Biodesulfurisation of DBT with Pseudomonas putida CECT5279 by resting cells: Influence of cell growth time on reducing equivalent concentration and HpaC activity

Dibenzothiophene (DBT) biodesulfurisation (BDS) route using a genetically modified organism, Pseudomonas putida CECT 5279, is studied. Tests of BDS with whole cells and with homogenized cells are carried out by taking samples of the cells during growth. The influence of the growth phases in the evolution of the intermediates of the 4S DBT desulfurising route is shown.Conversions of the five key compounds of the 4S route (DBT, DBTO, DBTO₂, HBPS and HBP) are measured. DBT conversion values are maximal with cells obtained after 30 h of growth time. HBP conversion values do not coincide with DBT conversion values, the maximum HBP production is obtained with cells grown for 10 h. A greater intermediate DBTO and DBTO₂ accumulation in broth is produced with cells obtained at 5 and 10 h of growth time. Nevertheless, the accumulation in broth of HBPS, another intermediate, is considerably lower than that observed with cells obtained at 23, 30 and 45 h of growth time.Also, the concentration of the reducing equivalents (NADH and FMNH₂) and flavin-oxido-reductase activity inside the cells is measured. This showed that the concentration of the reducing equivalents and the activity of the HpaC enzyme in the P. putida cytoplasm do not limit BDS rate.The influence of 4S compound transport across cellular membrane is studied by comparison of results obtained by resting cell assays (whole cells) and with homogenized cells assays (disrupted cells). The results show that there is no accumulation of any compound inside the cells, and that the transport rate across the cellular membrane does not limit the overall biodesulfurisation rate.     

Desulfurization of dibenzothiophene using the 4S enzymatic route: Influence of operational conditions on initial reaction rates

The influence of operational conditions (pH, temperature and oxygen transfer rate) on the initial reaction rates of the four reactions involved in the 4S biodesulfurization route of dibenzothiophenes (DBT) has been studied. The bioprocess was carried out using a genetically modified organism, Pseudomonas putida CECT 5279. The rates of the four reactions were calculated from the rates of production of different compounds involved in the 4S pathway, by matrix manipulation. The initial (zero time) reaction rates showed a slight dependence on oxygen transfer rate. Temperature and pH were optimal at 30°C and 9, respectively, temperature being the most important variable. This study also identifies the last reaction as the limiting step in the pathway.     

Desulfurization of dibenzothiophene by lyophilized cells of Pseudomonas delafieldii R-8 in the presence of dodecane

Several parameters that influence the dibenzothiophene (DBT) desulfurization by lyophilized cells of Pseudomonas delafieldii R-8 were studied in the presence of dodecane. The aqueous media tested with pH range in 4.6–8.5 made no obvious difference on the desulfurization activity. The rate and extent of desulfurization were strongly dependent on the volume ratio of oil-to-water, DBT concentration and the cell concentration. The specific desulfurization rate of DBT and 4,6-dimethyl DBT (4,6-DMDBT) could reach 11.4 and 9.4 mmol sulfur kg⁻¹ dry cells (DCW) h⁻¹, respectively. The desulfurization pattern of DBT was represented by the Michaelis–Menten equation. The kinetic parameters, the limiting maximal velocity (V_max) and Michaelis constant (K_m), for desulfurization of DBT were calculated.     

Influence of inoculation with plant growth promoting rhizobacteria (PGPR) on tomato plant growth and nematode reproduction under greenhouse conditions

Numerous species of soil bacteria which flourish in the rhizosphere of plants or around plant tissues stimulate plant growth and reduce nematode population by antagonistic behavior. These bacteria are collectively known as PGPR (plant growth promoting rhizobacteria). The effects of six isolates of PGPR Pseudomonas putida, Pseudomonas fluorescens, Serratia marcescens, Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus cereus, were studied on tomato plant growth and root knot nematode reproduction after 45 days from nematode infection. The highest number of shoot dry weight/g (43.00 g) was detected in the plant treated with S. marcescens; then P. putida (34.33 g), B. amyloliquefaciens (31.66 g), P. fluorescens (30.0 g), B. subtilis (29.0 g), B. cereus (27.0 g) and nematode alone (untreated) 20 g/plant. While the highest number of plant height was observed when plant was treated with S. marcescens, P. fluorescens, P. putida, B. amyloliquefaciens and P. putida 52.66, 50.66, 48 and 48 cm respectively. No significant differences were seen between previous treatments but only had significant differences compared with untreated plant. The highest number of fruit/plant was observed when plants were treated with S. marcescens (10.66), then B. amyloliquefaciens (8.66), P. putida (8), P. fluorescens (8) and B. cereus (7.66). No significant differences between the last 4 treatments, but all had significant differences compared with untreated plants. The highest weight of plant yield (g) was observed with S. marcescens (319.6 g/plant) and the lowest weight of plant yield was observed in plants treated with nematode alone (untreated). On the other hand, the lowest numbers of J₂/10 g of soil (78), galls/root, (24.33) galls/root, egg masses/root (12.66) and egg/egg masses were observed in the plants treated with S. marcescens.     

Purification and characterization of a flavin reductase from the biodesulfurizing bacterium Mycobacterium goodii X7B

Dibenzothiophene (DBT) in fossil fuels can be efficiently biodesulfurized by a thermophilic bacterium Mycobacterium goodii X7B. Flavin reductase DszD, which catalyzes the reduction of oxidated flavin by NAD(P)H, is indispensable for the biodesulfurization process. In this work, a flavin reductase DszD in M. goodii X7B was purified to homogeneity, and then its encoding gene dszD was amplified and expressed in Escherichia coli. DszD is a homodimer with each subunit binding one FMN as cofactor. The K_m values for FMN and NADH of the purified recombinant DszD were determined to be 6.6 ± 0.3 μM and 77.9 ± 5.4 μM, respectively. The optimal temperature for DszD activity was 55 °C. DszD can use FMN or FAD as substrate to generate FMNH₂ or FADH₂ as product. DszD was coexpressed with DBT monooxygenase DszC, the enzyme catalyzing the first step of the biodesulfurization process. It was indicated that the coexpressed DszD could effectively enhance the DszC catalyzed DBT desulfurization reaction.     

Comparative assessment of in situ bioremediation potential of cadmium resistant acidophilic Pseudomonas putida 62BN and alkalophilic Pseudomonas monteilli 97AN strains on soybean

A study was undertaken to examine the effects of the acidophilic strain 62BN (pH 5.5) and alkalophilic strain 97AN (pH 9.0) on remediation of cadmium and their subsequent effects on soybean (Glycine max var. PS-1347) in acidic and alkaline soils, respectively. The effect of cadmium on soybean plants was studied in acidic (pH 6.3 ± 0.2) and alkaline (8.5 ± 0.2) soil amended with 124 μM CdCl₂ concentration, respectively, and the cadmium toxicity was evident from stunted growth, poor rooting, and cadmium accumulation in each case. Furthermore, 16S ribosomal DNA (rDNA) sequencing identified 62BN as a Pseudomonas putida strain and 97AN as a Pseudomonas monteilli strain. In situ studies showed that on seed bacterization, both the P. putida 62BN strain and P. monteilli 97AN strain were able to enhance plant growth in terms of agronomical parameters, in the presence of cadmium in acidic and alkaline soils, respectively. Apart from this, strain 62BN and 97AN reduced cadmium concentration in plant and soil significantly (p < 0.05) in their respective soil types. Further comparative analysis revealed that P. putida 62BN was more effective than P. monteilli 97AN strain in remediation of cadmium. The bacterial strains offer promise as inoculants to improve the growth of plants in the presence of toxic Cd concentrations in the environment with their optimum pH.     

Vibrio aestivus sp. nov. and Vibrio quintilis sp. nov., related to Marisflavi and Gazogenes clades,respectively

Two new Vibrio species, Vibrio aestivus and Vibrio quintilis, are described after a polyphasic characterization of strains M22^T, M61 and M62^T, isolated from seawater collected off a beach on the East coast of Spain (Valencia). All three strains are Gram negative, mesophilic, slightly halophilic, fermentative rods. V. aestivus (M22^T = CECT 7558^T = CAIM 1861^T = KCTC 23860^T and M61 = CECT 7559 = CAIM 1862 = KCTC 23861) is oxidase positive, reduces nitrates to nitrites, is negative for Voges Proskauer, arginine dihydrolase and indole and non hydrolytic on most substrates tested. The 16S rRNA gene sequences of M22^T and M61 are most similar to Vibrio marisflavi (97.1–97.2%) but phylogenetic analysis using NJ, MP and ML methods display Vibrio stylophorae (96.2% similarity) as sibling species. The three species form a deep clade in the genus Vibrio. Average Nucleotide Identity (ANI) values, determined as a measure of overall genomic resemblance, confirmed that strains M22^T and M61 are members of the same species, different to V. marisflavi CECT 7928^T.V. quintilis (M62^T = CECT 7734^T = CAIM 1863^T = KCTC 23833^T) is aerogenic, arginine dihydrolase and Voges Proskauer positive, oxidase negative and unable to reduce nitrate, traits shared by most species in the Gazogenes clade. It is unpigmented and does not grow on TCBS Agar. 16S rRNA gene similarities to its nearest species, Vibrio aerogenes and Vibrio mangrovi, are 97.6% and 96.0% respectively. Strain M62^T and V. aerogenes CECT 7868^T display ANI values well below the 95% boundary for genomic species.     

Detection and quantification of 4-substituted phenols: a comparison of mushroom tyrosinase and cell extracts of Pseudomonas putida F6

A comparison of the spectrophotometric detection and quantification of a number of 4-substituted phenols by two sources of the enzyme tyrosinase (Agaricus bisporus (mushroom) versus Pseudomonas putida) is described. Incubation of either source of tyrosinase with selected 4-substituted phenols results in the formation of coloured products that absorb light maximally within a narrow wavelength range (400–423 nm). The inclusion of the nucleophile 3-methyl-2-benzothiazolinone (MBTH) in the tyrosinase assay results in more intensely coloured products that also absorb light within a narrow wavelength range (440–475 nm). The molar extinction coefficient of the reaction products in the tyrosinase and tyrosinase–MBTH assay differed dramatically with values between 714–1580 and 14213–26563 M⁻¹ cm⁻¹, respectively. The addition of MBTH improved the sensitivity of the reaction between 1.3- and 100-fold, depending on the substrate and source of the enzyme. The limit of detection of 4-substituted phenols also varied according to substrate and the source of enzyme used in the assay. The lowest detectable concentration of 4-substituted phenol was 2.5 μM 4-hydroxyphenoxy acetic acid in the presence of mushroom tyrosinase and MBTH and 2.5 μM 2-(4-hydroxyphenyl) ethanol in the presence of cell extract of P. putida F6 and MBTH.     

Development of a microtitre plate method for determination of phenol utilization,biofilm formation and respiratory activity by environmental bacterial isolates

Twenty-five aerobic phenol-degrading bacteria, isolated from different environmental samples on phenol agar after several subcultures in phenol broth, utilized phenol (0.2 g l⁻¹) within 24 h, but removal of phenol was more rapid when other carbon sources were also present. A microtitre plate method was developed to determine growth rate, biofilm formation and respiratory activity of the strains isolated. Pseudomonas putida strains C5 and D6 showed maximum growth (as O.D. at 600 nm), P. putida D6 and unidentified bacterial strain M1 were more stable at high concentrations of phenol (0.8 g l⁻¹), and P. putida C5 formed the greatest amount of biofilm in 0.5 g phenol l⁻¹ medium. Measurement of dehydrogenase activity as reduction of triphenyl tetrazolium chloride supported data on growth rate and biofilm formation. The microtitre plate method provided a selective method for detection of the best phenol degrading and biofilm-forming microorganisms, and was also a rapid, convenient means of studying the effect of phenol concentration on growth rate and biofilm formation.     

Optimization and kinetic study on the synthesis of palm oil ester using Lipozyme TL IM

Enzymatic synthesis of palm oil esters (POE) was carried out via alcoholysis of palm oil (PO) and oleyl alcohol (OA) catalyzed by Lipozyme TL IM. The optimum reaction conditions were: temperature: 60 °C; enzyme load: 24.7 wt%; substrate ratio: 1:3 (PO/OA), impeller speed: 275 rpm and reaction time: 3 h. At the optimum condition, the conversion of POE was 79.54%. Reusability study showed that Lipozyme TL IM could be used for 5 cycles with conversion above 50%. The alcoholysis reaction kinetic follows the Ping-Pong Bi-Bi mechanism characterized by the V_max, K_m(PO), and K_m(OA) values of 32.7 mmol/min, 0.3147 mmol/ml and 0.9483 mmol/ml, respectively. The relationship between initial reaction rate and temperature was also established based on the Arrhenius law.     

Kinetics and mechanism of lipase catalyzed monoacylglycerols synthesis

Monoacylglycerols are increasingly used in several industrial applications as effective and cheap emulsifiers. In the present work monostearin synthesis has been studied, using lipase as a biocatalyst of the esterification reaction of stearic acid with (R,S)-1,2-O-iso-propylidene glycerol (solketal). The lipase from Candida antarctica (CaL B) was immobilized in AOT/isooctane water in oil microemulsions. Optimization of the reaction conditions have shown that the highest production (80% in 30 min) could be achieved at 40 °C, in microemulsions with relatively low water content (w_o = 8). Kinetic studies have shown that the esterification reaction of stearic acid with solketal catalyzed by CaL B occurs via the ordered bi–bi mechanism, in which inhibition by the acid was identified. Moreover, at high fixed solketal concentrations a negative cooperativity is pronounced, which means that binding of the alcohol lowers the affinity of the enzyme for binding of the acid. Values of all kinetic parameters have been determined.     

Intestinal and immunological effects of daily oral administration of Lactobacillus salivarius CECT5713 to healthy adults

There is an increasing interest in the intestinal and immunological effects of probiotics. The aim of the present study is to evaluate the tolerance and beneficial effects in healthy adults of the strain, Lactobacillus salivarius CECT5713 isolated from breast milk. A phase II, randomized, double-blinded, placebo-controlled human clinical trial was carried out in 40 healthy adults. The Probiotic group received a daily dose of 2 × 10⁸ CFU of L. salivarius CECT5713 in capsules during 4 weeks while volunteers of the control received only a placebo. Gastrointestinal and immunological parameters were analyzed. Results showed that L. salivarius CECT5713 was well tolerated and no adverse effects were detected. Consumption of the probiotic strain increased fecal lactobacilli counts (7.9 ± 0.1 vs. 7.05 ± 0.2 CFU/g feces, P = 0.001). Also, an improvement in the frequency of defecation (P = 0.04) was observed. Probiotic treatment induced significantly the percentage of NK cells and monocytes, as well as the plasmatic levels of immunoglobulins M, A and G, and the regulatory cytokine IL-10 (72.3 ± 11.7 in probiotic group vs. 27.3 ± 6.4 pg/mL in control group, P < 0.01). Thus, it can be concluded that daily administration of L. salivarius CECT5713 to healthy adults is safe and improve gut microbiota and different parameters related to immune response.     

Hydroxy functional acrylate and methacrylate monomers prepared via lipase—catalyzed transacylation reactions

Candida antarctica lipase B (CAL-B, Novozyme 435) catalyzes the transacylation of methyl acrylate and methyl methacrylate with diols and triols in 2-methyl-2-butanol at 50 °C. Under the experimental conditions, up to 70 mol% of the acyl donor methyl acrylate was converted. Methyl methacrylate is the less efficient acyl donor (up to 60 mol%) due to the higher sterical hindrance in the enzymatic transacylation. Under the reaction conditions high yields of the mono-acylated products are obtained, which contain minor amounts of bis(meth)acrylates. In addition it was observed that Novozyme 435 catalyzes regioselectively the acylation of the primary hydroxyl groups. In comparison with the chemical catalyzed route no selectivity was observed for unsubstituted diols. For substituted diols more mono-acylated product was formed in the lipase-catalyzed reaction than in the chemical catalyzed reaction.     

Synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester catalyzed by thermolysin variants with improved activity

Thermolysin is industrially used for the synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N-carbobenzoxy-l-aspartic acid (ZD) and l-phenylalanine methyl ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, I168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009;145:103–13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 °C, the molecular activity k_cat values of the variants were 1.6–3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant K_m values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 μM, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis.     

Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis

Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL⁻¹ in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.     

Analysis of Dibenzothiophene Desulfurization in a Recombinant Pseudomonas putida Strain

Biodesulfurization was monitored in a recombinant Pseudomonas putida CECT5279 strain. DszB desulfinase activity reached a sharp maximum at the early exponential phase, but it rapidly decreased at later growth phases. A model two-step resting-cell process combining sequentially P. putida cells from the late and early exponential growth phases was designed to significantly increase biodesulfurization.     

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l⁻¹) and rhamnolipids yield (4.1 g l⁻¹) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (μ) of 0.272 h⁻¹, specific product yield (q_p) of 0.318 g g⁻¹ h and volumetric productivity (P_V) of 0.024 g l⁻¹ h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l⁻¹ and surface tension as 31.2 mN m⁻¹.     

Biosynthesis of deoxynucleoside triphosphates,dCTP and dTTP: reaction mechanism and kinetics

The enzyme reaction mechanism and kinetics for biosyntheses of deoxycytidine triphosphate (dCTP) and deoxythymidine triphosphate (dTTP) from the corresponding deoxycytidine diphosphate (dCDP) and deoxythymidine diphosphate (dTDP) catalyzed by pyruvate kinase were studied. The kinetic model for the two synthetic reactions was found to follow the Bi–Bi random rapid equilibrium mechanism similar to that of the biosynthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP). Kinetic constants involved in the reactions including the maximum reaction velocity, the Michaelis–Menten constants, and the inhibition constants for dCTP and dTTP biosyntheses were experimentally determined. This enzyme reaction requires Mg²⁺ ion and the optimal Mg²⁺ concentration was also determined. The experimental results showed a good agreement with the simulation results obtained from the kinetic model developed. The kinetics of the four biosynthetic reactions for deoxynucleoside triphosphates (dNTP) including dATP, dGTP, dCTP, and dTTP from the corresponding deoxynucleoside diphosphates (dNDP) including dADP, dGDP, dCDP, and dTDP were analyzed. The results suggest that the binding kinetics of phosphoenolpyruvate (PEP) and pyruvate are similar for all four biosynthetic reactions. The affinity of the dNDP substrates to enzyme is of the same order of magnitude as the corresponding dNTP as inhibitors. The order of reactivity and substrate specificity for dNDP is dADP > dGDP > dCDP > dTDP in the pyruvate kinase (PK) reactions. The results obtained from this study can be applied to bioreactor design and production of dCTP and dTTP for biosynthesis of DNA at a significantly lower cost compared to the currently available chemical method.     

Characterization of a novel meta-fission product hydrolase from Dyella ginsengisoli LA-4

A novel meta-fission product hydrolase gene (mfphA) located in the bphX gene region of Dyella ginsengisoli LA-4 was successfully cloned and heterologously expressed in this study. The deduced amino acid sequence of MfphA showed 75% identity with the sequence of 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas putida UCC22. The results suggested that MfphA belonged to the α/β hydrolase family, which could hydrolyze the meta-fission products (MFPs) during the biodegradation process of monocyclic compounds. The His-Tag MfphA was purified, and the subunit molecular mass of MfphA was about 35.3 kDa by SDS-PAGE analysis. According to the apparent kinetic parameters, the specificity of MfphA was determined in the following order: 6-methyl-HODA > HODA > 5-methyl-HODA > 6-phenyl-HODA > 5-chloro-HODA (HODA, 2-hydroxy-6-oxohexa-2,4-dienoate). The activity of MfphA for HODA was higher than that for 6-methyl-HODA. It was shown that the maximum activity was at 70 °C, and MfphA was stable for storage at low temperature for 30 days.