Yeast protein recovery from

Use of Beta-Glucanases as an Aid in Protein Recovery from Yeast... 

In conjunction with research on protein extraction from yeast, we investigated methods for the maximum recovery of protein possessing good functional properties but low in nucleic acid. Therefore, we examined the feasibility of making the yeast protein resistant to proteolysis during extraction and nucleic acid reduction. Using established extraction procedures (76), we observed... 

From the applied point of view where cell wall lytic enzymes are to be used for improvement in protein recovery and protein processing from yeast cells, a number of areas are much in need for further research. One of the problems of using microbial enzymes for this purpose is their inducible nature. Investigations have shown that yeast cell walls are the best inducers, followed by /3-(l —>3)- and / -(1 6)-glucans, while... 

In subunit R2 of ribonucleotide reductase there is a tyrosyl radical (Y ) in close proximity to a di-iron cluster.100 In the protein from E. coli the EPR signal from Y can be observed up to room temperature. However, in the protein from yeast the Y signal broadens above 15 K and is not observable above about 60 K. Saturation recovery measurements at 140 GHz showed that at 60 K the spin-lattice relaxation rates for the Y signal in the yeast protein were about 2 orders of magnitude faster than for the E. coli protein. The temperature dependence of the relaxation enhancement was consistent with the activation energy for the first excited state of the di-iron cluster, so the relaxation enhancement was attributed to interaction with the di-iron cluster. Relaxation enhancements measured at 140 GHz showed little orientation dependence so the enhancement was assigned to isotropic exchange, which is different from the orientation-dependent dipolar interaction observed for the E. coli protein.100... 

An interesting illustration of the roles played by low-molecular-mass protein stabilizers in assisting cells to cope with heat stress is provided by work of Singer and Lindquist (1998a,b), who examined the complementary roles of the disaccharide trehalose and heat-shock proteins in recovery from thermal stress by the yeast Saccharomyces cerevisiae. Many types of yeast produce high concentrations of... 

Chemical modification of yeast protein has received limited attention though as described above it has potential as a method for facilitating recovery of yeast protein. Current studies are concerned with determination of the functional properties of proteins succinylated during the extraction. The composition of yeast proteins prepared by different methods is shown (Table 8). Noteworthy is the protein and nucleic acid concentration in the yeast isolate which differed from the concentrate in that cell wall material was removed by centrifugation. 

Inactivation of alcohol dehydrogenase from yeast with 14C-labeled [3-(3-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation showed the presence of 1-carboxymethyl histidine66. After inactivation of the enzyme with labeled [3-(4-bromoacetylpyridinio)-propyl]-adenosine pyrophosphate followed by oxidation, S-carboxymethyl cysteine was identified in the protein. In the case of glyceraldehyde-3-phosphate dehydrogenase, treatment with either coenzyme analogue leads to the modification of the cysteine residue. Treatment with [14C]nicotinamide-5-bromo-4-methylimidazole dinucleotide did not reveal any modified amino-acid-residues. The labeled nicotinamide residue split off during the recovery of the inactivated enzyme. Attempts to synthesize an inactivator labeled with a 14C-acetyl residue did not give satisfactory yields. If the enzyme-coenzyme derivative was treated with tritiated sodium boron hydride, tritium could be introduced (Fig. 22). Studies with... 

The structured model is consistent with features of lytic enzyme action and yeast structure reported in the literature. The sequential removal of the two wall layers, followed by protoplast rupture, accurately describes the early lag in protein and carbohydrate release. The presence of residual solids at long reaction times was accounted for stabilization of protoplasts by substances released from lysed cells. The structured model can be used to estimate the effects of several process alternatives, as shown in a simulation of a process for recovery of site-linked enzymes from yeast. 

Hostettmann K, Marston A, Hostettmann M (1998) Preparative chromatography techniques applications in natural product isolation. Springer, Berlin, Heidelberg, New York, 244 pp Hua ZC (1997) Renaturation and purification of recombinant tissue-type plasminogen activator expressed in E. coli. lUBMB Life 41(4) 815-820 Huang RB, Andrews BA, Asenjo JA (1991) Diflerential product release (DPR) of proteins from yeast a new technique for selective product recovery from microbial cells. Biotechnol Bioeng 38 977-985... 

Certain polypeptides resulting from protease digestion of proteins contribute to the typical taste of savoury foods. The DNA sequence coding for an octapep-tide known as beefy meaty peptide was cloned into yeast as a fusion with the yeast a factor to be secreted as free octapeptide into the medium which facilitated its recovery [37]. Alternatively, intracellular expression of tasty peptides... 

For the conditions in Fig. 9, improvements of 35-50% in the permeate flux were observed when a secondary membrane was used, owing to a reduction in the protein fouling of the primary membrane. Little or no flux recovery was observed with each backpulse, as might be expected from the relatively low resistance of the yeast layer and the irreversible nature of the protein fouling. The flux continuously declined with time owing to irreversible fouling, though the rate of decline was reduced by the SMY. 

Feedstocks of yeast cell suspensions are relatively common starting material, as the yeast cell usually exports the expressed recombinant protein efficiently in the culture medium. A process was developed for the recovery of human serum albumin produced in Pichia pastoris using a 5 cm diameter column run in expanded-bed mode and scaled up directly to a 100 cm diameter column using 150 liters of cationic exchangers. Using this size of column, it was possible to apply a 1900 liter cell suspension. The yield of human serum albumin varied from 83 to 91% over four different runs.22... 

Succinylation reduced the isoelectric point of yeast proteins from pH A.5 to 4.0 and markedly improved their solubility in pH range 4.5 to 6. Heat denatured yeast proteins were facilely solubilized following succinylation (74). Succinylated yeast proteins were very stable to heat above pH 5, and remained soluble at temperatures above 80°C. As the degree of succinylation was increased the rate of precipitation of the derivatized protein increased in the neighborhood of the isoelectric point and much larger protein floes were obtained facilitiating their recovery (74). 

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