Yeast cells disruption

Biotechnology Cell disruption Yeast disruption for enzyme extraction... 

Method of cell disruption yeast suspension 50% wet weight is passed through a bead mill using 0.5 mm glass beads... 

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,... 

The subcellular location of PG was studied in cells disrupted by osmotic lysis through formation and disruption of sphaeroplasts from self-induced anaerobically-grown cells. A discontinuous sucrose-density gradient produced four bands labelled I, II, III and IV. Band I included many vesicles and a peak of alkaline phosphatase activity (a vacuolar marker in yeasts), NADPH cytochrome c oxidoreductase activity, an endoplasmic reticulum marker, and... 

The ability to derive the intrinsic mechanical properties of yeast cells should now allow the mechanisms of high-pressure homogenisation to be determined unambiguously, and a priori predictions of the extent of cell disruption to be made for given homogeniser conditions. This should allow better process optimisation. 

To localize the precursors of Acp, the low molecular weight compounds present in yeast cells were isolated by cell disruption, centrifugation and ultrafiltration (Schieberle, P., in preparation). Boiling and continous extraction of a phosphate buffer solution containing the compounds of a molecular weight lower than 1000 produced substantial amounts of Acp. Furthermore, the free proline content of the yeast used in these experiments was analysed and calculated to be more than 200 mg/kg yeast. 

The only disadvantage of the succinylation procedure (which is practical and amenable to conventional cell disruption processes) is that the final product is a succinylated protein. Succinyl groups cannot be removed from the succinylated proteins under mild conditions. This could be a problem if succinylated yeast protein was a major source of dietary proteins. Therefore we explored the feasibility of using reversible modifying reagents (citraconic anhydride and maleic anhydride) to separate proteins from NAs and subsequently remove the modifying groups under mild acidic conditions. 

Isolation of Proteins with a Reduced Nucleic Acid Level. The procedure is virtually identical to that described for succinylation of yeast proteins (87). In a typical experiment proteins, together with NA, were extracted from the disrupted yeast cells at pH 8.5-9.0 and centrifuged at 15,000 rpm for 30 min at 5°C. Citraconic anhydride then was added in small increments to the supernatant with constant stirring while the pH was maintained between 8.0-8.5 by adding 3.5IV NaOH. After the stabilization of the pH, the pH of the solution was decreased to 4.2 to precipitate the proteins. Protein then was separated by centrifugation, dissolved in water (pH adjusted 8.5), dialyzed extensively against water (pH 8.5) at 5°C, and lyophilized. 

Another use of cell disruption as a step in the analytical process is for obtaining a suspension of single cells — that can be used under optimal fermentation conditions — by ultrasonic disruption of cells manufactured in active dry wine yeast. Their potential was confirmed by comparing the elution profiles of non-sonicated and sonicated yeast sample dispersions obtained using two different field flow fractionation techniques [88]. 

Comparison of Maltodextrin and Dextran ATPS. Direct comparisons between DP 10/PEG and dextran/PEG systems for enzyme purification were made by extracting YADH from both YEC and a crude slurry of disrupted yeast cells (YH). These data are shown in Table 2. The Kf and Kadh were virtually the same for either dextran/PEG or MD/PEG and YEC. The partition coefficient for total protein, Kpro/ in the dextran/PEG system was 2-3 times higher, while the purification factors were 50% greater in the DP 10/PEG system. The partition coefficients for both total protein and YADH drop dramatically, however, while the purification factors in both cases are increased relative to the same parameters for YEC. 

Lin et al. (55,63) have described a method to lyse baker s yeast cells using subcritical and SCF CO2 (25-35°C, 7-34 MPa). In a batch process, pressurized CO2 was allowed to permeate the cells for a fixed time, followed by rapid depressurization. The rapid depressurization resulted in explosive expansion of the CO2 within the cells, leading to cell rupture and release of the cellular components. SCF CO2 at 35°C and 20 MPa was the most effective for enzyme release. Higher temperatures were more effective at cell disruption, but they also resulted in lower activity of the released enzymes. Enzyme release from baker s yeast cells was further enhanced with the addition of a cell-wall-lytic enzyme, (3-glucuronidase, to the cell mass before pressurization (55). 

Cereghino GPL, Cereghino JL, Ilgen C et al. (2002) Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris. Curr Opin Biotechnol 13 329-332 Chan WK, Belfort M, Belfort G (1991) Protein overproduction in Escherichia coli RNA stabilization, cell disruption and recovery with a cross-flow microfiltration membrane. J Biotechnol... 

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