Synthesis and enzymic hydroxylation of protocollagen model peptide containing a hydroxyproline residue

1. The walls of Micrococcus sp. A1contain about 43% of a phosphorylated polymer. It was extracted with cold trichloroacetic acid and purified by chromatography on DEAE-cellulose. 2. The polymer contained equimolar amounts of d-glucose, N-acetylgalactosamine and phosphate, and was readily hydrolysed under gentle acidic conditions to a phosphorylated disaccharide. 3. Chemical and enzymic degradation indicated that this was 3-O-alpha-d-glucopyranosyl-N-acetylgalactosamine with a phosphomonoester group at the 6-position on the glucose. 4. Related degradation of the polymer itself indicated that the repeating structure was the disaccharide with a phosphodiester residue joining the 1-position on galactosamine to the 6-position on glucose in a neighbouring unit. This polymer is thus another example of the increasing number of microbial wall polymers or teichoic acids possessing sugar 1-phosphate linkages.     

Microbial models of mammalian metabolism. Aromatic hydroxylation

The potential for selected microorganisms to hydroxylate aromatic substrates in a manner analogous to mammalian systems has been studied. Based on literature precedence and prior experience, 11 microorganisms were chosen from among a variety of genera of fungi and bacterial species and were incubated with 13 model compounds including acetanilide, acronycine, aniline, anisole, benzene, benzoic acid, biphenyl, chlorobenzene, coumarin, naphthalene, nitrobenzene, trans-stilbene, and toluene. In most instances, the microbial model system yielded patterns of phenolic metabolites similar to those reported with cytochrome P₄₅₀ monooxygenases of hepatic microsomes and/or in vivo mammalian systems. Furthermore, N-acetylation of aniline, N-deacetylation of acetanilide, and O-demethylation of anisole were found with certain organisms. The potential usefulness of microbial systems for the synthesis of preparative quantities of mammalian metabolites of foreign organic compounds is discussed.     

The pathway of betalain biosynthesis: Effect of cytokinin on enzymic oxidation and hydroxylation of tyrosine in Amaranthus tricolor seedlings

The effect of exogenously added tyrosine or l -3-(3,4-dihydroxyphenyl) alanine on the accumulation of betacyanin in response to cytokinin in Amaranthus tricolor (L.) var. tricolor half-seedlings depends on the age of the seedlings and the treatment of the seedlings prior to induction of pigment by benzyladenine. Neither extracted polyphenol oxidase, peroxidase or tyrosine hydroxylase activity, nor in vivo tyrosine hydroxylation is increased in response to exposure of seedlings to cytokinin. However a small percentage of the polyphenol oxidase activated or unmasked by Triton X-100 treatment of membrane fractions is increased after cytokinin treatment of half-seedlings for 22 h. It is concluded that cytokinin control may be on a multi-enzyme membrane-located complex involving part of the polyphenol oxidase activity of the tissue (possibly an isoenzyme), and that the majority of the polyphenol oxidase activity in Amaranthus is a constitutive membrane enzyme which is not involved in betacyanin synthesis. Although cytokinins do not affect in vivo tyrosine hydroxylation, this activity follows closely the accumulation of betacyanin which is first detectable about 6.5 h after the application of cytokinin. Only a very low level of in vivo hydroxylation can be demonstrated in half-seedlings treated for 6 h either with or without cytokinin but it begins to increase shortly after this time. A large increase in this activity by 16 h (independent of cytokinin) can be almost completely (79%) prevented by chloramphenicol (300 μM) although the drug increases accumulation of betacyanin. At this concentration about 30% of the protein synthesis in inhibited. In vitro tyrosine hydroxylation is, however, not reduced in half-seedlings treated with chloramphenicol during 16 h induction nor is extractable polyphenol oxidase reduced. It is concluded that chloramphenicol is inhibiting the synthesis of some protein essential for in vivo hydroxylation other than the activity measured during in vitro hydroxylation and that the inhibition of synthesis of 79% in vivo hydroxylation still leaves enough activity for maximum betacyanin synthesis.     

Kinetic models for enzymic cellulose degradation in aqueous two-phase systems

Cellulose materials can readily be degraded into cellobiose and glucose by hydrolysis of the enzymes cellulase and beta-glucosidase in aqueous media. Product inhibition does, however, retard the reaction rate and reduce productivity. This may be avoided by carrying out the degradation of cellulose in an aqueous two-phase system, which permits the enzymes and the substrate to be partitioned to one phase and the products to be extracted into a second phase. In addition, two-phase systems also allow recycling of the enzymes. Here, three models previously developed for "one-phase" enzymic degradation are compared to data from enzymic degradation in an aqueous two-phase system. The models tested agreed relatively well with batch experiments during a period of 200 h. For one of the models tested, continuous degradation also gave accurate agreement.     

The enzymic hydrolysis of amygdalin

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond beta-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the beta-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components.