258 research outputs found
Characterisation, cloning and production of industrially interesting enzymes : gluconolactone oxidase of Penicillium cyaneo-fulvum and gluconate 5-dehydrogenase of Gluconobacter suboxydans
The work covered in this thesis is focused on the development of technology for bioconversion of glucose into D-erythorbic acid (D-EA) and 5-ketogluconic acid (5-KGA). The task was to show on proof-of-concept level the functionality of the enzymatic conversion or one-step bioconversion of glucose to these acids. The feasibility of both studies to be further developed for production processes was also evaluated.
The glucose - D-EA bioconversion study was based on the use of a cloned gene encoding a D-EA forming soluble flavoprotein, D-gluconolactone oxidase (GLO). GLO was purified from Penicillium cyaneo-fulvum and partially sequenced. The peptide sequences obtained were used to isolate a cDNA clone encoding the enzyme. The cloned gene (GenBank accession no. AY576053) is homologous to the other known eukaryotic lactone oxidases and also to some putative prokaryotic lactone oxidases. Analysis of the deduced protein sequence of GLO indicated the presence of a typical secretion signal sequence at the N-terminus of the enzyme. No other targeting/anchoring signals were found, suggesting that GLO is the first known lactone oxidase that is secreted rather than targeted to the membranes of the endoplasmic reticulum or mitochondria. Experimental evidence supports this analysis, as near complete secretion of GLO was observed in two different yeast expression systems. Highest expression levels of GLO were obtained using Pichia pastoris as an expression host. Recombinant GLO was characterised and the suitability of purified GLO for the production of D-EA was studied. Immobilised GLO was found to be rapidly inactivated during D-EA production. The feasibility of in vivo glucose - D-EA conversion using a P. pastoris strain co-expressing the genes of GLO and glucose oxidase (GOD, E.C. 1.1.3.4) of A. niger was demonstrated.
The glucose - 5-KGA bioconversion study followed a similar strategy to that used in the D-EA production research. The rationale was based on the use of a cloned gene encoding a membrane-bound pyrroloquinoline quinone (PQQ)-dependent gluconate 5-dehydrogenase (GA 5-DH). GA 5-DH was purified to homogeneity from the only source of this enzyme known in literature, Gluconobacter suboxydans, and partially sequenced. Using the amino acid sequence information, the GA 5-DH gene was cloned from a genomic library of G. suboxydans. The cloned gene was sequenced (GenBank accession no. AJ577472) and found to be an operon of two adjacent genes encoding two subunits of GA 5-DH. It turned out that GA 5-DH is a rather close homologue of a sorbitol dehydrogenase from another G. suboxydans strain. It was also found that GA 5-DH has significant polyol dehydrogenase activity. The G. suboxydans GA 5-DH gene was poorly expressed in E. coli. Under optimised conditions maximum expression levels of GA 5-DH did not exceed the levels found in wild-type G. suboxydans. Attempts to increase expression levels resulted in repression of growth and extensive cell lysis. However, the expression levels were sufficient to demonstrate the possibility of bioconversion of glucose and gluconate into 5-KGA using recombinant strains of E. coli.
An uncharacterised homologue of GA 5-DH was identified in Xanthomonas campestris using in silico screening. This enzyme encoded by chromosomal locus NP_636946 was found by a sequencing project of X. campestris and named as a hypothetical glucose dehydrogenase. The gene encoding this uncharacterised enzyme was cloned, expressed in E. coli and found to encode a gluconate/polyol dehydrogenase without glucose dehydrogenase activity. Moreover, the X. campestris GA 5-DH gene was expressed in E. coli at nearly 30 times higher levels than the G. suboxydans GA 5-DH gene. Good expressability of the X. campestris GA-5DH gene makes it a valuable tool not only for 5-KGA production in the tartaric acid (TA) bioprocess, but possibly also for other bioprocesses (e.g. oxidation of sorbitol into L-sorbose).
In addition to glucose - 5-KGA bioconversion, a preliminary study of the feasibility of enzymatic conversion of 5-KGA into TA was carried out. Here, the efficacy of the first step of a prospective two-step conversion route including a transketolase and a dehydrogenase was confirmed. It was found that transketolase convert 5-KGA into TA semialdehyde. A candidate for the second step was suggested to be succinic dehydrogenase, but this was not tested.
The analysis of the two subprojects indicated that bioconversion of glucose to TA using X. campestris GA 5-DH should be prioritised first and the process development efforts in future should be focused on development of more efficient GA 5-DH production strains by screening a more suitable production host and by protein engineering.Erytorbiinihappo on C-vitaminiinin stereoisomeeri, jota käytetään elintarvikkeissa antioksidanttina. Erytorbiinihappoa tuotetaan teollisuudessa moniportaisella prosessilla, joka on samankaltainen kuin C-vitamiinin valmistuksessa käytettävä Reichstein-prosessi. Luonnossa ainoastaan Penicillium-homeiden on havaittu tuottavan erytorbiinihappoa. Synteesireitti glukoosista erytorbiinihapoksi on lyhyt, sisältäen vain kaksi entsyymiä, glukoosioksidaasin, joka hapettaa glukoosin glukono-1,5-laktoniksi (joka vesiliuoksessa spontaanisti tasapainottuu glukono-1,4-laktonin ja glukonihapon kanssa) ja glukonolaktonioksidaasin, joka hapettaa glukono-1,5- ja glukono-1,4-laktonin edelleen erytorbiinihapoksi. 1960- ja 1970-luvuilla kehiteltiin menetelmiä erytorbiinihapon tuottamiseksi Penicillium-homeilla, mutta kannattavaa prosessia ei ole onnistuttu rakentamaan.
Tartarihappo on toinen elintarviketeollisuuden kannalta melko keskeinen orgaaninen happo, jota käytetään paitsi happamuuden säätelyaineena myös raaka-aineena emulgointiaineiden valmistuksessa. Tartarihappoa tuotetaan nykyisin pääasiassa viiniteollisuuden sivutuotteena. Koska viinintuotannon vuotuinen sadonvaihtelu heijastuu voimakkaasti myös tartarihapon markkinahintaan, uusia menetelmiä hapon tuottamiseksi on etsitty vakaamman hintatason takaamiseksi. Tartarihappoa voidaankin valmistaa 5-ketoglukonihaposta hapettamalla esimerkiksi jalometallien tai vanadaatin avulla. 5-ketoglukonihappoa voidaan valmistaa glukoosista fermentoimalla käyttäen 5-ketoglukonihappoa tuottavia Glukonobacter- tai Acetobacter-kantoja.
Tässä väitöstutkimuksessa on selvitetty mahdollisuuksia tuottaa erytorbiinihappoa, 5-ketoglukonihappoa ja tartarihappoa geneettisesti muokatuilla tuotantomikrobeilla. Työssä on tuotettu ja puhdistettu erytorbiinihapon ja 5-ketoglukonihapon synteesejä katalysoivat entsyymit glukonolaktonioksidaasi ja glukonaatti-5-dehydrogenaasi. Osittaisen aminohapposekvenssien avulla entsyymejä koodaavat geenit on kloonattu cDNA- ja genomikirjastoista. Kumpaakin entsyymiä on ylituotettu heterologisessa isäntämikrobissa (Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris). Rekombinanttisesti tuotettuja entsyymejä on karakterisoitu ja niiden sekä keinotekoisen metaboliareitin omaavien tuotantomikrobien soveltuvuutta kyseisten orgaanisten happojen teolliseen tuotantoprosessiin on arvioitu.
Sekä erytorbiinihappoa että 5-ketoglukonihappoa tuotettiin sekä rekombinanttisten entsyymien että geneettisesti modifioitujen tuotantoisäntien avulla. Glukonolaktonioksidaasia ylituotettiin 72-kertaisesti P. pastoriksessa verrattuna villityypin Penicillium cyaneo-fulvum-kantaan. Rekombinanttisesti tuotettu glukonolaktonioksidaasi immobilisoitiin ja immobilisoidulla entsyymillä onnistuttiin tuottamaan erytorbiinihappoa glukonihaposta. P. pastoris-kanta, jossa ylituotettiin sekä Aspergillus nigerin glukoosioksidaasia että glukonolaktonioksidaasia, konvertoi fermantaatiossa glukoosia erytorbiinihapoksi 6% saannolla. Tutkimusessa glukonolaktonioksidaasin havaittiin olevan erittyvä entsyymi, toisin kuin aiemmin on luultu ja se onkin toistaiseksi ainoa tunnettu erittyvä sokerilaktonioksidaasi. Geenisekvenssin perusteella havaitiin, että P. cyaneo-fulvumin glukonolaktonioksidaasi on ainoastaan kaukainen sukulainen muille tunnetuille sokerilaktonioksidaaseille muodostaen oman erittyvien laktonioksidaasien alaryhmän.
Glukonaatti-5-dehydrogenaasin ylituotto E. colissa osoittautui vaikeaksi ja rekombinanttista entsyymiä onnistuttiin tuottamaan ainoastaan lähes samalla tasolla kuin villityypin Gluconobacter suboxydansissa. Koska E. colissa ei kuitenkaan ole sivuaktiivisuutena glukonaatti-2-dehydrogenaasia, ovat 5-ketoglukonaatin maksimisaannot rekombinanttikannalla teoriassa parempia kuin G. oxydanssissa, jolla fermentoitaessa osa glukonihaposta päätyi 2-ketoglukonihapoksi. Geneettisesti modifioidulla E. colilla onnistuttiin konvertoimaan 75% glukonihaposta 5-ketoglukonihapoksi. Glukonaatti-5-dehydrogenaasin geenisekvekssin avulla löytyi kuitenkin mielenkiintoinen homologinen karakterisoimaton entsyymi Xanthomonas campestriksesta. Tämän entsyymin geenin kloonaus ja ylituotto onnistui selvästi paremmin ollen 25-kertainen villityyppiin verrattuna. Karakterisoitaessa entsyymi osoittautuikin glukonaatti-5-dehydrogenaasiksi eikä todennäköiseksi glukoosidehydrogenaasiksi, kuten se oli sekvensointiprojektin yhteydessä GenBank:iin nimetty. Kumpikin entsyymi osoittautui kuitenkin omaavan huomattavasti laajemman substraattispesifisyyden, pystyen käyttämään substraattinaan glukonihapon lisäksi monia erilaisia polyoleja, kuten sorbitoli ja arabitoli. Suurimmat aktiivisuudet mitattiin käyttäen substraattina 2,3-butandiolia. Entsyymien substraattispesifisyys vastaa hyvin empiirisiin havaintoihin perustuvaa Bertrand-Hudsonin sääntöä sekundääristen polyolien hapettamisesta etikkahappobakteereilla. Luultavasti glukonaatti-5-dehydrogenaasi vastaa pääosin tästä säännöstä. Laajasta substraattispesifisyydestä johtuen glukonaatti-5-dehydrogenaasin teolliset sovellutukset ovat paljon aiemmin oletettua laajemmat ja sille voi löytyä lisää sovelluksia mm. C-vitamiinin ja joidenkin hienokemikaalien teollisessa tuotannossa
Transcriptome and proteome analysis of xylose-metabolising Saccharomyces cerevisiae
Increasing concern about global climate warming has accelerated research into renewable energy sources that could replace fossil petroleum-based fuels and materials. Bioethanol production from cellulosic biomass by fermentation with baker s yeast Saccharomyces cerevisiae is one of the most studied areas in this field. The focus has been on metabolic engineering of S. cerevisiae for utilisation of the pentose sugars, in particular D-xylose that is abundant in the hemicellulose fraction of biomass. Introduction of a heterologous xylose-utilisation pathway into S. cerevisiae enables xylose fermentation, but ethanol yield and productivity do not reach the theoretical level.
In the present study, transcription, proteome and metabolic flux analyses of recombinant xylose-utilising S. cerevisiae expressing the genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis and the endogenous xylulokinase were carried out to characterise the global cellular responses to metabolism of xylose. The aim of these studies was to find novel ways to engineer cells for improved xylose fermentation. The analyses were carried out from cells grown on xylose and glucose both in batch and chemostat cultures. A particularly interesting observation was that several proteins had post-translationally modified forms with different abundance in cells grown on xylose and glucose. Hexokinase 2, glucokinase and both enolase isoenzymes 1 and 2 were phosphorylated differently on the two different carbon sources studied. This suggests that phosphorylation of glycolytic enzymes may be a yet poorly understood means to modulate their activity or function.
The results also showed that metabolism of xylose affected the gene expression and abundance of proteins in pathways leading to acetyl-CoA synthesis and altered the metabolic fluxes in these pathways. Additionally, the analyses showed increased expression and abundance of several other genes and proteins involved in cellular redox reactions (e.g. aldo-ketoreductase Gcy1p and 6-phosphogluconate dehydrogenase) in cells grown on xylose. Metabolic flux analysis indicated increased NADPH-generating flux through the oxidative part of the pentose phosphate pathway in cells grown on xylose.
The most importantly, results indicated that xylose was not able to repress to the same extent as glucose the genes of the tricarboxylic acid and glyoxylate cycles, gluconeogenesis and some other genes involved in the metabolism of respiratory carbon sources. This suggests that xylose is not recognised as a fully fermentative carbon source by the recombinant S. cerevisiae that may be one of the major reasons for the suboptimal fermentation of xylose. The regulatory network for carbon source recognition and catabolite repression is complex and its functions are only partly known. Consequently, multiple genetic modifications and also random approaches would probably be required if these pathways were to be modified for further improvement of xylose fermentation by recombinant S. cerevisiae strains.Ilmaston lämpeneminen ja fossiilisten polttoaineiden rajallisuus on lisännyt kiinnostusta uusiutuvista raaka-aineista valmistettaviin polttoaineisiin ja tuotteisiin. Polttoaine-etanoli, joka on tuotettu fermentoimalla kasviperäistä selluloosaa ja hemiselluloosaa sisältävää kasvimateriaalia, on paljon tutkittu vaihtoehto fossiilisille polttoaineille. Kaikkien kasvimateriaalissa olevien sokereiden hyödyntäminen on tärkeää etanolin tuotannon taloudelliselle kannattavuudelle. Laajalti etanolin tuottoon käytettävä leivinhiiva S. cerevisiae fermentoi kuitenkin tehokkaasti vain glukoosia ja heksoosisokereita eikä kykene käyttämään hemiselluloosassa suhteellisen runsaana esiintyvää viisihiilistä pentoosisokeria ksyloosia. Ksyloosin käytön mahdollistavat entsyymit ksyloosireduktaasi ja ksylitolidehydrogenaasi, joita ilmentävät geenit on siirretty leivinhiivaan Pichia stipitis -hiivasta. Ksyloosia käyttävät muuntogeeniset leivinhiivakannat tuottavat ksyloosista etanolia, mutta etanolin saanto ja tuottonopeus eivät vielä yllä toivotulle tasolle.
Tässä työssä on etsitty uusia keinoja parantaa leivinhiivan ksyloosin fermentointikykyä vertaamalla ksyloosia käyttävän leivinhiivan ksyloosiaineenvaihduntaa glukoosiaineenvaihduntaan DNA-sirujen, proteomiikan ja aineenvaihduntavuoiden mallintamisen avulla. Tulokset osoittavat, että ksyloosi ei kykene glukoosin tavoin vaimentamaan leivinhiivassa sitruunahappokierron, glyoksylaattisyklin ja glukoneogeneesin proteiineja ilmentäviä geenejä eikä useita muita geenejä, jotka liittyvät muiden hiilen lähteiden kuin sokereiden käyttöön. Ksyloosin käyttö muutti myös asetyyli-CoA:n valmistukseen liittyvien geenien ilmentymistä sekä asetyyli-CoA:n aineenvaihduntareitillä olevien proteiinien määriä. Lisäksi ksyloosin käyttö aktivoi geenejä, joiden ilmentyminen on aiemmin liitetty hiilen lähteen ja ravinteiden loppumiseen.
Mielenkiintoinen havainto oli, että glykolyysiin osallistuvat entsyymit heksokinaasi, glukokinaasi ja enolaasi fosforyloituivat eri tavoin ksyloosilla ja glukoosilla kasvaneissa soluissa, mikä viittaa siihen, että näiden entsyymien aktiivisuutta ja/tai toimintaa eri olosuhteissa säädellään fosforylaation avulla. Tutkimuksessa havaittiin myös, että ksyloosilla kasvaneissa soluissa useiden solujen hapetus- ja pelkistysreaktioihin osallistuvien proteiinien määrä ja niitä ilmentävien geenien transkriptio oli lisääntynyt. Lisäksi aineenvaihduntavuoiden mallinnus osoitti, että hiilivirta pentoosifosfaattireitin sen osan kautta, joka tuottaa NADPH:ta, oli vilkastunut ksyloosia käyttävissä soluissa verrattuna glukoosilla kasvaneisiin soluihin. Nämä tulokset viittaavat muuntogeenisen hiivan pyrkimykseen lisätä solun sisäisen NADPH:n määrää ksyloosireduktaasientsyymin tarpeisiin.
Kaiken kaikkiaan soluissa havaitut muutokset viittaavat siihen, että S. cerevisiae ei kykene tunnistamaan ksyloosia glukoosin kaltaiseksi täysin fermentoitavaksi hiilen lähteeksi. Tämä on luultavasti yksi syistä miksi etanolin saanto ja ksyloosin fermentointinopeus eivät yllä optimaaliselle tasolle. Hiilen käyttöön liittyvän aineenvaihduntaverkon toiminnan säätely on mutkikasta ja vain osittain tunnettua. Onkin todennäköistä, että etanolin tuotannon tehostamiseksi ksyloosista säätelyverkkoa muuttamalla tarvitaan useiden säätelyyn osallistuvien geenien ilmentymisen muuttamista tai geenien sattumanvaraista mutageneesiä
Inactivation of poly(3-hydroxybutyrate) (PHB) biosynthesis in ‘Knallgas’ bacterium Xanthobacter sp. SoF1
Aerobic hydrogen-oxidizing ‘Knallgas’ bacteria are promising candidates for microbial cell factories due to their ability to use hydrogen and carbon dioxide as the sole energy and carbon sources, respectively. These bacteria can convert atmospheric CO2 to chemicals which could help to mitigate climate change by replacing fossil fuel-based chemicals. A known method to enhance the product yield is to disrupt competing metabolic pathways in the host organism. One such pathway in many ‘Knallgas’ bacteria is polyhydroxybutyrate (PHB) biosynthesis. In this study, the PHB biosynthesis genes of a non-model ‘Knallgas’ bacterium Xanthobacter sp. SoF1 were identified. Consequently, the phaA, phaB and phaC genes were individually deleted and the resulting knockouts were evaluated for their ability to produce PHB in autotrophic shake flask and small-scale bioreactor cultivations. The results demonstrate that PHB production was inactivated in the phaC1 knockout strain, which advances the development of Xanthobacter sp. SoF1 as a production host. Graphical Abstract: [Figure not available: see fulltext.].</p
Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae
<p>Abstract</p> <p>Background</p> <p>Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of <it>Saccharomyces cerevisiae </it>for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising <it>S. cerevisiae </it>grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose.</p> <p>Results</p> <p>Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators <it>HAP4</it>, <it>CAT8 </it>and <it>SIP1-2 </it>and <it>4</it>. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor <it>RGT1 </it>and its target genes <it>HXT2-3</it>, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells.</p> <p>Conclusion</p> <p>The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant <it>S. cerevisiae </it>strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.</p
Intraoperative colonic pulse oximetry in left-sided colorectal surgery : can it predict anastomotic leak?
An anastomotic leak is a fairly common and a potentially lethal complication in colorectal surgery. Objective methods to assess the viability and blood circulation of the anastomosis could help in preventing leaks. Intraoperative pulse oximetry is a cheap, easy to use, fast, and readily available method to assess tissue viability. Our aim was to study whether intraoperative pulse oximetry can predict the development of an anastomotic leak. The study was a prospective single-arm study conducted between the years 2005 and 2011 in Helsinki University Hospital. Patient material consisted of 422 patients undergoing elective left-sided colorectal surgery. The patients were operated by one of the three surgeons. All of the operations were partial or total resections of the left side of the colon with a colorectal anastomosis. The intraoperative colonic oxygen saturation was measured with pulse oximetry from the colonic wall, and the values were analyzed with respect to post-operative complications. 2.3 times more operated anastomotic leaks occurred when the colonic StO(2) was Low intraoperative colonic StO(2) values are associated with the occurrence of anastomotic leak. Despite its handicaps, the method seems to be useful in assessing anastomotic viability.Peer reviewe
Stoma reversal after Hartmann's procedure for acute diverticulitis
Background: Hartmann's procedure is a treatment option for perforated acute diverticulitis, especially when organ dysfunction(s) are present. Its use has been criticized mostly out of fear of high permanent stoma rate. The aim of this study was to investigate the rate of stoma reversal, reasons behind non-reversal, and safety of reversal surgery. Methods: This was a single-center retrospective study of patients undergoing urgent Hartmann's pro-cedure due to acute diverticulitis between the years 2006 and 2017 with follow-up until March 2021. Results: A total of 3,319 episodes of diverticulitis in 2,932 patients were screened. The Hartmann's procedure was performed on 218 patients, of whom 157 (72%) had peritonitis (48 (22%) with organ dysfunction). At 2-years, 76 (34.9%) patients had died with stoma, 42 (19.3%) were alive with stoma, and 100 (45.9%) had undergone stoma reversal. The survival of patients with and without reversal were 100% and 42.7% at 1-year, 96.0% and 35.0% at 2-years and 88.9% and 20.7% at 5-years, respectively. The risk factors for nonreversal were old age, a need for outside assistance, low HElsinki Staging for Acute Diverticulitis stage, and higher C-reactive protein level upon hospital admission. The most common reasons for nonreversal in surviving patients were patient not willing to have the operation 18 (41%) and dementia 10 (23%). Twelve (12%) patients had a major complication after reversal (Clavien-Dindo IIIb-IV) and 90-day mortality after reversal was 0%. Conclusion: After the Hartmann's procedure for acute diverticulitis, one-third died, half underwent stoma reversal, and one-fifth did not undergo stoma reversal within 2 years. Patients who survive with stoma are either not willing to have reversal or have severe comorbidities excluding elective surgery. The Hartmann's procedure remains a viable option for high-risk patients with perforated acute diverticulitis. (c) 2022 The Author(s). Published by Elsevier Inc.Peer reviewe
UJIAN NASIONAL MENGKEBIRI KEDAULATAN GURU
Stardisasi telah menjadi pilihan Kebijakan pendidikan di Indonesia, sebagai memenuhi dari pentingnya lokal, nasional dan global. ISO 9000 adalah sebagai standardisasi internasional kualitas pendidikan yang telah dikompromikan oleh banyak negara di dunia telah memberikan pelayanan terbaik dan memberikan harapan konsumen pendidikan. Standarisasi kelulusan siswa di Indonesia adalah Ujian Nasional, oleh karena pelaksanaan ini telah kehilangan nilai kejujuran, kebaikan dan karakter pendidikan kita . Karena alasan pengguna standardisasi Ujian Nasional tidak sebagai salah satu di semua ukuran fundamental sukses nasional pendidikan di Indonesia. Ujian Nasional telah kehilangan kedaulatan guru karena tidak memberikan autoritas sama sekali untuk guru untuk memberikan nilai kepada siswa mereka. Pemerintah perlu mengambil kebijakan lain untuk mengubah Ujian Nasional yang satu dapat menciptakan kreativitas siswa dan guru untuk tidak membunuh demokrasi kitaStardisasi telah menjadi pilihan Kebijakan pendidikan di Indonesia, sebagai memenuhi dari pentingnya lokal, nasional dan global. ISO 9000 adalah sebagai standardisasi internasional kualitas pendidikan yang telah dikompromikan oleh banyak negara di dunia telah memberikan pelayanan terbaik dan memberikan harapan konsumen pendidikan. Standarisasi kelulusan siswa di Indonesia adalah Ujian Nasional, oleh karena pelaksanaan ini telah kehilangan nilai kejujuran, kebaikan dan karakter pendidikan kita . Karena alasan pengguna standardisasi Ujian Nasional tidak sebagai salah satu di semua ukuran fundamental sukses nasional pendidikan di Indonesia. Ujian Nasional telah kehilangan kedaulatan guru karena tidak memberikan autoritas sama sekali untuk guru untuk memberikan nilai kepada siswa mereka. Pemerintah perlu mengambil kebijakan lain untuk mengubah Ujian Nasional yang satu dapat menciptakan kreativitas siswa dan guru untuk tidak membunuh demokrasi kit
Concurrent heavy use of general and oral health services among Finnish adults
The aim was to identify heavy users of public health (PHS) and public oral health (POHS) services and combined and concurrent users of these services.\nNumbers of 18+ year-old patients and their visits to POHS (12,124 patients) and PHS (28,479 patients) were collected from two patient registers in a Finnish town in 2013. The combined dataset consists of 32,481 patients. Using a highest decile criterion for both for POHS and PHS, those patients who had made 8 or more visits were categorised as heavy users. Patients who had made total of 10 or more visits to the POHS and/or PHS were categorised as combined heavy users. Patients who had made 8 or more visits to both the POHS and PHS were categorised as concurrent heavy users (195 patients).\nHeavy users of POHS were more often men and those of PHS more often women. Combined heavy users were likely to be women and to be older. The combined heavy users accounted for 40% of all visits of POHS and/or PHS. Among them 30% did not have any POHS visits and 4% did not have any PHS visits. Concurrent heavy use was rare, involving 0.06% of all patients, but made 3.4% of all visits.\nOf the patients making 10 or more POHS and/or PHS visits, only five percent were concurrent heavy users of both services. As many non-communicable diseases share common risk factors the combined heavy users of PHS should be directed to use POHS and vice versa
Physiological effects of over-expressing compartment-specific components of the protein folding machinery in xylose-fermenting Saccharomyces cerevisiae
Molecular and physiological basis of Saccharomyces cerevisiae tolerance to adverse lignocellulose-based process conditions
Lignocellulose-based biorefineries have been gaining increasing attention to substitute current petroleum-based refineries. Biomass processing requires a pretreatment step to break lignocellulosic biomass recalcitrant structure, which results in the release of a broad range of microbial inhibitors, mainly weak acids, furans, and phenolic compounds. Saccharomyces cerevisiae is the most commonly used organism for ethanol production; however, it can be severely distressed by these lignocellulose-derived inhibitors, in addition to other challenging conditions, such as pentose sugar utilization and the high temperatures required for an efficient simultaneous saccharification and fermentation step. Therefore, a better understanding of the yeast response and adaptation towards the presence of these multiple stresses is of crucial importance to design strategies to improve yeast robustness and bioconversion capacity from lignocellulosic biomass. This review includes an overview of the main inhibitors derived from diverse raw material resultants from different biomass pretreatments, and describes the main mechanisms of yeast response to their presence, as well as to the presence of stresses imposed by xylose utilization and high-temperature conditions, with a special emphasis on the synergistic effect of multiple inhibitors/stressors. Furthermore, successful cases of tolerance improvement of S. cerevisiae are highlighted, in particular those associated with other process-related physiologically relevant conditions. Decoding the overall yeast response mechanisms will pave the way for the integrated development of sustainable yeast cell--based biorefineries.This study was supported by the Portuguese Foundation for Science and Technology (FCT) by the strategic funding of UID/BIO/04469/2013 unit, MIT Portugal Program (Ph.D. grant PD/BD/128247/
2016 to Joana T. Cunha), Ph.D. grant SFRH/BD/130739/2017 to Carlos E. Costa, COMPETE 2020 (POCI-01-0145-FEDER-006684), BioTecNorte operation (NORTE-01-0145-FEDER-000004), YeasTempTation (ERA-IB-2-6/0001/2014), and MultiBiorefinery project (POCI-01-0145-FEDER-016403). Funding by the Institute for Bioengineering and Biosciences (IBB) from FCT (UID/BIO/04565/2013) and from Programa Operacional Regional de Lisboa 2020 (Project N. 007317) was also receiveinfo:eu-repo/semantics/publishedVersio
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