Galactosidases

The scope of the present review will be limited to the enzyme and protein characteristics of galactosidases, and their occurrence, isolation, characterization, and properties as catalytic agents. The topic of induced enzyme-synthesis will only be touched on, and reference will be made to original and review articles on the subject. The biological role played by these enzymes will not be discussed. Throughout the article, all glycosides discussed will be understood to be pyranoid, unless otherwise noted. 

Among the animals, it is found in snails Helix pomatia), and in the intestines of dogs, rabbits, calves, sheep, and goats. - In the calf, a 

Procedure Take 0.5 ml. of o-nitrophenyl fl-D-galactoside solution, 0.125 ml. of the salt solution, 0.1-0.5 ml. of the enzyme solution, and make to 2.5 ml. with buffer. The buffer, sodium chloride, and substrate solutions are measured into a photometer cell (d — 1 cm.), and brought to the required temperature (20°). The reaction is started with enzyme and is followed by noting the optical density at 405 m/i (against water) every 30 seconds. The increase of optical density (AE) in 2 minutes is taken for calculation of the turnover number. The molecular extinction coefficient of o-nitrophenol at 405 mp and pH 7.6 is 3.1 X 10 cm /mmole.  

Procedure A diluted bacterial suspension (5 ml., containing 0.2 mg. of dry bacteria per ml.) is shaken with 0.1 ml. of toluene for 15 minutes in a J,-shapcd tube fixed on a rotator inclined at an angle of 5°. This suspension (0.1-0.5 ml.) is then added to 4.0 ml. of the solution of o-nitrophenyl 3-D-galactoside and phosphate buffer is added to make 5.0 ml. The reaction mixture is incubated at 40°, and the optical density (at 405 mp) is noted (against water) after 15 minutes. The optical density of a reagent blank, run side-by-side is subtracted from the test value. 

and Reithel, who crystallized /3-galactosidaso from E. coli, ML 308, defined the unit of enzyme activity as the amount bringing about the hydrolysis of 10 mole of o-nitrophenyl /3-n-galactoside per minute at 10 M substrate concentration in 0.05 M sodium phosphate buffer, pH 7.0, at 25°. 

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Beta-cydodextnn Beta-galactosidase Beta-globin Betaine... 

If an antibody to the protein of interest is available, it is sometimes possible to use vector sequences, eg, the beta-galactosidase promoter sequence, to direct the transcription of the passenger DNA into messenger RNA and the translation of that mRNA into protein which can be recognized by the antibody. Although this method is somewhat less reHable than the use of nucleic acid probes, specialized vectors are available for this purpose. 

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Lactase (P-galactosidase) is produced commercially from the lactose fermenting Klujveromjcesfragilis. The enzyme has a pH optimum of 6—7 and is used ia the hydrolysis of lactose ia milk or skim milk. 

Procedures of the beta-galactosidase activity measuring using colour reaction with ONPG and X-Gal without cells permeabilization were developed and the detection limit at the level of 4 ppb has been achieved. The influence of the foreign ions (phosphate, sulphate, carbonate et. al) was studied. 

Goebel and Avery J Exptl Medicine 50 521 7929 Snyder and Link J Am Chem Soc 75 1758.] chromogenic substrate for P-galactosidases [Buoncore et al. J Appl Biochem 2 390 1980]. 

What is the amino acid sequence of the fusion protein Where is the junction between /3-galactosidase and the sequence encoded by the insert (Consult the genetic code table on the inside front cover to decipher the amino acid sequence.)... 

Husum et al. found that the hydrolytic activities of P-galactosidase from E. coli and the protease subtilisin in a 50 % aqueous solution of the water-miscible ionic liquid [BMIM][Bp4] were comparable to those in 50 % aqueous solutions of ethanol or acetonitrile (Entry 9) [37]. 

The widely commercially exploited guar GaM has been the subject of some studies dealing with chemical or enzymic modifications aimed to extend the apphcation range of this polysaccharide. Specific oxidation on the C-6 position of the Galp side chain units was performed by /1-galactosidase [241,430]. 

Several lipases were more efficient than PLE and subtilisin Carlsberg for the desymmetrization of an N-t-butoxycarbonyl (Boc) meso-piperidine diester (Figure 6.13). The (3R)-monoester was converted into optically pure isogalactofagomme, a potent galactosidase inhibitor [60]. 

As in the alkaline phosphatase example above, p-galactosidase, an enzyme with a molecular weight of approximately 360,000, has also been incorporated into a polyanhydride and released in a well-controlled fashion. As is shown in Fig. 14, the release of 3-galactosidase was quite linear over most of the time examined, and was complete, reaching 100% release in about 800 hr. This experiment utilized 5% loaded, compression-molded wafers of PCPP-SA 9 91, 1.4 cm in diameter and 0.5 mm thick, weighing 50 mg. 

FiCURE 14 Release of 3-galactosidase from compression-molded discs of PCPP-SA 9 91. Details were as described in the text. 

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