Carbon metabolism influenced for promoters and temperature used in the heterologous protein production using Pichia pastoris yeast

Abstract Nowadays, it is necessary to search for different high-scale production strategies to produce recombinant proteins of economic interest. Only a few microorganisms are industrially relevant for recombinant protein production: methylotrophic yeasts are known to use methanol efficiently as the sole carbon and energy source. Pichia pastoris is a methylotrophic yeast characterized as being an economical, fast and effective system for heterologous protein expression. Many factors can affect both the product and the production, including the promoter, carbon source, pH, production volume, temperature, and many others; but to control all of them most of the time is difficult and this depends on the initial selection of each variable. Therefore, this review focuses on the selection of the best promoter in the recombination process, considering different inductors, and the temperature as a culture medium variable in methylotrophic Pichia pastoris yeast. The goal is to understand the effects associated with different factors that influence its cell metabolism and to reach the construction of an expression system that fulfills the requirements of the yeast, presenting an optimal growth and development in batch, fed-batch or continuous cultures, and at the same time improve its yield in heterologous protein production.

Biopharmaceuticals from microorganisms: from production to purification

ABSTRACT The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN , , and ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

Biopharmaceuticals from microorganisms: from production to purification

The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN , , and ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.(AU)

Recombinants proteins for industrial uses: utilization of Pichia pastoris expression system

The innovation in industrial process with impact in the efficient production is the major challenge for actual industry. A high numerous of enzymes are utilized in at different level of process, the search for new alternatives with better characteristic has become a field of study of great interest, the recombinant protein achievement in a different host system is an alternative widely assessed for production of this. The microorganism Pichia pastoris has been used like a successful expression system in diverse areas, improved the yield and extraction-recovery of the product expressed. The reported of diverse authors in the production of enzymes with different application in industry is varied, in this review the different industry areas and the characteristic of the enzymes produced are detailed.(AU)

Optimization of clavulanic acid production by Streptomyces daufpe 3060 by response surface methodology

Clavulanic acid is a -lactam antibiotic which has a potent -lactamase inhibiting activity. In order to optimize its production by the new isolate Streptomyces DAUFPE 3060, the influence of two independent variables, temperature and soybean flour concentration, on clavulanic acid and biomass concentrations was investigated in 250 mL-Erlenmeyers according to a 2² central composite design. To this purpose, temperature and soybean flour (SF) concentration were varied in the ranges 26-34°C and 10-50 g/L, respectively, and the results evaluated utilizing the Response Surface Methodology. The experimental maximum production of clavulanic acid (629 mg/L) was obtained at 32°C and 40 g/L SF after 48 h, while the maximum biomass concentration (3.9 g/L) at 30°C and 50 g/L soybean flour, respectively. These values are satisfactorily close to those (640 mg/L and 3.75 g/L, respectively) predicted by the model, thereby demonstrating the validity of the mathematical approach adopted in this study.

Kinetic analysis of batch ethanol acetylation in isothermal non-stationary multiphase systems by lyophilized mycelium of Aspergillus oryzae

A relatively complex network of reactions has been investigated, using as a network model the isothermal batch esterification of acetic acid with ethanol in n-heptane catalyzed by lyophilized mycelium of Aspergillus oryzae. The kinetic analysis was firstly carried out on the whole system, without any simplification, by means of the well-known integral method. Owing to the poor results obtained by this way, we developed an alternative approach, combining initial rates and integral analysis and reducing the number of empirical parameters to be determined by the use of equilibrium data. All the values of the parameters calculated according to this "composite" approach to kinetic analysis well correlate with experimental data.

Partial purification of glucose-6-phosphate dehydrogenase by aqueous two-phase poly(ethyleneglycol)/phosphate systems

Glucose-6-phosphate dehydrogenase (G6PDH) is an important enzyme used in biochemical and medical studies and in several analytical methods that have industrial and commercial application. This work evaluated the extraction of G6PDH in aqueous two-phase system (ATPS) of poly(ethyleneglycol) (PEG)/phosphate buffer, using as enzyme source a medium prepared through commercial baker's yeast disruption. Firstly, the effects of PEG molar mass on the enzyme partition and of homogenization and rest on the system equilibrium were investigated. Afterwards, several ATPS were prepared using statistical analysis (2² factorial design). The results, including kinetic and thermodynamic parameters for the G6PDH activity, showed partial purification of this enzyme in ATPS composed of 17.5% (w/w) PEG400 and 15.0% (w/w) phosphate. A high enzymatic recovery value (97.7%), a high partition coefficient (351), and an acceptable purification factor (2.28 times higher than in cell homogenate) were attained from the top phase. So, it was possible to attain an effective enzyme pre-purification by separating some contaminants with a simple method such as liquid-liquid extraction in aqueous two-phase systems (ATPS).

Glicose-6-fosfato desidrogenase (G6PDH) é uma importante enzima usada em estudos bioquímicos e médicos, bem como em diversos métodos analíticos com aplicação comercial e industrial. Neste trabalho foi avaliado a extração da G6PDH em sistemas de duas fases aquosas (ATPS) constituídos por poli(etilenoglicol) (PEG)/tampão fosfato, usando como fonte de enzima um meio preparado por rompimento de leveduras de panificação comercial. Inicialmente foram investigados os efeitos da massa molar do PEG na partição da enzima e da homogeneização e repouso no equilíbrio do sistema. Na sequência, diversos ATPS foram preparados usando análise estatística (planejamento fatorial 2²). Os resultados, incluindo parâmetros cinéticos e termodinâmicos para a atividade da G6PDH, indicaram parcial purificação desta enzima em ATPS constituídos por 17,5% (p/p) PEG400 e 15,0% (p/p) fosfato. Um alto valor de recuperação enzimática (97,7%), um alto coeficiente de partição (351), e um fator de purificação aceitável (2,28 vezes maior que em homogenato celular) foram obtidos na fase superior do sistema. Assim, foi possível alcançar uma pré-purificação eficaz da enzima separando alguns contaminadores aplicando um método simples tal como a extração líquido-líquido em sistemas bifásicos (ATPS).