Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. epsilon-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield, e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes.
SummaryCandida utilis was cultivated in a 5-literjar fermentor using ethanol as sole carbon source. Control of ethanol in the cultivation broth was performed by using an ethanol vapor monitoring instrument and an oxygen electrode coupled with two control circuits. By setting upper and lower bounds according to the predetermined conditions, a signal from a gas monitoring sensor switched the lower or higher bound relay governing the actuating or switch-off of the motor; this maintained a proper concentration of ethanol in the cultivation of ethanol in the system. The growth of cells was found to be satisfactory. Cell concentration reached 64 g/liter during a 20-hr cultivation. As the results of comparative experiments, the control mode using the gas monitoring instrument was found to be superior to that using dissolved oxygen as a controlling signal, especially at high cell concentration.
A new continuous affinity chromatography for enzyme purification and concentration was proposed. It combined the features of pH-parametric pumping and simulated moving bed operations. Adsorption and desorption were taking place simultaneously in the system. Regeneration of adsorbent was performed by successively shifting the adsorption and desorption sections from one column to another with a time controller. Performance tests for enzyme separation and purification were carried out by devising six-staged affinity chromatographic columns, 16cm ID x 40cm Ht, packed with lOcm height chitin-CHOM each for trypsin purification. Factors affecting the successful operations of the systems and separation efficiency of enzyme were investigated. The system was applied to the purification of trypsin from extract of porcine pancreas. It was found that fairly high separation efficiency can be achieved, SAR (specific activity ratio = specific activity of productlspecific activity of feed) of eluant was as high as 3.9. Thus it is considered as a promising method of enzyme purification. For purification of c~d e enzyme, SMB (Simulated Moving Bed) can be incorporated with a washing section between the adsorption and desorption sections for washing out the impure proteins contaminated into the columns.
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