Enzymes cell disruption

A 1000-litre fermenter has been used to produce a continuous feed of Escherichia coli containing a high level of j3-D alactosidase. Investigation of the individual unit operations for the isolation of the enzyme, cell disruption, nucleic acid removal, protein precipitation and solid-liquid separation after each stage, permitted operation of a semi-continuous process which would yield 130 g protein hr comprising 23% j3-D-galactosidase. 

Biotechnology Cell disruption Yeast disruption for enzyme extraction... 

These units have been used to disrupt bacterial cells for release of enzymes. (See Cell Disruption. )... 

Chemical lysis, or solubilization of the cell wall, is typically carried out using detergents such as Triton X-100, or the chaotropes urea, and guanidine hydrochloride. This approach does have the disadvantage that it can lead to some denaturation or degradation of the produci. While favored for laboratory cell disruption, these methods are not typically used at the larger scales. Enzymatic destruction of the cell walls is also possible, and as more economical routes to the development of appropriate enzymes are developed, this approach could find industrial application. Again, the removal of these additives is an issue. 

CASE STUDY PROCESS INTEGRATION OF CELL DISRUPTION AND FLUIDISED BED ADSORPTION FOR THE RECOVERY OF LABILE INTRACELLULAR ENZYMES... 

Keywords cell disruption process integration fluidised bed adsorption intracellular enzymes protein recovery... 

The primary purification of the enzyme G3PDH was exploited herein as a preliminary study to investigate and demonstrate the feasibility of the integrated operation of cell disruption by bead milling and immediate product capture by fluidised bed adsorption (panel A in Figure 17.6). Yeast G3PDH binds nicotinamide adenine dinucleotide (NAD) as a cofactor,... 

From the above description it will be appreciated that the efficiency of release of nutrients from ingested plant material is dependent upon the ease with which the digestive enzymes can penetrate the cell wall to release the nutrients so that they can diffuse out of the structure to be absorbed. Thus tissue maturity, cooking, macerating, mastication and mode of tissue failure, all of which control particle size, cell wall softening or cell disruption, are key features which regulate nutrient release. 

Balasundaram B, Pandit AB (2001) Significance of location of enzymes on their release during microbial cell disruption. Biotech Bioeng 75 607-614... 

Farkade VD, Harrison STL, Pandit AB (2005) Heat induced translocation of proteins and enzymes within the cells an effective way to optimize the microbial cell disruption process. Biochem Eng J 23 247-257... 

Table 6.1 Some chemical, physical and enzyme-based techniques that may be employed to achieve microbial cell disruption...

Various enzymes are produced intracellularly. Hence, following cell harvesting, an efficient disruption process to disintegrate the cell to release the intracellular proteins is needed. Some types of cells are broken readily by gentle treatment, while others are very resistant to breakage. A number of cell disruption methods have been developed ... 

More recently the biotransformation of limonene by another Pseudomonad strain, P. gladioli was reported [76,77]. P. gladioli was isolated by an enrichment culture technique from pine bark and sap using a mineral salts broth with limonene as the sole source of carbon. Fermentations were performed during 4-10 days in shake flasks at 25°C using a pH 6.5 mineral salts medium and 1.0% (+)-limonene. Major conversion products were identified as (+)-a-terpineol and (+)-perillic acid. This was the first time that the microbial conversion of limonene to (+)-a-terpineol was reported, see pathway 4. The conversion of limonene to a-terpineol was achieved with an enzyme, a-terpineol dehydratase (a TD), by the same group [78]. The enzyme, purified more than tenfold after cell-disruption of Pseudomonas gladioli, stereospecifically converted (4 )-(+)-limonene to (4/ )-(+)-a-terpineol or (4S)-(+)-limonene to (4S)-(+)-a-terpineol. a-Terpineol is widely distributed in nature and is one of the most commonly used perfume chemicals [27]. 

Many biological cells contain degradative enzymes (proteases) that catalyze the hydrolysis of peptide linkages. In the intact cell, functional proteins are protected from these destructive enzymes because the enzymes are stored in cell organelles (lysosomes, etc.) and released only when needed. The proteases are freed upon cell disruption and immediately begin to catalyze the degradation of protein material. This detrimental action can be slowed by the addition of specific protease inhibitors such as phenylmethyl-sulfonyl fluoride or certain bioactive peptides. These inhibitors are to be used with extreme caution because they are potentially toxic. 

Cell disruption techniques are used to recover materials produced within the cell, for example, industrial enzymes and some pharmaceutical proteins. Generally this stage of bioseparation will follow cell recovery, for example, by centrifugation, and precede the isolation of the desired product from the cell debris which is also produced during the disruption process. 

Intracellular Products. Intracellular production of bioproducts is less preferable but sometimes the only way to produce certain compounds in appreciable amounts. In this case, cell disruption is required for recovery. High-pressure homogenization, bead mills, and chemical or enzymatic disruption of the cell wall with lysozyme or similar enzymes can be used to achieve cell breakage. In the case of small molecules, organic solvent extraction has also been described. If cell debris remains in the centrate, it must be removed by methods described above, thus adding extra steps to the process. 

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