En aquest treball s'exposen dues línies de recerca centrades en el procés de producció d'una lipasa de Rhizopus oryzae (ROL) en Pichia pastoris: la utilització de diferents tècniques de monitoratge i l'aplicació d'estratègies de cultiu que permetin millorar la producció de la ROL en una soca Mut+ d'aquest sistema d'expressió. En els cultius discontinus alimentats amb P. pastoris cal disposar d'una mesura en línia i en temps real de la concentració de substrat, el metanol. Per això es va desenvolupar un analitzador d'injecció seqüencial. Es va comprovar que aquest té una freqüència d'anàlisi òptima per a cultius amb la soca Muts però baixa per a la soca Mut+. Amb aquesta última soca, es van utilitzar i comparar dos mesuradors comercials en fase gas. Es va avaluar també la fluorimetria com a tècnica de monitoratge per a la determinació de tres variables clau: la biomassa, el substrat (glicerol/metanol) i la proteïna recombinant.Inicialment es va fer un seguiment de l'evolució de la biomassa a partir del senyal de fluorescència off-line del triptòfan. Es pot predir la biomassa correctament però es van evidenciar diferents desavantatges: la necessitat de dilució de les mostres i d'un sistema de presa de mostra específic. Per això es va decidir aplicar i avaluar la fluorimetria multivariable in situ. Amb la utilització d'una sonda fluorimètrica in situ, combinada amb mètodes quimiomètrics de tractament de dades multivariables, es va aconseguir predir la biomassa i el substrat a partir de la determinació del senyal de diversos fluoròfors. No es va aconseguir una bona predicció de la producció de ROL. Per millorar el seguiment de la proteïna, es va fusionar a la GFP. Es van detectar dos inconvenients: els nivells de producció disminuïen respecte a la producció de ROL únicament i la riboflavina, que Pichia excreta al medi, interferia amb el senyal de fluorescència del mutant de GFP escollit. Tant amb la filtració de la mostra abans de la determinació del senyal de GFP com si s'hagués escollit un mutant de GFP amb emissió més allunyada de la riboflavina es podria haver evitat aquest inconvenient i fer un seguiment de la ROL excretada. Es pot seguir la ROL intracel·lular.Quant a la producció de la ROL, es va estudiar l'efecte del nivell de metanol residual en cultius discontinus alimentats amb la soca Mut+. Existeix una concentració òptima entorn els 2.5 g·l-1 de metanol al medi. A concentracions superiors es va apreciar inhibició per substrat. Es va observar un fenomen de limitació per transferència d'oxigen al final del cultiu i una important disminució de la viabilitat cel·lular.Per això, es van avaluar estratègies de cultiu alternatives. Primer es va aplicar una estratègia de metanol limitant (MLFB) per evitar la limitació d'oxigen al final d'un cultiu a 2.5 g·l-1 de metanol residual (MNLFB). Es va millorar la productivitat un 40%. En segon lloc, es va aplicar una estratègia de temperatura limitant per avaluar el seu efecte sobre la producció. No es van millorar els resultats. Finalment, es va aplicar una estratègia MNLFB a 2.5 g·l-1 de metanol però amb un medi amb una menor osmolaritat i una fase final amb limitació de temperatura per evitar la limitació d'oxigen. Es va aconseguir reduir la mortalitat cel·lular però la productivitat disminuïa respecte el cultiu on s'aplica una fase de MLFB al final de la inducció. Tot i això, es va obtenir un producte final un 30% més pur en quant a activitat lipolítica respecte la proteïna total. Posteriorment es va procedir a escalar la producció en planta pilot. Es va observar limitació en la transferència d'oxigen en fases inicials de la inducció. Això va originar l'aparició d'un subproducte, associat a una reducció en la producció de ROL extracel·lular. En millorar la transferència d'oxigen es va minimitzar aquesta limitació i va millorar la producció. No es van aconseguir els mateixos nivells de productivitat que a escala laboratori però es continua treballant en la millora de la transferència de matèria per assolir-los. / In this work two research lines, applied to the production of a Rhizopus oryzae lipase (ROL) in Pichia pastoris, are shown: the application of different monitoring and cultivation techniques to improve ROL production in a P. pastoris Mut+ strain.During P. pastoris fed-batch cultures, methanol needs to be on line measured and monitored in real time. For this purpose, a sequential injection analyzer was developed. Although it presented a suitable analysis frequency for a Muts strain it was too low for a Mut+ strain. When the later was used, two different methanol commercial sensors in the outlet gas steams were utilized and compared. Fluorometry was also evaluated as a monitoring technique for three key variables: biomass, substrate (glycerol/methanol) and recombinant protein. Initially, biomass was followed by the off-line determination of tryptophan's fluorescence. Biomass was correctly predicted with this system but different disadvantages appeared: the need of a sample dilution procedure and a specific sampling device. Therefore, multivariable in situ fluorometry was subsequently evaluated. By means of an in situ multivariable fluorimetric probe, combined with chemometric methods to data processing, biomass and substrate prediction was properly achieved. ROL production could not be satisfactorily estimated.To improve ROL monitoring, it was fusioned to the green fluorescent protein (GFP). Two main disadvantages were found: production levels were lower when compared to solely ROL expression and riboflavin, naturally excreted by the yeast, interfered to GFP's signal. This problem could be solved with sample filtration prior to GFP's measurement and also if a different GFP mutant had been chosen with emission signal further to riboflavin's.With respect to ROL production, the effect of methanol concentration was studied in Mut+ fed-batch cultures. There is an optimal methanol concentration about 2.5 g·l-1. Substrate inhibition was observed at higher methanol levels. Oxygen transfer limitation at the end of the induction phase and an important cell viability decrease were also found. Therefore, alternative culture techniques were evaluated. First a methanol limited fed-batch phase (MLFB) was applied when oxygen limitations appeared at the end of a 2.5 g·l-1 methanol fed-batch phase (MNLFB). Productivity increased up to 40% with this strategy. Secondly, a temperature limited fed-batch was applied. No better results were obtained compared to the reference MNLFB culture. Finally, a 2.5 g·l-1 methanol fed-batch was applied with a lower salt content medium and a final temperature limited phase when oxygen limitation appeared. Cell death was reduced but productivity decreased with respect to the reference MNLFB culture. However, a 30% purer lipase was obtained in terms of lipase activity to total protein.Thereafter, the scaling of the production process in a pilot plant was evaluated. Oxygen limitation was found in the early induction phase. This caused a byproduct secretion associated to a ROL production decrease. When oxygen transfer was enhanced, byproduct secretion was reduced and ROL production improved. Similar laboratory scale productivities were not achieved but oxygen mass transfer is being further enhanced to reach the objective levels.

In recent years. as industrialized society developed. people have made a lot of environment pollution problems owing to overusing fossil fuel, moreover. fossil fuel is going to deplete. As the result. the study of the substitute energy is promoted. Ligonocellulose (lignin, cellulose and hemicelluloses are included) are the most plentiful renewable resources in the nature, and it have high economic values which extensively use on food, paper and energy. Recent years, it is a hot issue that decomposing cellulose into the minimum unit called glucose, and further, fermenting into alcohol to generate biomass energy. This research goal is cloning Arabidopsis endo-1,4-£]-glucanase and transfer to Escherichia coli(DH5£\). Selection pPICZ£\A the success transferr DNA Fragment. Then to sequence and because of cell in pPICZ£\A have expression in Pichia. pastoris. AOX1 promoter have Mass productions Arabidopsis endo-1,4-£]-glucanase gene in Pichia pastoris. Carries on the extracellular expression by the methyl alcohol induction way. Can obtain recombinant DNA protein. However the Western blotting analysis demonstration has this enzyme active protein At4g11050 pellet Molecular weight about 89 KDa. Again by way of congo red and Dye-CMC assay activeness of the examination enzyme protein. The result discovers At4g11050 in the pellet cell activity compares with negative control has the obvious activity.

Generation of alcohol for biofuels from fermentation of sugar or starch has several economic disadvantages such as high cost of sugar processing and land usage competing with staple food. The solution may reside in hydrolysis of cellulose from crop waste such as stalks of rice and corn or non-crop plants such as weeds or wood. Our goal is to identify cellulases that can degrade cellulosic biomass more efficiently. Studies of microbial Family 9 glycoside hydrolase (GH9) proteins, including both endo-glucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), have shown that they function through an inverting mechanism to cleave the 1, 4-£]-glucosidic bond between two unsubstituted Glc units. The main function of plant glycoside hydrolases are involved in polysaccharide metabolism of cell wall during cell growth. Twelve Arabidopsis thaliana (Columbia) endo-1,4-£]-glucanases that belong to the GH9, were cloned and expressed in Pichia pastoris in order to produce cellulases to facilitate efficient bio-alcohol production. The recombinant proteins do not show in vitro endo-1, 4-£]-glucanase activity, but we can detect the recombinant proteins expression in supernatant or in pellet. The lack of enzymatic activity from recombinant proteins is probably due to improper folding or glycosylation, or fast degradation resulted from the above reasons. Other bioreactor will be tested in the future. Genetic engineering to modify Arabidopsis thaliana (Columbia) endo-£]-1, 4-glucanases is another approach to produce functional cellulases with economic efficiency that can be adapted to industrial scale for alcohol generation. On the other hand, we use semi-quantitative PCR method to study the Arabidopsis GH9 genes expression level in different tissue. At4g39000 and At3g43860 were found only in flowers and inflorescence, and At1g65610 expression in roots and shoots of the amount of more. Other genes in different tissues, was no found significant difference.

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, December 2003. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--Oregon Graduate Institute, 2000.

Vienas pagrindinių biomedicininės paskirties baltymų gamybos iššūkių yra pigių ir saugių ekspresijos sistemų, tinkamų glikoproteinų sintezei, paieška bei esamų sistemų tobulinimas. Vaistai, sukurti baltymų pagrindu, sudaro apie ketvirtadalį naujai patvirtinamų vaistų rinkos, o apie 60% jų sudaryti iš glikoproteinų. Dabar glikoproteinų sintezei naudojamos žinduolių kultūros turi keletą trūkumų. Jose gaunamų rekombinantinių baltymų kaina yra didelė, ribotas tūrinis našumas, ląstelės lėtai dauginasi ir auga, būna užkrėstos retrovirusais, gaunamas heterogeniškas produktas ir užima daug laiko sukurti stabilią ląstelių liniją. Itin intensyviai vykstantis tinkamų ekspresijos sistemų kūrimas kol kas nedavė norimų rezultatų. Mielių, kaip ir augalų bei vabzdžių, ekspresijos sistemos, dėl keletos priežasčių yra įvardijamos kaip vienos pagrindinių kandidatų užimti šią vietą. Visų pirma, mielės yra pripažintos kaip saugus organzimas, jų gentika, biochemija ir fiziologija yra gerai ištirta, be to, sėkmingai pradėti kurti rekombinantiniai mielių kamienai su sudėtingu žinduolių tipo N-glikozilinimu. Vis tik mielėse susintetintų glikoproteinų, tinkamų farmacijos pramonei, skaičius yra labai nedidelis, dažniausiai glikoproteinai nebūna tinkamai suvynioti ir modifikuoti, o esmininės priežastys, paaiškinančios mielių trūkumus sintetinant tokio tipo baltymus, nėra išaiškintos. Eukariotų genų inžinerijos laboratorijoje jau yra sukaupta nemaža patirties sintetinant mielėse virusinius glikoproteinus... [toliau žr. visą tekstą] / Growing market of the glycoprotein based drugs increases demands of safe, cheap and effective expression systems for production of glycoproteins. Mammalian cell cultures, which are being used for this purpose, are very expensive and ineffective. Yeasts are rising as one of the best alternatives. Well known genetics, biochemistry and physiology are only few advantages. Yeasts are also considered to be safe and easy to manipulate organism. Still, despite introducing humanized glycosylation pathways, yeast based expression systems are not able to produce glycoproteins for pharmaceutics, with a very few exceptions. So, further researches in adapting yeast for glycoproteins synthesis must be made. This work is directed for this purpose. In this work mumps, measles and influenza virus surface glycoproteins were expressed in yeast Pichia pastoris. Results show, that mumps virus hemagliutinin-neuraminidase is not synthesized in P.pastoris. Synthesis of measles virus (MeV) hemagliutinin (H) glycoprotein was not effective, with recombinant protein not possessing characteristics of native analogue. MeV-H was found in two forms: unglycosylated polypeptide precursor and glycosylated form, both aggregated and insoluble in non-ionic detergent. Increase in MeV-H expression level resulted in extensive accumulation of unglycosylated MeV-H protein precursors in the cytoplasm of yeast cells. Addition of S.cerevisiae α-factor secretion signal sequence to globular part of MeV-H made... [to full text]

No abstract available. / Thesis (M. Sc.)-University of Durban-Westville, 1994.

Thesis (MScIng)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Stuttgart, Univ., Diss., 2002.

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: expanded bed chromatography; Pichia pastoris. Includes bibliographical references (leaves 44-45).