Amylases purification

In 1833 an amylase from germinating barley was recovered and called diastase (1). Like malt itself, this product converted gelatinized starch into sugars, primarily maltose. Shordy thereafter, BerzeHus proclaimed the existence of non-living catalysts, and Schwaim (2) reported on his observation and purification of pepsin. 

If amylases are to be used as tools for the detailed study of the breakdown and structure of their substrates it is obviously important to separate them from other enzymes and from other naturally associated constituents which may influence the results. It is then equally important to study the properties of the purified amylase and to supply it with the chemical environment necessary to protect it from inactivation and to enable it to act efficiently. With beta amylases this ideal has often been approached. Beta amylases from several sources have been prepared by selective inactivation of other enzymes that accompany them in nature23 and highly active products have been obtained by extensive purification.20 24-26 Balls and his associates have recently reported the crystallization of beta amylase from sweet potato.27... 

The general status of the alpha amylases with respect to the above ideal is not so satisfactory. The difficulties involved in the purification of these amylases are increased because there are no simple criteria by which the investigator can make sure that the alpha amylase under consideration is not contaminated by beta amylase or by some other gluco-sidase.1 8,6 18 28 However, evidence is accumulating to indicate that these alpha amylases have important individual characteristics which will differentiate them from one another. Such characteristics should make these enzymes increasingly useful in the study of their substrates and thus amply repay continued efforts at their purification. 

Pancreatic amylase is very labile and sensitive to its chemical environment. Its lability is accelerated by purification and by such factors as dilution of its aqueous solutions, dialysis of its aqueous solutions against water, unfavorable hydrogen ion activities and unfavorable temperatures.29-31, 36,33 The loss of amylase activity in solutions of pancreatic amylase increases with increasing temperature and is very rapid between 50° and 60°. The inactivation of pancreatic amylase in aqueous solution may be retarded by the addition of certain anions, of which the chloride ion is outstanding 37-39 by the addition of certain cations, of which... 

Even after extensive purification, the amylase of Aspergillus oryzae is relatively stable in aqueous solutions held at ordinary room temperature. Its lability increases with increasing temperatures and becomes very rapid between 50° and 60°. This loss of activity may be retarded by the presence of substrate and by the presence of calcium ions.4070... 

Examination of the ratios of the dextrinogenic to the saccharogenic activities of malted barley extracts before and after treatment shows that the results of the Ohlsson procedures23 are not always predictable.8 The concentration of the amylases in the extracts, and the kinds and concentrations of substances which accompany them, influence the results. The presence or the absence of calcium ions is an important factor. Calcium ions increase the inactivation of beta amylase of malted barley and protect the alpha amylase from inactivation at unfavorable temperatures and also at unfavorable hydrogen ion activities.28 With purification, both amylases become increasingly thermolabile and increasingly sensitive to unfavorable hydrogen ion activities.78... 

Malto-oligosaccharide aldonolactones react with ethylenediamine to give Ar-(2-aminoethyl)aldonamides (113-115), which have been successfully grafted onto carriers via amide linkages. The malto-oligosaccharides were produced by degradation of amylose with alpha amylase. After purification of the oligosaccharides, they were converted into the lactones by hypoiodite or electrolytic oxidation. 

The purification of some enzymes inactivates them because substances essential for their activity but not classed as a prosthetic group are removed. These are frequently inorganic ions which are not explicit participants in the reaction. Anionic activation seems to be non-specific and different anions are often effective. Amylase (EC 3.2.1.1), for example, is activated by a variety of anions, notably chloride. Cationic activation is more specific, e.g. magnesium is particularly important in reactions involving ATP and ADP as substrates. In cationic activation it seems very likely that the cation binds initially to the substrate rather than to the enzyme. 

The pore size of porous titania can be up to 2000 A. Titania is used for the purification of proteins and as a support for bound enzymes. The purification of /1-lactoglobulin from cheese whey, of protease from pineapple, /5-lactamase, and amylase can be achieved with titania. The latter two purifications are impossible on alumina. Titania is also used as a support in peptide synthesis. The separation of plasmid DNA is shown in Figure 3.24. 

Table III compares the biochemical features of the B-amylase which was purified to homogeneity from C. thermosulfurogenes (74). The enzyme is a tetrameric glycoprotein with an apparent molecular weight of 210,000. The thermophilic B-amylase binds tightly to raw starch presumably by glycoconjugate forces and it is still active while bound to starch (75). This feature has been used for improved affinity purification of the enzyme using raw starch (76).

J. J. Marshall and C. M. Lauda, Purification and properties of phaseolamin, an inhibitor of a-amylase, from the kidney bean, Phaseolus vulgaris, J. Biol. Chem., 250 (1975) 8030-8037. 

Very recently, Lazarova and Tonova [53] reported an integrated process for extraction and stripping of a-amylase using RMs in a stirred cell with separated compartments for each process. A comparison between the classical process and the integrated process indicated a 1.27-fold enhancement in the enzyme purification by the latter. This integrated process was operated with 100 ml volume in... 

The purification of the pancreatic alpha amylase was effected on an affinity adsorbent prepared from enzymically degraded starch plus agarose activated with bisoxirane.14 The fractions from the affinity column were analyzed for protein components by u.v. absorbance, and for alpha amylase activity by incubating the fractions with starch and measuring the increase in reducing sugars. The results are shown in Fig. 5. 

Nirmala, M. and Muralikrishna, G. (2003b). Three a-amylases from malted finger millet (ragi, Eleusine coracana, Indaf-15)—Purification and partial characterization. Phytochemistry 62, 21-30. 

Saxena, L., Iyer, B. K., and Ananthanarayan, L. (2007). Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor process. Biochemistry 42, 491-495. 

There are several possible arrangements tolerating the presence of particles during adsorption of proteins to particulate matrices. Batch adsorption in stirred tanks is performed by contacting adsorbent particles with a cell containing suspension. After protein capture the adsorbent is separated from the broth and the protein of interest can be eluted. This procedure has been described for the isolation of antibiotics [12], the purification of ot-amylase from B. amylo-liquefaciens broth [13], and the isolation of the prothrombin complex from... 

Feller, G., T. Lonhienne, C. Deroanne, C. Libioulle, J. Van Beeummen, and C. Gerday. 1992. Purification, characterization, and nucleotide sequence of the thermolabile a-amylase from the Antarctic psychrotroph Alteromonas haloplanctis A23. Journal of Biology Cbemisty 267 5217-5221. 

A different method was developed for the purification of pancreatic amylase.4 This enzyme differs somewhat in its solubilities and considerably in the conditions governing its activity, from the corresponding enzyme of malt but the purified amylase preparations from the pancreas and from malt are much alike in respect to the chemical properties above mentioned. Pancreatic amylase is much less stable than malt amylase and much more quickly suffers a loss or diminution of enzymic activity in solution, which as in the case of malt amylase appears to be due, at least chiefly, to hydrolysis of the enzyme molecule. 

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