Kinetic model for DBT desulphurization by resting whole cells of Pseudomonas putida CECT5279

Bio-desulphurization kinetics of dibenzothiophene (DBT) using Pseudomonas putida CECT 5279, a genetically modified micro-organism (GMO), is studied. A kinetic model describing the 4S route of DBT desulphurization is proposed. Bio-desulphurization experiments have been carried out using resting whole cells of P. putida CECT 5279 obtained at different growth times as biocatalysts. The kinetic equations proposed for each reaction have been previously checked by studying each reaction of the 4S route individually, employing different substrates in different experiments. Finally, simple Michaelis–Menten kinetic equations for the three first reactions catalyzed by two mono-oxygenases (DszC and DszA) and a kinetic equation taking into account competitive inhibition due to product for the final reaction catalyzed by a desulfinase (DszB) have been adopted. DBT has been desulphurized using cells obtained at different growth times (5, 10, 23, 30 and 45 h). The overall kinetic model proposed involving the four reactions of the 4S route was fitted to all the experimental data yielding a set of kinetic parameters able to describe the system evolution. Cell age has influence on the rates of all the reactions: reactions (1), (2) and (3) present maximum rates for cell grown during 30 h, while reaction (4) shows a maximum rate for cells with around 10 h of growth time. However, affinities of each substrate and the inhibition constant of the last reaction are not influenced by the time of growth.     

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.     

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.     

Evaluation of the technical performance of three different commercial digital breast tomosynthesis systems in the clinical environment

The aim of this work was to research and evaluate the performance of three different digital breast tomosynthesis (DBT) systems in the clinical environment (Siemens Mammomat Inspiration, Hologic Selenia Dimensions, and Fujifilm Amulet Innovality). The characterization included the study of the detector, the automatic exposure control, and the resolution of DBT projections and reconstructed planes.The modulation transfer function (MTF) of the DBT projections was measured with a 1 mm thick steel edge, showing a strong anisotropy (30–40% lower MTF_0.5 frequencies in the tube travel direction). The in-plane MTF_0.5, measured with a 25 μm tungsten wire, ranges from 1.3 to 1.8 lp/mm in the tube-travel direction and between 2.4 and 3.7 lp/mm in the chest wall–nipple. In the latter direction, the MTF peak shift is more emphasized for large angular range systems (2.0 versus 1.0 lp/mm). In-depth resolution of the planes, via the full width at half maximum (FWHM) from the point spread function of a 25 μm tungsten wire, is not only influenced by angular range and yields 1.3–4.6 mm among systems. The artifact spread function from 1 mm diameter tungsten beads depends mainly on angular range, yielding two tendencies whether large (FWHM is 4.5 mm) or small (FWHM is 10 mm) angular range is used. DBT delivers per scan a mean glandular dose between 1.4 and 2.7 mGy for a 45 mm thick polymethyl methacrylate (PMMA) block.In conclusion, we have identified and analysed specific metrics that can be used for quality assurance of DBT systems.     

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.     

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.     

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.     

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.     

Integrated bioprocess for the oxidation of limonene to perillic acid with Pseudomonas putida DSM 12264

An efficient integrated bioprocess for the oxidation of limonene to perillic acid with Pseudomonas putida DSM 12264 was developed. Perillic acid is a promising candidate for natural preservation and pharmaceutical application. At elevated concentrations the monoterpenoic acid inhibits growth and biotransformation activity of P. putida DSM 12264. The maximum growth rate showed a linear decrease from μ = 1.43 h^?1 in the absence of perillic acid to complete inhibition at 165 ± 7 mM perillic acid. The maximum specific activity of limonene-transforming resting cells revealed an exponential decrease from almost 8 U/g cdw without perillic acid to <0.5 U/g cdw at >25 mM perillic acid. A method for in situ product recovery (ISPR) based on anion exchange resin was established to overcome product inhibition. A column containing a fluidized bed of Amberlite IRA 410 Cl was coupled to the bioreactor and enabled product removal by continuously recirculating the unfiltered broth through the ISPR unit. This led to a cumulative perillic acid concentration of 187 mM (31 g/L) after 7 days, which represents the highest product concentration achieved in a microbial monoterpene oxyfunctionalization so far. The ISPR approach reduced the further downstream processing steps needed which yielded a 93% pure product with a loss of 2%.     

The harmful alga Aureococcus anophagefferens utilizes 19′-butanoyloxyfucoxanthin as well as xanthophyll cycle carotenoids in acclimating to higher light intensities

Aureococcus anophagefferens is a picoplanktonic microalga that is very well adapted to growth at low nutrient and low light levels, causing devastating blooms (“brown tides”) in estuarine waters. To study the factors involved in long-term acclimation to different light intensities, cells were acclimated for a number of generations to growth under low light (20 μmol photons m^? 2 s^? 1), medium light (60 or 90 μmol photons m^? 2 s^? 1) and high light (200 μmol photons m^? 2 s^? 1), and were analyzed for their contents of xanthophyll cycle carotenoids (the D pool), fucoxanthin and its derivatives (the F pool), Chls c₂ and c₃, and fucoxanthin Chl a/c polypeptides (FCPs). Higher growth light intensities resulted in increased steady state levels of both diadinoxanthin and diatoxanthin. However, it also resulted in the conversion of a significant fraction of fucoxanthin to 19′-butanoyloxyfucoxanthin without a change in the total F pool. The increase in 19′-butanoyloxyfucoxanthin was paralleled by a decrease in the effective antenna size, determined from the slope of the change in F₀ as a function of increasing light intensity. Transfer of acclimated cultures to a higher light intensity showed that the conversion of fucoxanthin to its derivative was a relatively slow process (time-frame of hours). We suggest the replacement of fucoxanthin with the bulkier 19′-butanoyloxyfucoxanthin results in a decrease in the light-harvesting efficiency of the FCP antenna and is part of the long-term acclimative response to growth at higher light intensities.     

Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous two-phase systems

The kinetically controlled synthesis of cephalexin in aqueous two-phase systems was studied, using immobilized penicillin acylase, 7-amino 3-desacetoxycephalosporanic acid as nucleophile and phenylglycine methyl ester as acyl donor. The organic phases used were 80% (v/v) polyethyleneglycol 400 and 600 and the aqueous phase was 2.5 M (NH₄)₂SO₄. 7-amino 3-desacetoxycephalosporanic acid and cephalexin partition coefficients were determined at pH 7.4 and 7.8, at 14 °C and 20 °C. Highest partition coefficient for cephalexin was obtained for polyethyleneglycol 400–(NH₄)₂SO₄ at pH 7.4 and 20 °C, while the lowest partition coefficient for 7-amino desacetoxycephalosporanic acid was obtained in the same system at pH 7.8 and 14 °C. No significant effect of pH was observed on conversion yield and productivity of cephalexin synthesis; however, higher values were obtained with polyethyleneglycol 400 as organic phase. Higher conversion yields with both biphasic systems were obtained at the lowest temperature, where product hydrolysis was lower; volumetric productivity was higher for the fully aqueous medium (control), being higher at 20 °C. All parameters of synthesis were improved at higher substrates concentrations, obtaining conversion yields of 78.2% and 65.4%, with 60 mM 7-amino desacetoxycephalosporanic acid for the polyethyleneglycol 400–(NH₄)₂SO₄ system and the control, respectively.     

Estudios de la viabilidad y la vitalidad frente al congelado de la levadura probiótica Saccharomyces boulardii: efecto del preacondicionamiento fisiológico

The aim of this study was to evaluate the vitality and viability of the probiotic yeast Saccharomyces boulardii after freezing/thawing and the physiological preconditioning effect on these properties. The results indicate that the specific growth rate (0.3/h^?1) and biomass (2-3 × 10⁸ cells/ml) of S. boulardii obtained in flasks shaken at 28 °C and at 37 °C were similar. Batch cultures of the yeast in bioreactors using glucose or sugar-cane molasses as carbon sources, reached yields of 0.28 g biomass/g sugar consumed, after 10 h incubation at 28 °C; the same results were obtained in fed batch fermentations. On the other hand, in batch cultures, the vitality of cells recovered during the exponential growth phase was greater than the vitality of cells from the stationary phase of growth. Vitality of cells from fed-batch fermentations was similar to that of stationary growing cells from batch fermentations. Survival to freezing at –20 °C and subsequent thawing of cells from batch cultures was 0.31% for cells in exponential phase of growth and 11.5% for cells in stationary phase. Pre-treatment of this yeast in media with water activity (a_w) 0.98 increased the survival to freezing of S. boulardii cells stored at –20 °C for 2 months by 10 fold. Exposure of the yeast to media of reduced a_w and/or freezing/thawing process negatively affected cell vitality. It was concluded that stress conditions studied herein decrease vitality of S. boulardii. Besides, the yeast strain studied presented good tolerance to bile salts even at low pH values.     

ATP sensitizes H460 lung carcinoma cells to cisplatin-induced apoptosis

Platinum resistance of cancer cells may evolve due to a decrease in intracellular drug accumulation, decreased cell permeability or by an increased deactivation of the drug by glutathione (GSH). The aim of this study was (1) to investigate the effect of adenosine 5′-triphosphate (ATP) on the cytotoxicity of cisplatin in a large cell lung carcinoma cell line (H460), and (2) to examine the potential involvement of increased cisplatin uptake, GSH depletion and pyrimidine starvation by ATP in this effect. H460 cells were harvested and seeded (5% CO₂; 37 °C). Subsequently, cells were incubated with medium or ATP followed by an incubation with cisplatin. Cytotoxicity screening was analyzed by the sulforhodamine B (SRB) colorimetric assay, lactate dehydrogenase and caspase-3/7 activity. Pre-incubation for 72 h with 0.3 and 3 mM ATP strongly enhanced the anti-proliferative potency of cisplatin 2.9- and 7.6-fold, respectively. Moreover, after incubation of H460 cells with 0.3 mM ATP the intracellular platinum concentration increased, indicating increased cisplatin uptake by ATP. ATP, despite lowering the LD₅₀ of cisplatin, did not modulate GSH levels in H460 cells. ATP itself showed a biphasic effect on H460 cell growth: 0.3 mM inhibited H460 cell growth via the pyrimidine starvation effect, activation of caspase-3/7 and LDH leakage, while 3 mM ATP showed no effect on cell growth. In conclusion, ATP sensitizes the H460 cells to cisplatin-induced apoptosis. The effect of 0.3 mM ATP is not due to GSH depletion but involves increased cisplatin uptake and pyrimidine starvation due to ATP conversion to adenosine followed by cellular uptake.     

Kinetic analysis of photosynthetic growth and photohydrogen production of two strains of Rhodobacter Capsulatus

Two mutants of Rhodobacter Capsulatus (JP91 and IR3), a photosynthetic purple non-sulfur bacterium, were grown in a batch photobioreactor under illumination with 30 mmol l⁻¹ dl-lactate and 5 mmol l⁻¹ l-glutamate as carbon and nitrogen source, respectively. Bacterial growth was measured by monitoring the increase in absorbance at 660 nm. The photosynthetic growth processes under different cultivated temperatures are well fitted by a specific logistic model to analyze the kinetics of photosynthetic growth of two strains, thus the apparent growth rates (k) of these photosynthetic bacteria, the variations of cell dry weight (CDW) as well as their relationship with temperature are obtained. In present work, k is (0.1465 ± 0.0146), (0.2266 ± 0.0207) and (0.3963 ± 0.0257) h⁻¹ for JP91 and (0.1117 ± 0.0122), (0.1218 ± 0.0133) and (0.2223 ± 0.0152) h⁻¹ for IR3 at 26, 30 and 34 °C, respectively. And the difference between CDW_max and CDW₀ is (0.8997 ± 0.0097), (0.8585 ± 0.0093) and (0.9241 ± 0.0099) g l⁻¹ for JP91 and (0.8167 ± 0.0089), (0.7878 ± 0.0086) and (0.8358 ± 0.0091) g l⁻¹ for IR3 at 26, 30 and 34 °C, respectively. Also real-time monitoring of hydrogen production rates is acquired by recording the flow rates of photohydrogen for these two strains under different temperatures. The effects of temperature on the bacteria growth, hydrogen production capability and substrate conversion efficiency are discussed based on these results. The most preferment temperature, 30 °C, showed good substrate conversion efficiency of 52.7 and 68.2% for JP91 and IR3, respectively.     

Development of a recombinant Escherichia coli-based biocatalyst to enable high styrene epoxidation activity with high product yield on energy source

A highly active recombinant whole-cell biocatalyst, Escherichia coli pETAB2/pG-KJE1, was developed for the efficient production of (S)-styrene oxide from styrene. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL–GroES and DnaK–DnaJ–GrpE). The styrene monooxygenases were produced to ca. 40% of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180 U/g CDW. The high StyAB activity in turn appeared to direct cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40%. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 6.4 mmol/g dry cells/h (106 U/g dry cells) and 67 mM/h (1110 U/L_aq), respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance.     

Effects of osmotic pressure on recombinant BHK cell growth and von willebrand factor (vWF) expression

The effects of osmotic pressure were investigated on cell growth and von willebrand factor (vWF) expression in batch culture, pulse culture and adaptive culture of recombinant baby hamster kidney (rBHK) cells. Intracellular contents of some amino acids including aspartic acid, glycine, arginine, alanine, valine and serine in adaptive culture showed a significant increase with environmental osmotic pressure and became steady after 6 h adaptation. There was little change in intracellular concentrations of amino acids in a control cultivation under 330 mOsmol kg⁻¹. With the increase of osmotic pressure from 330 to 350 mOsmol kg⁻¹, the specific growth rate of rBHK cells remained kept constant. However, the growth of rBHK cells was seriously inhibited under 370 mOsmol kg⁻¹. When gradually increasing the osmotic pressure from 370 to 470 mOsmol kg⁻¹ over more than 6 h, the specific growth rate of rBHK cells could increase by 40% in comparison with that when directly increasing within the same range. High osmotic pressure hardly effected any change in the percent of both cells during G₀/G₁ phase and apoptotic cells in the cell population, but the percentage of cells during S phase in the cell population increased. Higher osmotic pressure (470 mOsmol kg⁻¹) could inhibit the expression of vWF, although at 370 mOsmol kg⁻¹ the specific production rate of vWF was 47% higher than that in 330 mOsmol kg⁻¹.     

Specific activity and viability of Nitrosomonas europaea during discontinuous and continuous fermentation

The energy conservation and number of viable cells of Nitrosomonas europaea fluctuate dramatically during cultivation. In discontinuous culture the specific activity (SA) reaches its maximum after 9 h with about 2700 nmol O₂ (mg protein)^?1 min^?1, where the highest number of viable N. europaea cells is detectable after 21 h with 2 × 10⁸ cell ml^?1. Afterwards, both SA and viable cell number immediately start to decrease. Accordingly, the exponential growth turns into a linear growth, whereby the number of viable cells permanently decreases. The exponential growth phase can be extended from about 21 to 38 h by increasing the concentration of CO₂ or trace elements. In continuous fermentation of N. europaea, SA of about 2500 nmol O₂ (mg protein)^?1 min^?1 and viable cell number of 2.5 × 10⁸ cell ml^?1 is detectable at dilution rates between 1 and 1.8 day^?1. At dilution rates below 1 day^?1, SA and number of viable cells are reduced. The minimal doubling time is 13 and 15 h during continuous and discontinuous fermentation, respectively. Consequently, cell production of N. europaea should be performed in continuous fermentation. When bacteria are grown in discontinuous systems, they should be harvested in the early exponential growth phase.     

Development of a monolith based immobilized lipase micro-reactor for biocatalytic reactions in a biphasic mobile system

This paper reports a simple method for producing macroporous silica-monoliths with controllable porosity that can be used for the immobilization of lipases to generate an active and stable micro-reactor for biocatalysis. A range of commercially available lipases has been examined using the hydrolysis reactions of 4-nitrophenyl butyrate in water–decane media. The kinetic studies performed have identified that a similar value for k_cat is obtained for the immobilized Candida antarctica lipase A (0.13 min⁻¹) and the free lipase in solution (0.12 min⁻¹) whilst the immobilized apparent Michaelis constant K_m (3.1 mM) is 12 times lower than the free lipase in solution (38 mM). A 96% conversion was obtained for the immobilized C. antarctica lipase A compared to only 23% conversion for the free lipase. The significant higher conversions obtained with the immobilized lipases were mainly attributed to the formation of a favourable biphasic system in the continuous flowing micro-reactor system, where a significant increase in the interfacial activation occurred. The immobilized C. antarctica lipase A on the monolith also exhibited improved stability, showing 64% conversion at 80 °C and 70% conversion after continuous running for 480 h, compared to 40 and 20% conversions under the same temperature and reaction time for the free lipase.