0-Galactosidase

Galactosidase, also known as galactosidase hydrolase, includes both the cz-galactosidase, and j8-galactosidase, and some of them can be used for the transfer of galactosyl to prepare branched-CDs. However, the source, substrates and enzymatic properties of these two enzymes vary greatly and are introduced as follows. 

Independent lines of evidence establish a galactosyl-enzyme intermediate [Eq. (3)] in /3-galactosidase catalysis [rather than direct displacement, as in Eq. 

Fortunately, the limitation is relatively easily overcome by measuring the ratio of products in the presence two competing glycosyl acceptors [Eq. (5)]. Here, if a common glycosyl-enzyme intermediate exists, the ratio of products is constant, independent of the leaving group and independent of the ratedetermining step. 

Stokes and Wilson (729) used the approach to established the participation of a galactosyl-enzyme in j8-galactosidase solvolysis. They found the enzyme reaction in 0.247 M methanol and 0.171 M ethanol produced a constant ratio of phenol alkyl-/3-galactoside, with eight arylgalactoside substrates in the former case and four in the latter. 

Galactosyl-enzyme degradation is at least partly rate determining in hydrolysis of very reactive (more acidic leaving group) arylgalactosides such as 2,4- and 

5-dinitrophenylgalactopyranoside, and formation of the complex is rate determining for slower substrates such as phenyl- and p-nitrophenylgalactopyranoside 81, 126, 127, 132). The distinction is important since the latter substrates more directly report characteristics during glycoside bond scission in the chemical step of the reaction. 

Commercially available (3-gal usually is isolated from Escherichia coli and has a pH optimum at 7—7.5. By contrast, mammalian (3-galactosidases usually have a pH optimum within the range 5.5—6 thus interference from endogenous p-gal during immu-nohistochemical staining can be avoided. 

Due to the relatively high molecular weight of the enzyme, conjugates formed with 

Although numerous research articles have been written describing the preparation and use of antibody conjugates with (3-gal, the enzyme remains a minor player in ELISA procedures. Less than 1 % of all commercial ELISA products utilize this enzyme. 

P-Gal has a molecular weight of 540,000 and is composed of four identical subunits of MW 135,000, each with an independent active site (Melchers and Messer, 1973). The enzyme has divalent metals as cofactors, with chelated Mg+2 ions required to maintain active site conformation. The presence of NaCl or dilute solutions (5 percent) of low-molecular-weight alcohols (methanol, ethanol, etc.) causes enhanced substrate turnover. P-Gal contains numerous sulfhy-dryl groups and is glycosylated. 

Due to the relatively high-molecular-weight of the enzyme, conjugates formed with antibodies and P-gal tend to be much bulkier than those associated with AP or horseradish peroxidase. For this reason, antibody conjugates made with P-gal may have more difficulty penetrating tissue structures during immunohistochemical staining techniques than those made with the other enzymes. 

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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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