Helix pomatia enzyme

The action of the sulfatases in the Helix pomatia enzyme is insufficient to achieve complete hydrolysis at least of the 3/8-hydroxy-A steroids in umbilical cord blood and infant urine, and a further stage involving a solvolytic procedure (B40) is essential (S9, Sll). 

At least three, 3j8-hydroxy-A steroids have been shown to be present in urine in diconjugated form of these 16 S-OH-DHA and 21-OH-preg-nenolone are hydrolyzed by the Helix pomatia enzyme, but androstenediol (17a) is freed only by solvolysis (Sll, Fig. 5). The proportions of the individual 3j8-hydroxy-A steroids found in umbilical cord plasma mono-and diconjugated are shown in Table 15. Very little is free, but Simmer and co-workers (S21) have established a concentration of free 16a-OH-DHA of approximately 0.4 jag/100 ml. 

Urine (25 ml) was adjusted to pH 5.2 with acetic acid or NaOH, and 0.5 ml of phosphate buffer, pH 5.2, was added. To 10 ml of this solution were added 0.25 ml of Helix pomatia enzyme preparation and 4 drops of CHCI3. The mixture was incubated at 37 "C for 24 h and then extracted twice with 20 ml of EtOAc. The combined EtOAc layers were extracted twice with 20 ml of NaOH (0.1 M) and twice with 20ml of H2O. The EtOAc layer was dried over MgS04. The solvent was removed until 0.25-0.5 ml remained. The residue was transferred quantitatively, using the minimum amount of EtOAc, to a small test the tube with a Teflonstopper and a pear-shaped base. Internal standards and reference compounds were added, and EtOAc was removed under a stream of... 

The enzymatic hydrolysis is the method of choice when undesired reactions are to be avoided. /Tglucosidases (or /J-glucuronidases) from Helix pomatia digestive juice are now commercially available. They usually show a good hydrolytic performance, even if sometimes need long time reactions (days). Other enzymes are reported to have been used to specifically hydrolize saccharidic bonds like hesperidinase, /J-xylosidase, j3-galactosidase, and mixed crude enzymes like cellulase. 

Barry114 isolated a disaccharide, laminaribiose, on the partial hydrolysis of laminarin by enzymes (digestive juice of the snail Helix pomatia or H. aspersa) or by acid (seven hours with N oxalic acid on the water-bath). He showed that this disaccharide consisted solely of D-glucose and considered it to be 3-j3-D-glucopyranosyl-D-glucopyranose (XXIX). 

Acceptor specificity of a different type was observed by Wallenfels and coworkers. They found that different, isomeric, transfer products were formed at different rates with enzyme preparations from E. coli, calf intestine. Helix pomatia, and Aspergillus oryzae. These results have already been discussed. 

Bile acid glucuronides may be hydrolyzed by -glucuronidase (EC 3.2.1.31) from bovine liver or the digestive juice of Helix pomatia (S41). The latter enzyme preparation also contains sulfatase activity (S41), but this is likely to be an aryl sulfatase which may not use bile acid sulfates as substrates. 

Bacterial lipopolysaccharides have been degraded by enzymic preparations from Helix pomatia, resulting in an extensive loss of anticomplementary activity. The lipid A moiety of the lipopolysaccharide appeared to be the main site of attack with little evidence for the degradation of the polysaccharide component. 

Oyster glycogen has been treated with cyanogen bromide to form a soluble cyclic imidocarbonate derivative which was covalently linked to phosphory-lase b. The enzyme conjugate had enhanced thermal stability and decreased stability to low temperature, while other of its properties were not altered. Glycogen cyclic imidocarbonate yielded on reaction with 1,6-diaminohexane an insoluble product which provided an effective affinity support for adsorption at 0 °C of Helix pomatia a-amylase. Desorption of the enzyme was effected by increasing the temperature. 

The specificity of conduritol B epoxide is such that -glucosidases from widely differing sources have been found to react with loss of enzymic activity from various Aspergillus species, > yeast, snail Helix pomatia) sweet almonds, and mammals. The only exceptions have been the / -glucosidases from garbanzo plants Cicer arietum L.) and from Alocasia macrorrhiza The only other enzymes that have been found to be covalently inhibited are a-glucosidase from yeast Saccharo-myces cerevisiae) and the sucrase-isomaltase complex from rabbit small intestine. ... 

Helix pomatia contains an enzyme capable of splitting chitin to N-acetyl-glucosamine. The work of Zechmeister, T6th, and Vajda indicates the possibility that chitobiose (4-glucosamine D-glucosaminide) may be an intermediate because Helix digestive tracts also contain a chitobiase. 

Beta-glucuronidase Beta-glucuronidase from helix pomatia at 2,701,900 Fishman units per gram (Sigma Aldrich, Oakville, ON, Canada) (see Note 1). Dissolve 0.1 g of enzyme in 12 mL of 0.2 M ammonium acetate buffer. Vortex vigorously and mix for 30 min. Spin and pipette off supernatant for use. Store protected from light at 4°C. 

P-glucuronidase from E. coli is recommended for deconjugation of steroids. The same enzyme from Helix pomatia should be avoided because it converts 3-beta-5-ene-steroids into 3-oxo-4-ene-steroids. P-glucuronidase from Helix pomatia also converts 3-beta-hydroxy-5-alpha-reduced steroids and 3-beta-hydroxy-5-beta-reduced steroids to 3-oxo-5-alpha-reduced steroids and 3-beta-oxo-5-beta-reduced steroids, respectively. 

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