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El objetivo general de la presente tesis doctoral es el desarrollo de herramientas para la obtencion, produccion y aplicacion de dos enzimas glicosidicas: �¿-L-arabinofuranosidasa proveniente del hongo Aspergillus niger (Abf) y �À-D-glucosidasa (Bgl), proveniente de la levadura Candida molischiana. Estas hidrolasas se emplean en la liberacion de azucares en procesos de conversion de biomasa y en la industria alimentaria, pero tambien en la sintesis de aminoglicosidos, glicoconjugados y oligosacaridos, compuestos de alto valor anadido para la industria quimico-farmaceutica. Las enzimas se han expresado en la levadura metilotrofica Pichia pastoris, y se han purificado para caracterizar sus propiedades bioquimicas. Asimismo, se ha comprobado su capacidad para catalizar reacciones de transglicosilacion con alto rendimiento. En relacion a su produccion, se ha establecido y validado un modelo basado en restricciones del metabolismo de Pichia pastoris, evaluando su consistencia mediante analisis de flujos metabolicos posibilistico. El modelo permite estimar la tasa de crecimiento y la distribucion de flujos intracelulares a partir de unos pocos flujos extracelulares medidos experimentalmente. Adicionalmente, el modelo se ha extendido para estimar la productividad de proteina recombinante, y se ha empleado para analizar diferentes condiciones de cultivo de las cepas transgenicas que sobreproducen las enzimas Abf y Bgl. Finalmente, las enzimas se han inmobilizado en organosilicas bimodales de la familia UVM-7. Los biocatalizadores resultantes se han caracterizado bioquimica y fisico-quimicamente y se han evaluado en diferentes aplicaciones de interes biotecnologico. / Tortajada Serra, M. (2012). Process development for the obtention and use of recombinant glycosidases: expression, modelling and immobilisation [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16800 / Palancia

This thesis focuses on the study of the influence of selected parameters on the course of microbial cultivation and evaluation of bioprocess. It is divided into two parts. The first part deals with the production of mutant forms of the protein cryptogein yeast Pichia pastoris. The theoretical part summarizes the findings of the yeast P. pastoris and its expression. It also deals with cryptogein that induces defense reactions in plants. In the experimental part was produced mutant cryptogein X24, in which the concentration of each fraction and the ability to transfer sterols. The second part of this thesis is focused on aerobic and anaerobic oxidation of elemental sulfur by Acidithiobacillus ferrooxidans. In the theoretical section, our knowledge on A. ferrooxidans, its metabolism and the importance of ATP in cell metabolism was summarized. In the experimental part, the above bioprocess was monitored using pH, biomass concentration, the rate of oxidation of elemental sulfur the cellular ATP content.

Producing pharmaceuticals in Escherichia coli inevitably comes with an extensive purification process. This is because many of the native proteins of E. coli are immunogenic to humans, especially the heat and pH resistant endotoxins located in the membrane of E. coli. These native proteins drive up the cost of the purification, which led to a request from the biopharmaceutical company Affibody AB. They want a review on the possibilities of producing their unique Affibody®-molecules in a new, less problematic host cell. Based on a previous bachelor project, Affibody AB chose Pichia pastoris as the candidate. P. pastoris is a methylotrophic yeast that is increasing in use when it comes to producing pharmaceuticals. In this review, multiple ways of utilizing P. pastoris are presented. The process proposals are based on 4 different promoters, pAOX1, pAOX2, pFLD1 and the pGAP. The AOX1- and AOX2-promoters and the FLD1-promoter are inducible promoters that require an inducer-molecule. An inducible promoter presents the best control of the process. The GAP-promoter is a constitutive promoter, meaning that the gene is expressed continuously. A constitutive promoter provides a process which requires fewer steps and ingredients. If the Affibody®-molecules were to be produced with P. pastoris as the host cell, the products would contain less immunogenic substances. Further, P. pastoris is also a very effective option when it comes to producing protein extracellularly. This would ultimately lead to a purification process that requires less resources.

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK∼ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (kcat/KM=6.83 × 106 M-1 sec-1) and DDDDR (kcat/ KM=1.89 × 107 M-1 sec-1) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge-charge interactions in the extended substrate binding pocket.

The serine protease enteropeptidase exhibits a high level of substrate specificity for the cleavage sequence DDDDK∼ X, making this enzyme a useful tool for the separation of recombinant protein fusion domains. In an effort to improve the utility of enteropeptidase for processing fusion proteins and to better understand its structure and function, two substitution variants of human enteropeptidase, designated R96Q and Y174R, were created and produced as active (>92%) enzymes secreted by Pichia pastoris with yields in excess of 1.7 mg/Liter. The Y174R variant showed improved specificities for substrates containing the sequences DDDDK (kcat/KM=6.83 × 106 M-1 sec-1) and DDDDR (kcat/ KM=1.89 × 107 M-1 sec-1) relative to all other enteropeptidase variants reported to date. BPTI inhibition of Y174R was significantly decreased. Kinetic data demonstrate the important contribution of the positively charged residue 96 to extended substrate specificity in human enteropeptidase. Modeling shows the importance of the charge-charge interactions in the extended substrate binding pocket.

Recombinant human mast cell chymase (rhChymase) was expressed in secreted form as an active enzyme in the SuperMan5 strain of GlycoSwitch® Pichia pastoris, which is engineered to produce proteins with (Man) 5(GlcNAc)2 Asn-linked glycans. Cation exchange and heparin affinity chromatography yielded 5 mg of active rhChymase per liter of fermentation medium. Purified rhChymase migrated on SDS-PAGE as a single band of 30 kDa and treatment with peptide N-glycosidase F decreased this to 25 kDa, consistent with the established properties of native human chymase (hChymase). Polyclonal antibodies against hChymase detected rhChymase by Western blot. Active site titration with Eglin C, a potent chymase inhibitor, quantified the concentration of purified active enzyme. Kinetic analyses with succinyl-Ala-Ala-Pro-Phe (suc-AAPF) p-nitroanilide and thiobenzyl ester synthetic substrates showed that heparin significantly reduced KM, whereas heparin effects on kcat were minor. Pure rhChymase with Asn-linked glycans closely resembles hChymase. This bioengineering approach avoided hyperglycosylation and provides a source of active rhChymase for other studies as well as a foundation for production of recombinant enzyme with human glycosylation patterns.

Human mast-cell chymase (EC 3.4.21.39) is a chymotrypsin-like serine protease that is stored in and released from mast-cell granules. This enzyme has been expressed in Pichia pastoris by homologous recombination of the cDNA coding for the mature active chymase into the Pichia genome. Cells producing the highest levels of recombinant human chymase were selected by activity screening and they were grown in a fermentor. Methanol induction resulted in the secretion of active chymase into the Pichia growth media and increasing levels of enzyme were detected in the media for 5 days. Active enzyme was purified from the culture media with a 22 % yield of activity by a simple two-step procedure involving hydrophobic-interaction chromatography followed by affinity chromatography on immobilized heparin. The major peak from the heparin column contained a single band of 30.6 kDa on SDS/PAGE. The purified recombinant human chymase was 96% active and the yield was 2.2 mg/l of growth media.

The methylotrophic yeast. Pichia pastoris, is a powerful, adaptable, and inexpensive recombinant expression system commonly used to secrete heterologous protein. Although P. pastoris is a popular host organism, secretion inefficiency continues to be a major hurdle in its ability to produce high levels of foreign protein. Optimization of cis- and trans-acting factors has greatly enhanced the secretory capabilities of P. pastoris, however protein-specific engineering of a host organism is costly and not always effective. P. pastoris' secretion inefficiency is commonly due to trans-acting factors. Strains of S. cerevisiae have been engineered, through random genomic mutation, that are capable of overcoming these /ram-acting factors to secrete high levels of foreign protein. The Lin-Cereghino laboratory at University of the Pacific has developed a screen to identify mutations in P. pastoris capable of circumventing secretion obstacles. The P. pastoris genome was randomly disrupted through restriction enzyme-mediated integration of an antibiotic resistance marker. Supersecretion mutants were identified by their ability to secrete β-galactosidase, a reporter enzyme not natively secreted by P. pastoris. Sixteen β-galactosidase secretion (bgs) mutants were initially isolated by the Lin-Cereghino lab. This research focused on characterizing one of the resultant bgs mutants, ///. Initial sequencing and alignment studies identified the predicted LI1p sequence to be homologous to S. cerevisiae protein kinase C (PKC). Considering the role of PKC in the Cell Wall Integrity pathway of S. cerevisiae. the cell wall and secretory organelles of III were closely examined using transmission electron microscopy. Additionally, a qualitative alkaline phosphatase assay was used to evaluate the cell wall integrity of ///. Finally, the secretory phenotype of 111 was examined using a group of structurally and functionally diverse reporter proteins. In characterizing the bgs mutant, III, this research contributes to an understanding of cellular components that limit protein secretion in the yeast, P. pastoris.

The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.