Chymotrypsin, purification

Trypsin and chymotrypsin. Purification of both trypsin and chymo-trypsin by affinity chromatography is now a routine tool in many laboratories. Most commercial trypsin preparations are contaminated with trace amounts of chymotrypsin which restricts the use of the enzyme in protein structural studies. However, the soya trypsin inhibitor-agarose media provides a convenient column for separation of the enzymes. 

For desymmetrization of diesters 3 via their hydrolysis in water, pig Hver esterase [12], o -chymotrypsin [12, 13a], and Candida antarctica Hpase (CAL-B) [14] were successfully used. However, further studies showed that respective anhydrides 5 can be used as substrates for enzyme-catalyzed desymmetrization in organic solvents [15]. The desired monoesters 4 were obtained in high yield in this way, using immobilized enzymes Novozym 435 or Chirazyme L-2 (Scheme 5.3). After the reaction, enzymes were filtered off, organic solvents were evaporated, and the crude products were crystalHzed. This was a much simpler experimental procedure in which control of the reaction progress was not necessary, and aU problems associated with extraction of products from aqueous phase and their further purification were omitted [15]. 

SEC is a useful tool for monitoring enzyme reactions, as seen in Figure 4.21, where the speed of decomposition of /Mactoglobulin by a-chymotrypsin is shown. SEC is widely used for purification of proteins, but the separation is due only to the difference in molecular mass. Therefore, ion-exchange liquid chromatography is combined with SEC to improve the selectivity. 

Extraction and purification of proteins by employing RMs have been extensively studied using model systems (proteins from commercial suppliers) such as cytochrome C, ribonuclease, a-chymotrypsin, etc. However, very few reports are available on the extraction studies of proteins from the real systems of fermentation broths using RMs [ 15,43]. As we know that fermentation broths containing proteins are very much more complicated media compared to model proteins and mixture of model proteins, there is a need to investigate extraction behavior of proteins present in these real systems. 

Fig. 8. Purification of chain C by chromatography on Sephadex G-50 (79). Sepha-dex G-50 column (1.8 X 35 cm) equilibrated with 0.05 M HCl. Lyophilized aqueous extract of 30 mg of DFP-inhibited, performic acid-oxidized a-chymotrypsin (A4) is dissolved in 1 ml 0.05 M HCl, put into the column, and eluted (5 ml/hr) by 0.05 M HCl. Solid line, absorption of the fractions at 280 mu. Dotted line, absorption at 230 mil. Ordinates, optical density. Abscissas, volume of eluatein milliliters. A, chain A C, chain C.

The GMP activity that is used in alginate synthesis is associated with the algA gene product, which also catalyzes the PMI reaction. The first indications that AlgA is bifunctional came when the protein was purified. The isomerase and pyrophosphorylase activities co-purified, and their activities remained in constant ratio throughout the purification. Brief treatment with chymotrypsin cleaved approximately 1 kDa from the C-terminus of AlgA the remaining protein retained GDP-pyrophosphorylase activity, but the phosphoman-nose isomerase activity was lost. ... 

Although [Cr(OH2)6] and [Cr(OH)(OH2)5] continue to be the main starting species for anation studies (Table 6.5), there is a growing interest in the use of other aqua ions, such as ci5-[Cr(big)2(OH2)2] " or [Cr(ox)2(OH2)2] . In view of the interest in the biochemical role of Cr(III) in the Glucose Tolerance Factor (GTF), several Cr(III) aqua ions have been reacted with insulin and the products characterized after purification by dialysis. The chymotrypsin catalyzed hydrolysis of these Cr(III)-insulin complexes has also been investigated. " ... 

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