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Biotransformation of caffeoyl quinic acids from green coffee extracts by Lactobacillus johnsonii NCC 533

Rachid Bel-Rhlid — 2013-05-21T00:00:00Z

The potential of Lactobacillus johnsonii NCC 533 to metabolize chlorogenic acids from green coffee extract was investigated. Two enzymes, an esterase and a hydroxycinnamate decarboxylase (HCD), were involved in this biotransformation. The complete hydrolysis of 5-caffeoylquinic acid (5-CQA) into caffeic acid (CA) by L. johnsonii esterase occurred during the first 16 h of reaction time. No dihydrocaffeic acid was identified in the reaction mixture. The decarboxylation of CA into 4-vinylcatechol (4-VC) started only when the maximum concentration of CA was reached (10 mumol/ml). CA was completely transformed into 4-VC after 48 h of incubation. No 4-vinylphenol or other derivatives could be identified in the reaction media. In this study we demonstrate the capability of L. johnsonii to transform chlorogenic acids from green coffee extract into 4-VC in two steps one pot reaction. Thus, the enzymatic potential of certain lactobacilli might be explored to generate flavor compounds from plant polyphenols.

Tailoring fungal morphology of Aspergillus niger MYA 135 by altering the hyphal morphology and the conidia adhesion capacity: biotechnological applications

Verónica Colin — 2013-05-20T00:00:00Z

Current problems of filamentous fungi fermentations and their further successful developments as microbial cell factories are dependent on control fungal morphology. In this connection, this work explored new experimental procedures in order to quantitatively check the potential of some culture conditions to induce a determined fungal morphology by altering both hyphal morphology and conidia adhesion capacity. The capacity of environmental conditions to modify hyphal morphology was evaluated by examining the influence of some culture conditions on the cell wall lytic potential of Aspergillus niger MYA 135. The relative value of the cell wall lytic potential was determined by measuring a cell wall lytic enzyme activity such as the mycelium-bound beta-N-acetyl-D-glucosaminidase (Mb-NAGase). On the other hand, the quantitative value of conidia adhesion was considered as an index of its aggregation capacity. Concerning microscopic morphology, a highly negative correlation between the hyphal growth unit length (lHGU) and the specific Mb-NAGase activity was found (r = -0.915, P < 0.001). In fact, the environment was able to induce highly branched mycelia only under those culture conditions compatible with specific Mb-NAGase values equal to or higher than 190 U gdry.wt-1. Concerning macroscopic morphology, a low conidia adhesion capacity was followed by a dispersed mycelial growth. In fact, this study showed that conidia adhesion units per ml equal to or higher than 0.50 were necessary to afford pellets formation. In addition, it was also observed that once the pellet was formed the lHGU had an important influence on its final diameter. Finally, the biotechnological significance of such results was discussed as well.

On the potential application of polar and temperate marine microalgae for EPA and DHA production

Peter Boelen — 2013-05-14T00:00:00Z

Long chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are considered essential omega-3 fatty acids in human nutrition. In marine microalgae EPA and/or DHA are allegedly involved in the regulation of membrane fluidity and thylakoid membrane functioning. The cellular content of EPA and DHA may therefore be enhanced at low temperature and irradiance conditions. As a result, polar and cold temperate marine microalgal species might potentially be suitable candidates for commercial EPA and DHA production, given their adaptation to low temperature and irradiance habitats.In the present study we investigated inter- and intraspecific EPA and DHA variability in five polar and (cold) temperate microalgae. Intraspecific EPA and DHA content did not vary significantly in an Antarctic (Chaetoceros brevis) and a temperate (Thalassiosira weissflogii) centric diatom after acclimation to a range of irradiance levels at two temperatures. Interspecific variability was investigated for two Antarctic (Chaetoceros brevis and Pyramimonas sp. (Prasinophyceae)) and three cold-temperate species (Thalassiosira weissflogii, Emiliania huxleyi (Prymnesiophyceae) and Fibrocapsa japonica (Raphidophyceae)) during exponential growth. Interspecific variability was shown to be much more important than intraspecific variability. Highest relative and absolute levels of DHA were measured in the prymnesiophyte E. huxleyi and the prasinophyte Pyramimonas sp., while levels of EPA were high in the raphidophyte F. japonica and the diatoms C. brevis and T. weissflogii. Yet, no significant differences in LC-PUFA content were found between polar and cold-temperate species. Also, EPA and DHA production rates varied strongly between species. Highest EPA production rate (174 mug L-1 day-1) was found in the Antarctic diatom Chaetoceros brevis, while DHA production was highest in the cold-temperate prymnesiophyte Emiliania huxleyi (164 mug L-1 day-1). We show that, following careful species selection, effective mass cultivation of marine microalgae for EPA and DHA production may be possible under low temperature and irradiance conditions.

Cross-linked enzyme aggregates (CLEAs) of selected lipases: a procedure for the proper calculation of their recovered activity

María Guauque Torres — 2013-05-12T00:00:00Z

In the last few years, synthesis of carrier-free immobilized biocatalysts by cross-linking of enzyme aggregates has appeared as a promising technique. Cross-linked enzyme aggregates (CLEAs) present several interesting advantages over carrier-bound immobilized enzymes, such as highly concentrated enzymatic activity, high stability of the produced superstructure, important production costs savings by the absence of a support, and the fact that no previous purification of the enzyme is needed. However, the published literature evidences that a) much specific non-systematic exploratory work is being done and, b) recovered activity calculations in CLEAs still need to be optimized. In this context, this contribution presents results of an optimized procedure for the calculation of the activity retained by CLEAs, based on the comparison of their specific activity relative to their free enzyme counterparts. The protocol implies determination of precipitable protein content in commercial enzyme preparations through precipitation with ammonium sulphate and a protein co-feeder. The identification of linear ranges of activity versus concentration/amount of protein in the test reaction is also required for proper specific activity determinations. By use of mass balances that involve the protein initially added to the synthesis medium, and the protein remaining in the supernatant and washing solutions (these last derived from activity measurements), the precipitable protein present in CLEAs is obtained, and their specific activity can be calculated. In the current contribution the described protocol was applied to CLEAs of Thermomyces lanuginosa lipase, which showed a recovered specific activity of 11.1% relative to native lipase. The approach described is simple and can easily be extended to other CLEAs and also to carrier-bound immobilized enzymes for accurate determination of their retained activity.

Organization and characterization of a biosynthetic gene cluster for bafilomycin from Streptomyces griseus DSM 2608

Jae Yoon Hwang — 2013-05-10T00:00:00Z

Streptomyces griseus DSM2608 produces bafilomycin, an antifungal plecomacrolide antibiotic. We cloned and sequenced an 87.4-kb region, including a polyketide synthase (PKS) region, methoxymalonate genes, flavensomycinate genes, and other putative regulatory genes. The 58.5kb of PKS region consisting 12 PKS modules arranged in five different PKS genes, was assumed to be responsible for the biosynthesis of plecomacrolide backbone including 16-membered macrocyclic lactone. All the modules showed high similarities with typical type I PKS genes. However, the starting module of PKS gene was confirmed to be specific for isobutyrate by sequence comparison of an acyltransferase domain. In downstream of PKS region, the genes for methoxymalonate biosynthesis were located, among which a gene for FkbH-like protein was assumed to play an important role in the production of methoxymalonyl-CoA from glyceryl-CoA. Further the genes encoding flavensomycinyl-ACP biosynthesis for the post-PKS tailoring were also found in the upstream of PKS region. By gene disruption experiments of a dehydratase domain of module 12 and an FkbH-like protein, this gene cluster was confirmed to be involved in the biosynthesis of bafilomycin.

Production of ethanol and arabitol by Debaryomyces nepalensis: influence of process parameters

Himabindu Kumdam — 2013-05-09T00:00:00Z

Debaryomyces nepalensis, osmotolerant yeast isolated from rotten apple, is known to utilize both hexoses and pentoses and produce industrially important metabolites like ethanol, xylitol and arabitol. In the present study, the effect of different growth substrates, trace elements, nitrogen concentration and initial pH on growth and formation of ethanol and arabitol were examined. Optimum conditions for maximizing the product yields were established: glucose as carbon source, an initial pH of 6.0, 6 g/L of ammonium sulphate and addition of micronutrients. Under these best suited conditions, a concentration of 11g/L of arabitol and 19 g/L of ethanol was obtained in shake flask fermentations. The fermentation was scaled up to 2.5 L bioreactor and the influence of aeration, agitation and initial substrate concentration was also determined. Under optimal conditions (150 g/L glucose, 400 rpm and 0.5 vvm) ethanol concentration reached 52 g/L, which corresponds to a yield of 0.34 g/g and volumetric productivity of 0.28 g/L/h, whereas arabitol production reached a maximum of 14 g/L with a yield and volumetric productivity of 0.1 g/g and 0.07 g/L/h respectively.

Densitometry based microassay for the determination of lipase depolymerizing activity on polyhydroxyalkanoate

Diana Ch¿ng — 2013-05-08T00:00:00Z

A novel method for the assay of polyhydroxyalkanoate (PHA)-degrading ability of triacylglycerol lipases was developed. By applying the natural affinity of lipases towards hydrophobic interfaces, a sensitive and rapid densitometry analysis for the evaluation of hydrolytic activity of lipase droplets towards PHA-coated surface was successfully carried out. We found that 12 out of 14 tested lipases which are of fungal, bacterial and animal origin were able to hydrolyze P(3HB-co-92 mol% 4HB) thin film. The patterns and opacity of the hydrolysis spots of lipases on PHA films allowed easy comparison of PHA-hydrolytic strength of lipases. Lipase from the bacterium Chromobacterium viscosum exhibited the highest PHA-degrading activity. The hydrolytic activity of lipases on water insoluble PHA, emulsified p-nitrophenyl laurate and olive oil were also compared and interestingly some lipases showed better activity when PHA was used as a substrate.

Biotechnological potential of rhizobial metabolites to enhance the performance of Bradyrhizobium spp. and Azospirillum brasilense inoculants with soybean and maize

Bettina Marks — 2013-04-17T00:00:00Z

Agricultural sustainability may represent the greatest encumbrance to increasing food production. On the other hand, as a component of sustainability, replacement of chemical fertilizers by bio-fertilizers has the potential to lower costs for farmers, to increase yields, and to mitigate greenhouse-gas emissions and pollution of water and soil. Rhizobia and plant-growth-promoting rhizobacteria (PGPR) have been broadly used in agriculture, and advances in our understanding of plant-bacteria interactions have been achieved; however, the use of signaling molecules to enhance crop performance is still modest. In this study, we evaluated the effects of concentrated metabolites (CM) from two strains of rhizobia—Bradyrhizobium diazoefficiens USDA 110T (BD1) and Rhizobium tropici CIAT 899T (RT1)—at two concentrations of active compounds (10–8 and 10–9 M)—on the performances of two major plant-microbe interactions, of Bradyrhizobium spp.-soybean (Glycine max (L.) Merr.) and Azospirillum brasilense-maize (Zea mays L.). For soybean, one greenhouse and two field experiments were performed and effects of addition of CM from the homologous and heterologous strains, and of the flavonoid genistein were investigated. For maize, three field experiments were performed to examine the effects of CM from RT1. For soybean, compared to the treatment inoculated exclusively with Bradyrhizobium, benefits were achieved with the addition of CM-BD1; at 10–9 M, grain yield was increased by an average of 4.8%. For maize, the best result was obtained with the addition of CM-RT1, also at 10–9 M, increasing grain yield by an average of 11.4%. These benefits might be related to a combination of effects attributed to secondary compounds produced by the rhizobial strains, including exopolysaccharides (EPSs), plant hormones and lipo-chitooligosaccharides (LCOs). The results emphasize the biotechnological potential of using secondary metabolites of rhizobia together with inoculants containing both rhizobia and PGPR to improve the growth and yield of grain crops.

Change in product selectivity during the production of glyceric acid from glycerol by Gluconobacter strains in the presence of methanol

Shun Sato — 2013-04-02T00:00:00Z

To enhance the value-added use of methanol-containing raw glycerol derived from biodiesel fuel production, the effect of methanol supplementation on glyceric acid (GA) production by Gluconobacter spp. was investigated. We first conducted fed-batch fermentation with Gluconobacter frateurii NBRC103465 using raw glycerol as a feeding solution. GA productivity decreased with increasing dihydroxyacetone (DHA) formation when the raw glycerol contained methanol. The results of this experiment and comparative experiments using a synthetic solution modeled after the raw glycerol indicate that the presence of methanol caused a change in the concentrations of GA and DHA, two glycerol derivatives produced during fermentation. Other Gluconobacter spp. also decreased GA production in the presence of 1% (v/v) methanol. In addition, purified membrane-bound alcohol dehydrogenase (mADH) from Gluconobacter oxydans, which is a key enzyme in GA production, showed a decrease in dehydrogenase activity toward glycerol as the methanol concentration increased. These results strongly suggest that the observed decrease in GA production by Gluconobacter spp. resulted from the methanol-induced inhibition of mADH-mediated glycerol oxidation.

Importance of the ammonia assimilation by Penicillium purpurogenum in amino derivative Monascus pigment, PP-V, production

Teppei Arai — 2013-03-28T00:00:00Z

A fungal strain, Penicillium purpurogenum IAM 15392, produced the azaphilone Monascus pigment homolog when cultured in a medium composed of soluble starch, ammonium nitrate, yeast extract, and citrate buffer, pH 5.0. One of the typical features of violet pigment PP-V [(10Z)-12- carboxyl-monascorubramine] is that pyranoid oxygen is replaced with nitrogen. In this study, we found that glutamine synthetase (glnB) and glutamate dehydrogenase (gdh1) genes were expressed in the culture conditions conducive to PP-V production. Gln and Glu both support PP-V biosynthesis, but PP-V biosynthesis was much more efficient with Gln. We determined that synthesis of Gln by glutamine synthetase from ammonium is important for PP-V production.