In this article, we report the development and optimization of an industrial culture medium for the production of extracellular
lipase in the yeast Yarrowia lipolytica. Until now olive oil in combination with glucose was used as the carbon source and inducer for the production of lipase.
Our results demonstrate that methyloleate, a cheap hydrophobic compound, could efficiently substitute olive oil as the inducer
and carbon source for lipase production. A new process of lipase production was developed yielding a twofold increase in the
level of production compared with the levels in previous reports. 相似文献
Bacterial strains were selected from a desiccated polluted soil for their drought tolerance and their ability to grow on diesel
oil in view of incorporating them in a bioaugmentation product. These products are useful in case of recal citrant xenobiotic
pollution, where there is no intrinsic biodegradation activity in the soil. These strains grow on the easily degradable components
of diesel oil. In troduction of new catabolic genes into these desiccation-tolerant bacteria in order to improve their catabolic
functions was considered.
Plasmid-borne catabolic genes coding for enzymes in volved in the degradation of more recalcitrant compounds (Isopropylbenzene,
trichloroethene, 3-chloroben zoate, 4-chlorobiphenyl, biphenyl) were successfully introduced in some of the desiccation-tolerant
strains by means of natural conjugation. Strains exhibiting good tolerance to desiccation and able to grow on the new carbon
sources were obtained. The frequencies of integration of the plasmids ranged from 2×10−8 to 9.2 10−2 transconjugants/acceptor.
Drought-tolerance is indeed important for bioaugmentation because of its in trinsic ecological significance and because a
bioaugmentation starter has to be conditioned in a desic cated form to ensure good shelf-life. The conservation of the properties
during storage was evaluated by accelerated storage tests. 相似文献
In previous publications, we described the continuous production of D-fructose from enzymatic hydrolysis of inulin with immobilized permeabiliized cells ofKluyveromyces marxianus and the increase of productivity obtained by using a mutant selected by NTG action on the wild-type strain. By improving reactor performance, it has been possible to reach 2000 g/L/d of liberated sugar from an inulin solution with the mutant strain.
In addition, it has been shown that the KF 28 mutant was an invertase and pectinase hyperproducer. These enzymatic activities are also secreted by the speciesKluyveromyces marxianus. Therefore, we investigated the possibility of using immobilized cells of this yeast as a multipotential hydrolysis reactor. A sucrose hydrolysis reactor and a pectin hydrolysis reactor were set up. It is shown here that the majority of the optimized parameters from the inulin hydrolysis reactor can be transported directly to the other reactors. However, some parameters have to be adapted, especially for pectin hydrolysis.
Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular
lipase production. In this work, the development of a fed-batch process for the production of this enzyme in this strain was
described. A lipolytic activity of 2,145 U/mL was obtained after 32 h of batch culture in a defined medium supplemented with
10 g/L of tryptone, an enhancer of lipase expression. To maximize the volumetric productivity, two different fed-batch strategies
had been investigated. In comparison to batch process, the intermittent fed-batch strategy had not improved the volumetric
lipase productivity. In contrast, the stepwise feeding strategy combined with uncoupled cell growth and lipase production
phases resulted in a 2-fold increase in the volumetric lipase productivity, namely, the lipase activity reached 10,000 U/mL
after 80 h of culture. Furthermore, this lipase was purified to homogeneity by anion exchange chromatography on MonoQ resin
followed by gel filtration on Sephacryl S-100. This process resulted in an overall yield of 72% and a 3.5-fold increase of
the specific lipase activity. The developed process offers a great potential for an economic production of Lip2 at large scale
in Y. lipolytica LgX64.81. 相似文献
The thermodependence of growth kinetic parameters was investigated for the Antarctic psychrophilic strain Rhodotorula aurantiaca and a psychrotrophic strain of the same species isolated in Belgium (Ardennes area). Cell production, maximum growth rate
(μmax), and half-saturation constant for glucose uptake (Ks) of both yeasts were temperature dependent. For the two yeasts, a maximum
cell production was observed at about 0°C, and cell production decreased when temperature increased. The μmax values for both strains increased with temperature up to a maximum of 10°C for the psychrophilic strain and 17°C for the
psychrotrophic strain. For both yeasts, Ks for glucose was relatively constant at low temperatures. It increased at temperatures
above 10°C for the psychrophilic strain and 17°C for the psychrotrophic strain. Although its glucose affinity was lower, the
psychrotrophic strain grew more rapidly than the psychrophilicone. The difference in growth rate and substrate affinity was
related to the origin of the strain and the adaptation strategy of R. aurantiaca to environmental conditions. 相似文献
Biological sulfate reduction was investigated at the bench and pilot scales in order to determine optimum culture conditions. Efficient strains of sulfate-reducing bacteria (SRB) were selected by classical microbiological methods and by mutagenesis. Improvement factors, including stripping, scale-up, sulfate, and organic substrate concentrations, have been studied in batch bioreactors.
Two types of pilot-scale bioreactors have been adopted, the first being completely mixed with free cells and the second having two stages with immobilized cells on a fixed bed. An overall bioconversion capacity of 11 kg/m3·d of gypsum and 1.2 kg/m3·d of dissolved organic carbon has been achieved in the two-stage bioreactor.
The thermoresistance of a cold-adapted subtilisin dried by spray-drying was studied. Proteolytic activity of this enzyme was
measured before and after spray-drying. Without chemical additives, spray-drying yields ranged from 2–13%. The use of arabic
gum and lactose in the composition of the enzyme solutions allowed the strengthening of the enzyme structures and increased
water mobility in the product. Increase of water mobility led to a shorter residence time of the product in the spray-drier
and a net yield increase was obtained (yield higher than 50%). The effect of two selective mutations on the thermoresistance
to spray-drying of the cold-adapted subtilisin was also investigated. Mutation T85D (introduction of an additional link with
an ion Ca2+ necessary for enzyme activity, by substitution of Asp for Thr 85) had no effect on the thermoresistance of the subtilisin
to spray-drying. Mutation H121W (introduction of an additional aromatic link by substitution of Trp for His 121) reduced the
drying yield from 66% (not modified subtilisin) to 52%. This higher thermosensitivity could be explained by an increase of
the hygroscopic character of the modified subtilisin (mutation H121W). 相似文献
Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the
other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose
chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native Mr value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD+ less effectively than NADP+. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of
NAD+ or NADP+. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation
reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD+ and NADP+ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase
activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD+ or NADP+. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases. 相似文献
To gain insight into the interactions between fengycin and skin membrane lipids, mixed fengycin/ceramide monolayers were investigated using atomic force microscopy (AFM) (monolayers supported on mica) and surface pressure-area isotherms (monolayers at the air-water interface). AFM topographic images revealed phase separation in mixed monolayers prepared at 20 degrees C/pH 2 and composed of 0.25 and 0.5 fengycin molar ratios, in the form of two-dimensional (2-D) hexagonal crystalline domains of ceramide surrounded by a fengycin-enriched fluid phase. Surface pressure-area isotherms as well as friction and adhesion AFM images confirmed that the two phases had different molecular orientations: while ceramide formed a highly ordered phase with crystalline chain packing, fengycin exhibited a disordered fluid phase with the peptide ring lying horizontally on the substrate. Increasing the temperature and pH to values corresponding to the skin parameters, i.e., 37 degrees C/pH 5, was found to dramatically affect the film organization. At low fengycin molar ratio (0.25), the hexagonal ceramide domains transformed into round domains, while at higher ratio (0.5) these were shown to melt into a continuous fengycin/ceramide fluid phase. These observations were directly supported by the thermodynamic analysis (deviation from the additivity rule, excess of free energy) of the monolayer properties at the air-water interface. Accordingly, this study demonstrates that both the environmental conditions (temperature, pH) and fengycin concentration influence the molecular organization of mixed fengycin/ceramide monolayers. We believe that the ability to modulate the formation of 2-D domains in the skin membrane may be an important biological function of fengycin, which should be increasingly investigated in future pharmacological research. 相似文献