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Halbmayr E Mathiesen G Nguyen TH Maischberger T Peterbauer CK Eijsink VG Haltrich D 《Journal of agricultural and food chemistry》2008,56(12):4710-4719
This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( S?rvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry. 相似文献
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Vaaje-Kolstad G Westereng B Horn SJ Liu Z Zhai H Sørlie M Eijsink VG 《Science (New York, N.Y.)》2010,330(6001):219-222
Efficient enzymatic conversion of crystalline polysaccharides is crucial for an economically and environmentally sustainable bioeconomy but remains unfavorably inefficient. We describe an enzyme that acts on the surface of crystalline chitin, where it introduces chain breaks and generates oxidized chain ends, thus promoting further degradation by chitinases. This enzymatic activity was discovered and further characterized by using mass spectrometry and chromatographic separation methods to detect oxidized products generated in the absence or presence of H(2)(18)O or (18)O(2). There are strong indications that similar enzymes exist that work on cellulose. Our findings not only demonstrate the existence of a hitherto unknown enzyme activity but also provide new avenues toward more efficient enzymatic conversion of biomass. 相似文献
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Nguyen HA Nguyen TH Křen V Eijsink VG Haltrich D Peterbauer CK 《Journal of agricultural and food chemistry》2012,60(12):3275-3281
The lnbA gene of Lactococcus lactis ssp. lactis IL1403 encodes a polypeptide with similarity to lacto-N-biosidases and N-acetyl-β-D-hexosaminidases. The gene was cloned into the expression vector pET-21d and overexpressed in Escherichia coli BL21* (DE3). The recombinant purified enzyme (LnbA) was a monomer with a molecular weight of approximately 37 kDa. Studies with chromogenic substrates including p-nitrophenyl N-acetyl-β-D-glucosamine (pNP-GlcNAc) and p-nitrophenyl N-acetyl-β-D-galactosamine (pNP-GalNAc) showed that the enzyme had both N-acetyl-β-D-glucosaminidase and N-acetyl-β-D-galactosaminidase activity, thus indicating that the enzyme is an N-acetyl-β-D-hexosaminidase. K(m) and k(cat) for pNP-GlcNAc were 2.56 mM and 26.7 s(-1), respectively, whereas kinetic parameters for pNP-GalNAc could not be determined due to the K(m) being very high (>10 mM). The optimal temperature and pH of the enzyme were 37 °C and 5.5, respectively, for both substrates. The half-life of activity at 37 °C and pH 6.0 was 53 h, but activity was completely abolished after 30 min at 50 °C, meaning that the enzyme has relatively low temperature stability. The enzyme was stable in the pH 5.5-8 range and was unstable at pH below 5.5. Studies with natural substrates showed hydrolytic activity on chito-oligosaccharides but not on colloidal chitin or chitosan. Transglycosylation products were not detected. In all, the data suggest that LnbA's role may be to degrade chito-oligosaccharides that are produced by the previously described chitinolytic system of L. lactis. 相似文献
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Nguyen TT Mathiesen G Fredriksen L Kittl R Nguyen TH Eijsink VG Haltrich D Peterbauer CK 《Journal of agricultural and food chemistry》2011,59(10):5617-5624
Food-grade gene expression systems for lactic acid bacteria are useful for applications in the food industry. We describe a new food-grade host/vector system for Lactobacillus plantarum based on pSIP expression vectors and the use of the homologous alanine racemase gene (alr) as selection marker. A new series of expression vectors were constructed by exchanging the erythromycin resistance gene (erm) in pSIP vectors by the L. plantarum WCFS1 alr gene. The vectors were applied for the overexpression of β-galactosidase genes from L. reuteri L103 and L. plantarum WCFS1 in an alr deletion mutant of L. plantarum WCFS1. The expression levels obtained in this way, i.e. without the use of antibiotics, were comparable to the levels obtained with the conventional system based on selection for erythromycin resistance. The new system is suitable for the production of ingredients and additives for the food industry. 相似文献
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VRP Barros AYP Cavalcante TJS Macedo RS Barberino TLB Lins BB Gouveia VG Menezes MAA Queiroz VR Araújo RC Palheta Jr MCP Leite MHT Matos 《Reproduction in domestic animals》2013,48(6):1025-1033
The expression of melatonin type 1 (MT1) and FSH (FSHR) receptors in caprine ovaries and the effects of these hormones on the in vitro development of isolated pre‐antral follicles were evaluated. Follicles (≤200 μm) were cultured for 12 days in α‐MEM (control) or melatonin (100 or 1000 pg/ml) or sequential melatonin medium (100 pg/ml: from day 0 to day 6; 1000 pg/ml: from day 6 to day 12; experiment 1) and in control or sequential FSH (100 ng/ml from day 0 to day 6; 500 ng/ml from day 6 to day 12) or sequential melatonin or this latter plus sequential FSH (experiment 2). MT1 and FSHR expressions were observed in granulosa cells from secondary and antral follicles. The oocytes from primordial and primary follicles also express FSHR. Sequential melatonin increased the percentage of normal follicles and oocyte recovery compared with the control or melatonin (1000 pg/ml) at day 12. In experiment 2, all the treatments increased the normal follicles and growth compared with the control. In conclusion, this study demonstrated the presence of MT1 and FSHR in caprine ovaries. The addition of increased concentrations of melatonin (sequential medium) or FSH can be used to promote the in vitro development of caprine pre‐antral follicles. 相似文献
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Berit B. Aam Ellinor B. Heggset Anne Line Norberg Morten S?rlie Kjell M. V?rum Vincent G. H. Eijsink 《Marine drugs》2010,8(5):1482-1517
Chitooligosaccharides (CHOS) are homo- or heterooligomers of N-acetylglucosamine and D-glucosamine. CHOS can be produced using chitin or chitosan as a starting material, using enzymatic conversions, chemical methods or combinations thereof. Production of well-defined CHOS-mixtures, or even pure CHOS, is of great interest since these oligosaccharides are thought to have several interesting bioactivities. Understanding the mechanisms underlying these bioactivities is of major importance. However, so far in-depth knowledge on the mode-of-action of CHOS is scarce, one major reason being that most published studies are done with badly characterized heterogeneous mixtures of CHOS. Production of CHOS that are well-defined in terms of length, degree of N-acetylation, and sequence is not straightforward. Here we provide an overview of techniques that may be used to produce and characterize reasonably well-defined CHOS fractions. We also present possible medical applications of CHOS, including tumor growth inhibition and inhibition of TH2-induced inflammation in asthma, as well as use as a bone-strengthener in osteoporosis, a vector for gene delivery, an antibacterial agent, an antifungal agent, an anti-malaria agent, or a hemostatic agent in wound-dressings. By using well-defined CHOS-mixtures it will become possible to obtain a better understanding of the mechanisms underlying these bioactivities. 相似文献
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Lasing action in organic vertical-cavity surface-emitting laser (OVCSEL) structures is demonstrated. Optically pumped OVCSELs with an active layer composed of a thin-film organic semiconductor tris-(8-hydroxyquinoline) aluminum (Alq3) doped with DCM laser dye produced very narrow linewidth (0.2 +/- 0.1 angstrom), high-power (3 watts) emission that could be varied in different devices from orange to red. The efficient energy transfer from Alq3 to DCM results in a threshold input energy of 300 microjoules per square centimeter. An operational lifetime >10(6) laser pulses was achieved for a device operated well above threshold in atmosphere. The linewidths above threshold are Fourier transform-limited and could potentially be narrowed further. 相似文献
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Craig B. Faulds Samuel Collins James A. Robertson Janneke Treimo Vincent G.H. Eijsink Sandra W.A. Hinz Henk A. Schols Johanna Buchert Keith W. Waldron 《Journal of Cereal Science》2009
The impact of microbial proteases on the release of carbohydrates from BSG was studied. The proteases were able to release the non-cellulosic glucose, a portion of feruloylated arabinoxylan and over 50% of the protein from brewers' spent grain (BSG) after 24 h hydrolysis. The non-cellulosic glucose was derived from residual starch-derived products persisting in BSG after mashing. The proteases did not cleave the hydroxycinnamate ester linkages present on the arabinoxylan backbone, and thus do not behave as feruloyl esterases. However, the material solubilised from spent grain by the proteases contained up to 198 μg bound ferulic acid/g extract, which represented 8.6% of the total ferulic acid present in BSG. These results suggest that a portion of water-extractable feruloylated arabinoxylan and starch is trapped within the BSG matrix by a proteinaceous barrier. 相似文献
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JMS Santos VG Menezes RS Barberino TJS Macedo TLB Lins BB Gouveia VRP Barros LP Santos RJS Gonçalves MHT Matos 《Reproduction in domestic animals》2014,49(3):522-528
Studies with sheep are important to improve our knowledge about the factors that control folliculogenesis in mammals and to explore possible physiological differences among species. The aims of this study were to characterize FGF‐2 protein expression in ovine ovaries and to verify the effect of FGF‐2 on the morphology, apoptosis and growth of ovine pre‐antral follicles cultured in vitro. After collection, one fragment of ovarian tissue was fixed for histological analysis and TUNEL analysis (fresh control). The remaining fragments were cultured for 7 days in control medium (α‐MEM+) alone or supplemented with FGF‐2 at different concentrations (1, 10, 50, 100 or 200 ng/ml). After culturing, ovarian tissue was destined to histology and TUNEL analysis, and oocyte and follicle diameters were measured. The immunostaining for FGF‐2 was observed in oocytes from primordial, primary and secondary follicles, as well as in granulosa cells of secondary and antral follicles. The percentage of normal follicles was similar among control medium, 1 and 10 ng/ml FGF‐2, and significantly higher than those observed in 50, 100 or 200 ng/ml FGF‐2. A significant increase in follicle diameter was observed when tissues were cultured in 10, 50, 100 or 200 ng/ml FGF‐2 compared with the fresh control and the other treatments. Similar results were observed for oocyte diameter in tissues cultured with 50, 100 or 200 ng/ml FGF‐2 (p < 0.05). However, the percentage of apoptotic cells only decreased (p < 0.05) in ovarian tissues cultured in 1 or 10 ng/ml FGF‐2 compared with the control medium and other FGF‐2 treatments. In conclusion, this study demonstrated the presence of FGF‐2 in ovine ovaries. Furthermore, 10 ng/ml FGF‐2 inhibits apoptosis and promotes ovine follicle growth. As the sheep ovary is more similar to that of humans, the culture system demonstrated in this work seems to be an appropriate tool for studies towards human folliculogenesis. 相似文献