Branching enzymes purification

The enzyme from rabbit muscle may be extracted and crystallized readily, but many recrystallizations may be necessary to ensure freedom from such contaminants as alpha-amylase, debranching enzyme, and branching enzyme. Purification may be facilitated by the use of column chromatography on 0-(2-diethylaminoethyl) cellulose. The enzyme exists in two forms (a and h) that differ in their requirement for adenosine 5 -monophosphate as cofactor. The b form is inactive in the absence of cofactor, and may be converted into the active (adenosine 5 -monophos-phate-independent) a form by phosphorylation of a specific serine residue under the action of (contaminant-free) phosphorylase kinase with adenosine 5 -triphosphate as the phosphate donor. 

Borovsky, D., Smith, E. E., and Whelan, W. J. 1975. Purification and properties of potato 1,4-a-D-glucan 1,4-a-D-glucan, 6-a-(l,4-a-D-glucano)-transferase. Evidence a dual catalytic function in amylose branching enzyme. Eur. J. Biochem. 59, 615-625. 

Sun, C, Sathish, P, Ahlandberg, S, Dieber, A and Jansson, C. Identification of four starch-branching enzymes in barley endosperm partial purification of forms I, Ila and lib. 1997 New Phytol. 137 215-222. 

Mukerjea, R., Falconer, D.X, Yoon, S.H., and Robyt, J.F. 2010. Large-scale isolation, fractionation, and purification of soluble starch-synthesizing enzymes starch synthase and branching enzyme from potato tubers. Carbohydrate Research, 345, (11), 1555-1563. 

F. B. Caudwell, P. Cohen, Purification and subunit structure of glycogen-branching enzyme from rabbit skeletal muscle Eur. J. Biochem. 109 1980, 391-4. 

Branched cydodextrins are also used to increase the solubility of complexes. Two methods are used to make branched cydodextrins, an enzymic method and a pyrolytic method. In the enzymic method, a starch debranching enzyme, such as pul-lulanase, is added to a solution of cyclodextrin and a large excess of D-glucose or maltose to force the reaction to proceed in the reverse direction, i.e. to add rather than remove a branch.69 Since the equilibrium favors the debranching reaction, yields are low and the product typically contains —15% branched cyclodextrin and —85% glucose or maltose. Purification is difficult because of the high solubility of both the glucose or maltose and the branched cyclodextrin, but much of the unreacted cyclodextrin can be removed by crystallization. 

Strain NRRL B-512(F) produces large proportions of the extracellular enzyme dextransucrase,339 which is responsible for the synthesis of linear sequences ofa-D-(l —> 6)-linked D-glucosyl residues. The enzyme transfers the D-glucosyl group from a sucrose molecule to an enlarging dextran chain and liberates the D-fructose portion. As dextransucrase is an extracellular enzyme, production of dextran by cell-free, culture filtrates can result in enhanced yield and quality, and ease of purification of the product. By suitable adjustment of the conditions, products in a chosen molecular-weight range can be obtained. Formation of branches is not yet well understood, but the enzymes responsible will certainly be found. 

Morishige T, Dubouzet E, Choi K-B, Yazaki K, Sato F. Molecular cloning of columbamine O- methyltransferase from cultured Coptis japonica cells. Eur. J. Biochem. 2002 269 5659-5667. Hirata K, Poeaknapo C, Schmidt J, Zenk MH. 1,2-Dehydroreti-culine synthase, the branch point enzyme opening the morphinan biosynthetic pathway. Phytochemistry 2004 65 1039-1046. De-Eknamktil W, Zenk MH. Purification and properties of 1,2-dehydroreticuUne reductase from Papaver somniferum seedlings. Phytochemistry 1992 31 813-821. 

The number of papers and patents appearing in this field in recent years clearly indicate the existence of a new branch of biotechnology, known as "enzyme engineering," dealing with enzyme production, separation, purification and the development of their applications in enzymatic reactors. 

Komuniecki, R., Fekete, S. and Thissen, J. (1985) Purification and characterization of the 2-methyl-branched chain acyl CoA dehydrogenase, an enzyme involved in the enoyl CoA reduction in anaerobic mitochondria of the nematode Ascaris suum. J. Biol. Chem. 260 4770-4777. 

R-Enzyme was first isolated from broad beans and potatoes by Peat and coworkers. Purification was difficult in view of the relatively small amounts present and the large number of contaminating enzymes, particularly a maltodextrinyl transferase (D-enzyme), branching (Q-) enzyme, and alpha-amylase. However, a procedure was developed that gave preparations suflBciently pure to permit routine use of this enzyme during some ten years for debranching of amylaceous polysaccharides. ... 

The sequences of biochemical transformations involved in the synthesis of the aspartate family and branched-chain amino acids in multicellular plants are similar to those that occur in microorganisms. Support for this conclusion has been derived principally from isolation of a number of the requisite enzymes. Information on the kinetic and physical properties of enzymes is best achieved after extensive purification. In contrast, useful predictions of the physiological function of regulatory enzymes depend upon effective enzyme extraction and complete preservation of native properties. Since the latter objective has been emphasized during most investigations of enzymes associated with amino acid biosynthesis in plants, the bulk of our knowledge has been obtained from comparatively crude enzyme preparations. Results of both direct and competitive labeling experiments have added demonstrations of many of the predicted precursor-product relationships and a few metabolic intermediates have been isolated from plants. The nature of a number of intermediate reactions does, however, remain to be clarified notably, the reactions associated with the conversion of dihydropicolinate to lysine and those involved in the synthesis of leucine from 2-oxoisovalerate. 

Enzyme engineering represents a new branch of biotechnology that involves the production of enzymes, separation and purification, as well as the characterization and design of enzyme reactors. The application of enzymes has increased enormously in the last few years, coupled with a series of related developments, such as ... 

Dextransucrases.—Dextransucrase has been purified from Leuconostoc mesenteroides and examined. The structures of dextrans enzymatically synthesized therefrom were studied by periodate oxidation. It was found that the content of (1 -> 6)-glucosidic linkages in synthesized dextran was raised considerably as the purification of enzyme proceeded, and that the content of branch links was increased by adding to the purified enzyme a fraction obtained from the culture broth of the organism by chromatography. 

Bernback et al. [8] effected a purification of the pregastric lipase from calf pharyngeal tissues which was based on chromatographies on octyl-Sepharose and lentil-leetin-Sepharose followed by gel filtration. Dodecylsulfate polyacrylamide gel electrophoresis of the product revealed a molecular mass (M,) of 55 kDa and the enzyme protein was characterized as a glycoprotein with a high content of branched, aliphatic amino acid residues. The on gel filtration was 44-48 kDa. Timmermans et al. [9] have recently cloned the entire coding sequence for bovine pregastric lipase and showed that it exists as a mature 378-amino acid polypeptide with a molecular mass of 42.96 kDa. 

PCCase is a biotinylated protein that catalyzes a reaction required in the catabolism of amino acids and fatty acids of odd-numbered chain length, and in the catabolism and anabolism of branched-chain fatty acids. In order to characterize the structure of this enzyme from plants we undertook its purification. PCCase activity was purified from extracts of maize leaves by a four step scheme that included PEG precipitation, hydrophobic interaction chromatography, anion exchange chromatography and affinity chromatography. This purification scheme achieved a nearly 250-fold purification of PCCase activity. However, throughout this purification of PCCase, ACCase copurified. Indeed, SDS-PAGE analysis of the final purified PCCase preparation identified two biotinylated polypeptides of about 240 and 230 kDa. These polypeptides have previously been described as subunits of ACCase (7). Furthermore, mixed substrate kinetic studies (8) with the purified PCCase/ACCase preparation indicated that the carboxylation of propionyl-CoA and acetyl-CoA were carried out by the same enzyme. Furthermore, both PCCase and ACCase activities were similarly affected by a variety of inhibitors. 

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