Showing papers on "Downstream processing published in 2002"

An overview on fermentation, downstream processing and properties of microbial alkaline proteases

Abstract: Microbial alkaline proteases dominate the worldwide enzyme market, accounting for a two-thirds share of the detergent industry. Although protease production is an inherent property of all organisms, only those microbes that produce a substantial amount of extracellular protease have been exploited commercially. Of these, strains of Bacillus sp. dominate the industrial sector. To develop an efficient enzyme-based process for the industry, prior knowledge of various fermentation parameters, purification strategies and properties of the biocatalyst is of utmost importance. Besides these, the method of measurement of proteolytic potential, the selection of the substrate and the assay protocol depends upon the ultimate industrial application. A large array of assay protocols are available in the literature; however, with the predominance of molecular approaches for the generation of better biocatalysts, the search for newer substrates and assay protocols that can be conducted at micro/nano-scale are becoming important. Fermentation of proteases is regulated by varying the C/N ratio and can be scaled-up using fed-batch, continuous or chemostat approaches by prolonging the stationary phase of the culture. The conventional purification strategy employed, involving e.g., concentration, chromatographic steps, or aqueous two-phase systems, depends on the properties of the protease in question. Alkaline proteases useful for detergent applications are mostly active in the pH range 8–12 and at temperatures between 50 and 70°C, with a few exceptions of extreme pH optima up to pH 13 and activity at temperatures up to 80–90°C. Alkaline proteases mostly have their isoelectric points near to their pH optimum in the range of 8–11. Several industrially important proteases have been subjected to crystallization to extensively study their molecular homology and three-dimensional structures.

Enzymatic production of cyclodextrins

Abstract: Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly α-amylases. Due to its low solubility in water, separation and purification of β-CD is relatively easy compared to α- and γ-CD. In recent years more economic processes for γ-CD and especially α-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of α-CD production processes is presented.

Reverse micellar extraction for downstream processing of proteins/enzymes.

Abstract: New developments in the area of downstream processing are, hopefully, to fulfill the promises of modern biotechnology. The traditional separation processes such as chromatography or electrophoresis can become prohibitively expensive unless the product is of high value. Hence, there is a need to develop efficient and cost-effective downstream processing methods. Reverse micellar extraction is one such potential and a promising liquid-liquid extraction technique, which has received immense attention for isolation and purification of proteins/enzymes in the recent times. This technique is easy to scale-up and offers continuous operation. This review, besides briefly considering important physico-chemical and biological aspects, highlights the engineering aspects including mass transfer, mathematical modeling, and technology development. It also discusses recent developments in reverse micellar extraction such as affinity based separations, enzymatic reactions in reverse micelles coupled with membrane processes, reverse micellar extraction in hollow fibers, etc. Special emphasis has been given to some recent applications of this technique.

Large scale production and downstream processing of a recombinant porcine parvovirus vaccine.

Abstract: Porcine parvovirus (PPV) virus-like particles (VLPs) constitute a potential vaccine for prevention of parvovirus-induced reproductive failure in gilts. Here we report the development of a large scale (25 l) production process for PPV-VLPs with baculovirus-infected insect cells. A low multiplicity of infection (MOI) strategy was efficiently applied avoiding the use of an extra baculovirus expansion step. The optimal harvest time was defined at 120 h post-infection at the MOI used, with the cell concentration at infection being 1.5x10(6) cells/ml. An efficient purification scheme using centrifugation, precipitation and ultrafiltration/diafiltration as stepwise unit operations was developed. The global yield of the downstream process was 68%. Baculovirus inactivation with Triton X-100 was successfully integrated into the purification scheme without an increase in the number of process stages. Immunogenicity of the PPV-VLPs tested in guinea pigs was similar to highly purified reference material produced from cells cultured in the presence of serum-containing medium. These results indicate the feasibility of industrial scale production of PPV-VLPs in the baculovirus system, safety of the product, and the potency of the product for vaccine application.

Large-scale extraction of proteins

Abstract: The production of foreign proteins using selected host with the necessary posttranslational modifications is one of the key successes in modern biotechnology. This methodology allows the industrial production of proteins that otherwise are produced in small quantities. However, the separation and purification of these proteins from the fermentation media constitutes a major bottleneck for the widespread commercialization of recombinant proteins. The major production costs (50-90%) for typical biological product resides in the purification strategy. There is a need for efficient, effective, and economic large-scale bioseparation techniques, to achieve high purity and high recovery, while maintaining the biological activity of the molecule. Aqueous two-phase systems (ATPS) allow process integration as simultaneously separation and concentration of the target protein is achieved, with posterior removal and recycle of the polymer. The ease of scale-up combined with the high partition coefficients obtained allow its potential application in large-scale downstream processing of proteins produced by fermentation. The equipment and the methodology for aqueous two-phase extraction of proteins on a large scale using mixer-settlerand column contractors are described. The operation of the columns, either stagewise or differential, are summarized. A brief description of the methods used to account for mass transfer coefficients, hydrodynamics parameters of hold-up, drop size, and velocity, back mixing in the phases, and flooding performance, required for column design, is also provided.

Host selection as a downstream strategy: Polyelectrolyte precipitation of β‐glucuronidase from plant extracts

Abstract: Host selection can be a strategy to simplify downstream processing for protein recovery. Advancing capabilities for using plants as hosts offers new host opportunities that have received only limited attention from a downstream processing perspective. Here, we investigated the potential of using a polycationic precipitating agent ( polyethylenimine; PEI ) to precipitate an acidic model protein ( β- glucuronidase; GUS) from aqueous plant extracts. To assess the potential of host selection to enhance the ease of recovery, the same procedure was applied to oilseed extracts of canola, corn ( germ), and soy. For comparison, PEI precipitation of GUS was also evaluated from a crude bacterial fermentation broth. Two versions of the target protein were investigated — the wild-type enzyme (WTGUS) and a genetically engineered version containing 10 additional aspartates on each of the enzyme's four homologous subunits (GUSD10). It was found that canola was the most compatible expression host for use with this purification technique. GUS was completely precipitated from canola with the lowest dosage of PEI (30 mg PEI/g total protein), and over 80% of the initial WTGUS activity was recovered with 18-fold purification. Precipitation from soy gave yields over 90% for WTGUS but only 1.3-fold enrichment. Corn, although requiring the most PEI relative to total protein to precipitate ( 210 mg PEI/g total protein for 100% precipitation), gave intermediate results, with 81% recovery of WTGUS activity and a purification factor of 2.6. The addition of aspartate residues to the target protein did not enhance the selectivity of PEI precipitation in any of the systems tested. In fact, the additional charge reduced the ability to recover GUSD10 from the precipitate, resulting in lower yields and enrichment ratios compared to WTGUS. Compared to the bacterial host, plant systems provided lower polymer dosage requirements, higher yields of recoverable activity and greater purification factors. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 148–154, 2002.

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization.

Abstract: In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.

Process Design for Enzymatic Adipyl-7-ADCA Hydrolysis

Abstract: Adipyl-7-ADCA is a new source for 7-aminodeacetoxycephalosporanic acid (7-ADCA), one of the substrates for antibiotics synthesis. In this paper, a novel process for enzymatic 7-ADCA production is presented. The process consists of a reactor, a crystallization step, a membrane separation step, and various recycle loops. The reactor can either be operated batch-wise or continuously; with both types of processing high yields can be obtained. For batch reactors chemical degradation of 7-ADCA can be neglected. For continuous reactors, chemical stability of 7-ADCA is a factor to be taken into account. However, it was shown that the reaction conditions and reactor configuration could be chosen in such a way that also for continuous operation chemical degradation is not important. Downstream processing consisted of crystallization of 7-ADCA at low pH, followed by a nanofiltration step with which, at low pH, adipic acid could be separated from adipyl-7-ADCA and 7-ADCA. The separation mechanism of the nanofilter is based on size exclusion combined with charge effects. Application of this filtration step opens possibilities for recycling components to various stages of the process. Adipic acid can be recycled to the fermentation stage of the process while both adipyl-7-ADCA and 7-ADCA can be returned to the hydrolysis reactor. In this way, losses of substrates and product can be minimized.

Continuous annular chromatography.

Abstract: In recent years the demand for process scale chromatography systems in the industrial downstream process has been increasing steadily. Chromatography seems to be the method of choice when biological active compounds must be recovered from a mixture containing dozens of side products and contaminants as it is for example the case when processing fermentation broths. Since chromatography can solve almost any separation problem under mild operating conditions, a continuous chromatography system represents an extremely attractive and powerful option for such large-scale applications. The increasing number of biotechnological products forces system suppliers of the downstream processing side to develop new and improved high throughput purification technologies.

Cell separation, flocculation.

Abstract: Introduction Physicochemical Aspects of Aggregation Interactions between Particles Rate of Aggregation Surface Properties of Particles Brewing Yeast: A Model for the Implication of Specific Interactions in Flocculation Flocculation and Flocculence Yeast Behavior and Cell Surface Properties Genetic and Biochemical Aspects Toward a Comprehensive View of the Role of Specific and Nonspecific Interactions Flocculation in Downstream Processing Use of Colloidal Particles Use of Salts Use of Soluble Polymers Combined Use of Flocculating Agents Use of Specific Interactions Flocs as a State of Proliferating Biomass Continuous Ethanol Fermentation Activated Sludge Microbial Aggregates in Anaerobic Wastewater Treatment Conclusion Acknowledgments Bibliography Keywords: activated sludge; aggregation; brewing yeast; coagulation; downstream processing; flocculation; hydrophobicity; interfaces; separation; zeta potential

Expanded Bed Affinity Chromatography

Abstract: One of the promising methods that has the capability of handling large and dilute crude extracts for adsorptive purification of proteins and enzymes in a speedy fashion is fluidized bed or expanded bed adsorption. Fluidized or expanded bed adsorption has emerged over the last decade as an important technique that can exploit affinity interactions and yet handle particulate matter-containing feeds at high throughput rates while retaining most of the advantages offered by packed beds. This chapter is aimed at addressing some of the theoretical and practical aspects of expanded bed adsorption (EBA) technology. Particular attention is given to its use as an affinity separation method that can in most circumstances be used early on in a downstream processing (DSP) scheme. Details of the quantitative methods used to analyze the performance of an EBA operation and scale-up criteria are presented and discussed.

Kinetic modeling of the anion-exchange process of glucoamylases I and II from Aspergillus niger in batch stirred tank

Abstract: Purification of high added value products obtained by fermentation processes is a key research task in current biotechnology, especially the ones concerning proteins of very similar molecular structure such as isoenzymes. Purification and separation of biomolecules by commercial ion exchangers is an attractive alternative to affinity chromatography due to its availability and lower cost. For this reason and for its high uptake capacity there is an increasing interest in the application of ion exchangers in biotechnological downstream processing. In this paper, the kinetics of ion exchange of the two isoenzymes of glucoamylase from Aspergillus niger on the anion exchanger DEAE-Toyopearl 650 have been investigated in a batch stirred tank. The experimental results were fitted to a mathematical model accounting for both external fluid film mass transfer resistance and pore diffusivity. In addition, the kinetic parameters involved in the process have been obtained for each isoenzyme.

Advances in Matrices for Expanded Bed Adsorption

Abstract: Expanded bed adsorption (EBA) is a new technique for protein separation and purification, which sprung up in the past decade. EBA has been found to be widespread potential applications in the downstream processing of bioengineering. Some new properties of matrices must be added to this technique owing to its high requests. Advances in matrices for EBA, including some properties required?matrices in use as well as their preparation methods, are reviewed in this paper.

Systematic selection of down stream processing techniques.

Abstract: This project began as an extension of the work undertaken by Harrison, (1996) which looked at the large-scale downstream processing of antibody fragments. After this preliminary work a change was made to the structure of research, whereby I began to look at the theoretical basis for the systematic selection of downstream processing techniques. This thesis examines the reports of protein recovery in the literature as an attempt to create a means of choosing the most appropriate process. The work described focuses on the selection of downstream processing techniques for the purification of therapeutic proteins. Analysing the data collected from 96 protein purification papers published in 1994, attempted to find a factor that could be used to combine yield and purification factor, thereby generating an Operational Effectiveness. This is described as the fraction of contaminant removed for a 95% yield. A review of the most frequently used chromatography steps and the sequence for their use was also undertaken. The order of effectiveness for the steps was given as affinity chromatography = hydrophobic interaction chromatography > ion exchange = gel filtration > precipitation. The major costs of the production of therapeutic proteins incurred during downstream processing are also investigated. This was aided by communications with a number of European biotechnology companies, who provided costings from their companies for the manufacture of therapeutic protein drugs. The overall cost for the production of 100g of a monoclonal antibody was estimated at £100,000. The good manufacturing practice (GMP) requirements for antibody fragment production were also assessed, and shown to require at least 3 chromatography stages to meet the regulatory agencies.