-90 °C and pH range of 5-10. The produced biosurfactant could reduce the surface tension of water from 72 to 43 mN/m with a CMC of 1.21 mg/mL. The strain growing at a temperature of 45 °C produces a substantial amount of 5 g/L of biosurfactant in the medium supplemented with sunflower oil as the sole carbon source. Response surface methodology was employed to optimize the biosurfactant production using sunflower oil, sodium nitrate, and yeast extract as variables. The optimization resulted in 6.75 g/L biosurfactant production, i.e., 35% improved as compared to the unoptimized condition. Thin-layer chromatography, FTIR spectroscopy, 1H-NMR spectroscopy, and biochemical composition analysis confirmed the lipopeptide structure of the biosurfactant.

Cane molasses as a source of precursors in the bioproduction of tryptophan by Bacillus subtilis.

BACKGROUND AND OBJECTIVES: The essential amino acid L-tryptophan can be produced by a condensation reaction between indole and L-serine, catalyzed by B. subtilis with tryptophan synthase activity. Application of the tryptophan is widespread in the biotechnology domain and is sometimes added to feed products as a food fortifier. MATERIALS AND METHODS: The optimum concentration of the Iranian cane molasses was determined by measuring the amount of biomass after growth in 1 to 30 g/mL of molasses. The maximum amount of biomass was obtained in 10 g/mL molasses. Chromatographic methods, TLC and HPLC, were used to assay the amount of tryptophan produced in the presence of precursors of tryptophan production (indole and serine) and/or molasses. RESULTS: Our results indicate the importance of the Iranian cane molasses not only as carbon source, but also as a source of precursors for tryptophan production. CONCLUSION: This report evaluates the potential of cane molasses as an economical source for tryptophan production by B. subtilis, hence eliminating the requirement for additional serine and indole as precursors.

Application of biotransformation in flavor and fragrance industry.

The present study describes the isolation of microorganisms capable of producing alpha-pinene from beta-pinene. 24 (15 fungi, 9 bacteria) microorganisms were isolated from galbanum, gum and soil, were screened for their ability to transform beta-pinene to alpha-pinene. Biotransformation products were extracted with n-hexan and analyzed by gas chromatography. One microorganism (bacterial strain) were found. The biotransformation medium involved, phosphate buffer pH 6, 100 microL beta-pinene, 1 g biomass of microorganism, 37 degrees C, 150 rpm and 22 h. The experiments were performed in conical flasks. The optimum cell growth were obtained when 30 g L(-1) glycerin applied. The optimum conversion beta-pinene to alpha-pinene was obtained when 20 g L(-1) glycerin applied as carbon source for bacterial strain.