Thesis (doctoral)--Helsinki University of Technology, 2009. / Includes bibliographical references. Also available on the World Wide Web.

The yeast expression system Pichia pastoris was investigated as an encapsulation technology capable of serving as a rumen escape vehicle. Cellularly encapsulated protein is protected from the ruminal environment so long as the cell membrane, which surrounds and isolates the intracellular protein is physically intact. Intracellular expression of Green Fluorescent Protein (GFP) allows for the monitoring of cellular integrity as necessary for the protection of encapsulated protein from ruminal proteases. Upon cellular lysis GFP is exposed to extracellular proteases which result in both the proteolytic degradation of the protein-based GFP chromophore and its associated fluorescence. Visualization of rumen fluid under epifluorescent microscopy revealed a high level of background autofluorescence owing to the fluorescent plant particles, microbes, and fluorescent compounds therein. Visualization of GFP in rumen fluid can be optimized through GFP variant selection, filter set design, and light source selection based on bulb emission spectra. Incubation of intracellular GFP expressing P. pastoris in batch culture ruminal in vitro simulations demonstrated that 93%, 97%, and 25% of the P. pastoris inoculum maintained cellular integrity in clarified rumen fluid, bacterial fraction of rumen fluid, and whole rumen fluid, respectively, when incubated over 36 to 48 h. Continuous fermentation in vitro rumen simulations (Rusitec) demonstrated a P. pastoris escape rate of 19% when added daily to fully adapted Rusitec vessels having a dilution rate of 0.75d-1. Abomasal in vitro simulations demonstrated that 84% of the P. pastoris inoculum was lysed within 12 h, as necessary for the release of encapsulated protein. P.pastoris may be an effective post-fuminal delivery vehicle, provided that similar results are obtained in vivo. / xiv, 120 leaves : ill. ; 28 cm.

Mucopolysaccharidosis IIIB (MPS IIIB) is an autosomal recessive disorder of glycosaminoglycan (GAG) metabolism. Disruption of the gene encoding a-N-acetylglucosaminidase (Naglu) results in the inability to degrade the GAG heparan sulfate (HS). Consequently. undegraded HS builds up and results in the secondary accumulation of gangliosides and substantial changes in the expression of genes related to neural cell growth and function. Clinically, affected individuals display hyperactivity. insomnia and severe and progressive mental retardation. Currently. no treatment or cure is available for this devastating disorder which is ultimately fatal. Enzyme replacement therapy is one method being examined as an avenue for treatment of MPS IIIB. but it has yet to overcome difficult obstacles, such as production and targeted delivery. This thesis examines the use of the methylotrophic yeast Pichia pastoris as a host for the production of recombinant Naglu. A protein transduction domain (PTD) derived from the HIV-l Tat protein was fused to Naglu to circumvent the current problems faced in delivering this therapeutic enzyme. Expression of this fusion protein was tested in four different strains of Pichia. each with unique attributes. Though the Naglu produced was in an active recombinant form. it was not abundant and this has precluded further characterization. It is likely that inefficiency at the transcriptional/post-transcriptional level hindered higher expression levels. Optimization of these factors may well facilitate Naglu expression in Pichia pastoris. and ultimately allow for substantial enzyme production for use in replacement therapy.

Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder which is caused by a mutation in the gene encoding acid β-glucocerebrosidase (GBA, EC 3.2.1.45). Deficient activity in GBA leads to a wide variety of clinical phenotypes, including visceral symptoms such as hepatospenomegaly as well as neurological symptoms. Current enzyme replacement therapy is effective in treating visceral symptoms but cannot cross the blood-brain barrier to target neurological manifestations. Another drawback to current therapy is the high cost to patients due to present protein expression strategies. Recently, protein transduction domains, such as the synthetic PTD4 domain, have been proposed as a therapeutic strategy for drug delivery to the central nervous system. In the present study, we use an economical yeast expression system, Pichia pastoris, to produce a recombinant fusion protein GBA-PTD4, and semi-preparative hydrophobic interaction chromatography and gel filtration chromatography for purification. Results show that final preparations are near homogenous, with GBA-PTD4 accounting for approximately 76% of total protein and only one major contaminant. A cell line expressing GBA without a transduction domain was also created in anticipation of further cellular uptake studies. Future research will focus on large scale enzyme expression in fermentation systems and more direct purification methods such as immunoaffinity chromatography for better protein recovery. / Graduate

In order to establish the potential of an esterase from Lactobacillus casei for application in the dairy industry, this study was designed with the following objectives: (1) to determine the optimal cultural conditions for growth and enzyme production, (2) to construct a positive selection vector with low copy number for gene cloning, (3) to clone and sequence the gene encoding for esterase, (4) to biochemically characterize the purified recombinant esterase, and (5) to over-express an esterase of L. casei into Escherichia coli as well as into a methylotrophic yeast, Pichia pastoris using strong promoters. / Maximal growth and enzyme production in L. casei were obtained after 20 h in basal MRS medium containing 1% (w/v) lactose at pH 7.0, 30°C. Among various substrates (C2--C16) tested, the highest activity was towards C6 and C8. Although the enzyme was produced constitutively, tributyrin induced the enzyme production by a 2.5 fold. / A new E. coli and lactic acid bacteria shuttle vector with low copy and positive selection termed pCWL70 was constructed. High transformation efficiency and significant vector stability of pCWL70 were found in L. casei and Lactococcus lactis. / An esterase gene (estI) of L. casei CL96 was localized in a 3.3 kb Bam HI DNA fragment that contains an open reading frame of 1,800 bp. The open reading frame estI was isolated by PCR and subcloned into the E. coli expression vector pET29a, and Pichia expression vector pPICZ B, that allows the inducible expression under the control of the T7 promoter and an alcohol oxidase promoter (AOX1), respectively. E. coli BL21(DE3)pLysS containing estI expressed a novel 67.5 kDa protein corresponding to the EstI in N-terminal fusion with the S · tag peptide. / An esterase of L. casei CL96 was successfully over-expressed in E. coli up to 500 folds (about 25% of total cellular protein) as well as in the P. pastoris. In high cell density fed-batch fermentation, the recombinant Pichia strain containing linearized pCESTc was grown at pH 6.5 and 30°C, and the cell density peaked at about 180 h with 468 g wet cell weight per liter. The final yield was 3.7 x 106 units/ml, which is 980-folds higher than that of native L. casei cells. / The amino acid sequence of EstI indicated that the esterase is a member of a novel GHSMG family of lipolytic enzymes and S-formylglutathione hydrolases (FGH). The putative catalytic triad of EstI consists of residues Ser325, Asp516 and His558 as demonstrated by amino acid sequence alignments. (Abstract shortened by UMI.)

Frankfurt (Main), University, Diss., 2007.

München, Techn. Univ., Diss., 2002.

Techn. Hochsch., Diss., 2000--Aachen.

Techn. Universiẗat, Diss., 2004--Berlin.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2008. / Submitted by Jaqueline Oliveira (jaqueoliveiram@gmail.com) on 2008-12-11T19:02:46Z No. of bitstreams: 1 DISSERTACAO_2008_JulianaAmorimAraujo.pdf: 3037793 bytes, checksum: a7399845548557e765e50c02f6d3d986 (MD5) / Approved for entry into archive by Georgia Fernandes(georgia@bce.unb.br) on 2009-02-20T15:27:14Z (GMT) No. of bitstreams: 1 DISSERTACAO_2008_JulianaAmorimAraujo.pdf: 3037793 bytes, checksum: a7399845548557e765e50c02f6d3d986 (MD5) / Made available in DSpace on 2009-02-20T15:27:14Z (GMT). No. of bitstreams: 1 DISSERTACAO_2008_JulianaAmorimAraujo.pdf: 3037793 bytes, checksum: a7399845548557e765e50c02f6d3d986 (MD5) / A coagulação enzimática do leite para fabricação de queijo envolve a quebra de uma ligação peptídica da caseína por uma protease aspártica. Tradicionalmente, esta clivagem é feita em escala industrial pela ação da quimosina bovina que combina uma forte atividade de coagulação do leite com uma baixa atividade proteolítica. No presente estudo, é relatada a expressão, purificação e caracterização de quimosina recombinante bovina na levedura metilotrófica Pichia pastoris. Para alcançar altos níveis de produção, o cDNA do fragmento codante da pró-quimosina B bovina foi desenhado e sintetizado in vitro otimizando o codon usage para a expressão em P. pastoris. O gene sintético (CHYMb) foi clonado em vetores de expressão contendo os promotores PGK1 (expressão constitutiva) e AOX1 (expressão induzida) seguindo-se integração no genoma da levedura. Os clones transformantes com maior produção enzimática de cada sistema (P1 e A3, respectivamente) foram analisados em culturas simultâneas para comparação da produção enzimática. Maiores níveis de atividade de quimosina foram observados no clone sob o comando do promotor PGK1, que apresentou alta produção durante toda a cultura. Uma otimização na produção enzimática foi desenvolvida através de planejamentos fatoriais. A influência da densidade celular inicial, metanol e concentração da fonte de nitrogênio foram avaliados para aumentar a produção do clone A3. A interação dos fatores densidade celular inicial e concentração de metanol foi determinada como sendo o parâmetro mais importante, dobrando a produção. A influência de diferentes concentrações da fonte de nitrogênio e carbono, previamente selecionados (farelo de soja e açúcar invertido), foi também analisada para aumentar a produção enzimática no clone P1. Foi demonstrado por análise estatística que maiores concentrações de cada fator aumentam a produção. O perfil de atividade enzimática foi avaliada em fermentador com as condições determinadas nas otimizações. A atividade encontrada nos dois casos (A3 e P1) foi superior àquelas encontradas na fermentação em frascos. A quimosina recombinante foi posteriormente purificada apenas por uma etapa de cromatografia de exclusão molecular. Foi demonstrada a presença de enzima já processada no sobrenadante da cultura e uma fração de proteínas glicosiladas. A especificidade pela caseína foi demonstrada ser semelhante à quimosina comercial recombinante produzida por Aspergillus niger (Chr. Hansen). A alta produção de quimosina recombinante com alta especificidade em P. pastoris faz esse sistema de expressão atrativo para produção industrial desta enzima. _______________________________________________________________________________ ABSTRACT / Enzymatic milk coagulation for cheese manufacturing involves the cleavage of the scissile bond in casein by an aspartic protease. Bovine chymosin B is commonly used at the industrial level for milk coagulation because it combines a strong clotting activity with a low general proteolytic activity. In the present study, we report the expression, purification and characterization of recombinant chymosin B expressed in the methylotrophic yeast Pichia pastoris. To achieve high levels of production, the cDNA fragment coding for prochymosin was designed and synthesized based on codon usage optimization for expression in P. pastoris. The synthetic gene (CHYMb) was cloned into expression vectors based on the PGK1 promoter (constitutive expression) and AOX1 promoter (inducible expression). The resulted constructs were integrated in the yeast genome. Transformant clones producing high levels of enzyme activity from each system (P1 and A3, respectively) were analyzed in a simultaneous culture for comparison of enzymatic production. High levels of chymosin activity were observed in the clone controlled by the PGK1 promoter which showed high production levels throughout the culture. Optimization for enzymatic production was developed through factorial design. The influence of the initial cellular density, methanol and nitrogen source concentration were accessed for enzymatic production from clone A3. The interaction of the initial cellular density and methanol concentration was responsible for doubling enzyme production. The influence of different concentrations of nitrogen and carbon sources (soy extract and inverted sugar, respectively) was also observed to increase enzymatic production in clone P1. Statistic analysis demonstrated that higher concentrations of each factor increased enzyme production. The profile of enzymatic activity was evaluated in fermentors under the conditions determined during optimizations. In this case, the activities observed in clones A3 and P1 were superior to those found when theses clones were grown in shake flasks. The recombinant chymosin was purified by a single molecular exclusion chromatographic step. Purified enzyme derived from supernatant fraction was shown to be present in its processed form and a fraction of glycosylated protein was also detected. The specificity for casein was shown to be similar to that from commercial recombinant chymosin produced by Aspergillus niger (Chr. Hansen). The high production and high specificity of recombinant chymosin derived from P. pastoris renders this expression system as an attractive alternative for large scale industrial production of this enzyme.